JM

BES Vol. 14, No. 1, 2012 (ISSN 0972-3005)

Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 7-12
© Global Science Publications

UREA ACCUMULATION IN ACID WATER BY THE AFRICAN

LUNGFISH, PROTOPTERUS ANNECTENS (OWEN) OF
OGUTA LAKE, NIGERIA
A.I. OKAFOR

Department of Animal and Environmental Biology, Abia State, University, Uturu, Nigeria

(Received 1September 2011; Accepted 30 September 2011)

Key words : Acid water, Serum urea, pH, Protopterus annectens.

Abstract - Serum urea levels of the African lungfish, Protopterus annectens (Owen) following acclimation in
acid water was investigated in the laboratory. Adult specimens of the African lungfish, Protopterus
annectens (mean weight, 299.5g and mean length, 38.2cm) procured from Oguta lake at Oguta in Imo
State, Nigeria were subjected to acid water of the following pH values - 4.6, 5.0, 5.4,5.8, 6.2,6.6, 7.0 and 7.4
for a couple of weeks after which their serum urea levels were determined. The results revealed a
significant negative linear correlation (r = - 0.9435776 p<0.05) between water pH and mean serum urea
concentrations. The findings highlight some adjustments of the fish’s metabolic pathways in acid water
regimes.

INTRODUCTION compound that is excreted as a semisolid mass

Urea is a nitrogenous body waste product formed
from the catabolism of proteins (Frick and Wright,
2002; Tay, et al., 2003,2006). Amino nitrogen is
chiefly excreted as either ammonia, urea or uric
acid (Chew et al., 2004a; Welhrauch et al., 2009). The
basis for these differences lies in the physiology of
different organisms in relation to their habitats.
Since ammonia is freely soluble in water, most
aquatic teleosts quickly sweep it away and diluted
by the large volume of water that passes via the
gills. However, some formerly aquatic species that
have now evolved to live on dry land cannot afford
the expenditure of such volumes of water in order
to excrete ammonia. Rather, they evolved the
means of excreting amino nitrogen in the form of
urea, a neutral, highly soluble and nontoxic
molecule (Ip et al., 2001; Walsh et al. 2001;
Welhrauch et al., 2009).
In truly terrestrial vertebrates such as Aves and
Reptilia, the excretion of urea into urine requires
that some volumes of water be excreted as well.
Birds and lizards cannot afford that and so have
evolved the means of excreting amino nitrogen in
the form of uric acid, a relatively insoluble
containing very little water (Chew et al., 2004a).
The African lungfish, Protopterus annectens lives in
some shallow and swampy parts of some rivers
and lakes of West Africa. (Ndukuba et al. 2007;
Okafor 2008ab,2009; Hung et al 2009; Okafor and
Chukwu, 2010) Studies on its acid tolerance have
just begun (Okafor et al., 2006). The studies,
however, have established that the lowest water
pH at which P. annectens can tolerate is 4.6 (Okafor et
al., 2006). Therefore there is possibility of culturing
P. annectens in some moderate acid waters.
Consequently it is necessary to study some possible
excretory adjustments which the fish can make
whilst subjected to acid water regimes. Many
industrial and domestic effluents, when they enter
a water body can reduce that water pH
considerably by increasing the number of
hydrogen ions (Schofield, 1976; Cole, 1979).
The objective of this work is therefore to
determine precisely changes in serum urea levels of
P. annectens in waters of varied acid concentrations.
It is hoped that the information obtained from this
study would be quite useful in the culture of the
fish in acid waters or in areas where human
utilization of water contributes to high level of C02

*Corresponding author - E-mail : tconnection68@yahoo.com

8 OKAFOR

in that water body such as near sewage and some Table 1. The preparation of 3 litres each of citric acid /
industrial, domestic and even thermal effluents. disodium hydrogen orthophosphate buffer solutions of
various pH values
MATERIALS AND METHODS Volume of 0.2 Volume of pH of the
Molar disodium 0.1 Molar mixture
Live specimens of the African lungfish, P. annectens hydrogen ortho- citric acid at 260 C
phosphate C6H807.H20
obtained from Oguta lake at Oguta in Imo State,
Na2HPO4 (litres) (litres)
Nigeria were brought into Physiology laboratory
in Animal and Environmental Biology Department 1.40 1.60 4.6
of Abia State University, Uturu -Nigeria. 1.55 1.45 5.0
After the determination of their standard 1.67 1.33 5.4
lengths and weights, they were allowed to 1.81 1.19 5.8
acclimate at room temperature for 3 weeks inside 1.98 1.02 6.2
seven plastic tanks that measured 0.54 x0.38 2.18 0.82 6.6
x0.30m, which were neither aerated with air 2.47 0.53 7.0
2.73 0.27 7.4
pumps nor covered, and each of which contained
about 6 specimens with only 3L of dechlorinated
water. Consequently all specimens were mildly
The fish were fed on insect larvae, boiled rice and anaesthesized with chloroform and by the use of
beans and fish feed obtained from the Ministry of 5ml disposable syringes, about 1.0mL of blood was
Agriculture and Natural Resources, Owerri, in Imo drawn from the caudal blood vessels of each fish.
State, Nigeria ad libitum until used for the Each blood sample was centrifuged in a
experiment. The water in all tanks was changed Gallenkamp centrifuge at 3000 revolutions per
twice weekly to prevent accumulation of excess or minutes for 15 minutes. The residue was discarded
uneaten food, waste materials and the fish’s and the supernatant (serum) urea level was
mucous secretions. determined by the use of Diacetylmonoxime
Three litres each of the following pH buffer standard method ( Brewer et al., 1974; Baker and
solutions were prepared : 4.6, 5.0, 5.4, 5.8,6.2,6.6,7.0 Silverton, 1982).
and 7.4. This was carried out by mixing a certain
volume of 0.1M citric acid monohydrate (molecular RESULTS
mass, 210.14) with a corresponding volume of 0.2M
disodium hygrogen orthophosphate (molecular The mean percentage weight changes (PWC) at
mass 141.98). The actual pH of each prepared buffer various pH values are illustrated in Fig. 1.
solution was ascertained with a pH meter at 260C. Regression analyses shows a significant negative
(Table 1) pH buffer solutions below 4.6 were not correlation between water pH and PWC. (r = -0.898;
prepared because P. annentens does not, survive in p<0.05); and a significant negative correlation
water bodies below a pH of 4.6 (Okafor et al., 2006). between water pH and serum urea levels (r = -
The prepared buffer solutions were transferred 0.944, p<0.05 respectively (Figs. 1 and 2).
into 8 plastic tanks that measured 0.54 x 0.38 x
0.30m, each tank containing a particular pH buffer. DISCUSSION
24 healthy and active specimens of P. annectens
which had no obvious signs of external injuries or Protopterus, which has an excretory physiology
wounds were selected from amongst the survivors similar to that of other freshwater teleosts was able
of acclimation and introduced into the above eight to get rid of its nitrogenous metabolic wastes in the
tanks at a stocking rate of three specimens per tank. form of ammonia (Ip et al., 2005). However, the
All fishes were fed and all tanks were neither excretion of metabolic wastes in the form of
covered nor aerated with aerators throughout this ammonia entails that a relatively large volume of
period when they were immersed in acid waters of water be expended ( Hung et al., 2009; Meyerriecks,
various pH values. 2010). Many aquatic bony fishes can afford that
After 3 weeks of immersion in the above acid since ammonia is freely soluble in water and can be
waters, blood was extracted from all specimens so quickly swept away by the large volume of water
as to determine their serum urea levels. that passes through the gills. Ever since P. annectens
 Urea Accumulation in Acid Water by the African Lungfish, Protopterus annectens (Owen) 9

Fig. 1 The mean percentage changes in body weights at various pH values r= -0.944

Fig. 2 The mean serum urea levels (mg/dl) at various water pH values

was introduced into acid water, it was losing much
body water (Fig. 1) and thus had the problem of
increasing dehydration. The fish was now faced
with the threat of desiccation since no water was
ingested and still minute quantities of water were
lost via the lungs during breathing.
Consequently, it had to do something about this
continuous loss of body water through ammonia
excretion. On the other hand, ammonia is not
supposed to be accumulated in the body fluids
10 OKAFOR

Appendix: The percentage changes in body weights and serum urea levels at various pH values in specimens of
Protopterus annectens (O)

Sp(cm) S.L (cm) BWBIA (g) BWAIA (g) P.W.C. (%) S.U.L. mg/dl pH of water
medium

A 40.1 300.5 267.1 -11.1 285 4.6

B 41.0 303.8 274.6 -9.6 320 4.6
C 38.2 269.9 241.6 -10.5 260 4.6
D 40.5 284.6 268.1 -5.8 280 5.0
E 40.0 279.8 269.7 -3.6 240 5.0
F 37.2 260.8 239.4 -8.2 315 5.0
G 38.6 297.5 289.8 -2.6 265 5.4
H 33.9 179.5 175.5 -2.2 305 5.4
I 36.9 321.9 311.9 -3.1 175 5.4
J 36.6 242.4 236.6 -2.4 300 5.8
K 37.1 262.5 257.5 -1.9 265 5.8
L 38.4 301.2 297.9 -1.1 240 5.8
M 35.8 220.7 219.8 -0.4 185 6.2
N 40.7 393.4 390.2 -0.8 170 6.2
0 37.3 322.6 320.7 -0.6 205 6.2
P 42.0 398.7 399.5 0.2 95 6.6
Q 35.2 188.8 190.7 0.1 140 6.6
R 30.3 294.4 293.5 -0.3 115 6.6
S 34.7 185.0 185.6 0.3 28 7.0
T 41.9 491.2 493.7 0.5 29 7.0
U 40.3 307.2 308.4 0.4 25 7.0
V 41.8 477.5 482.8 1.1 28 7.4
W 36.9 296.6 298.1 0.5 29 7.4
X 41.0 306.5 308.3 0.6 30 7.4

Sp =Specimen ; SL = Standard lengths ; BWBIA = Body weights before introduction into acid water
BWAIA = Body weight after introduction into acid water ; PWC = Percentage weight change
S.U.L. = Serum urea level

since it is poisonous (Ip et al., 2001; Chew et al.,
2004a). The best option then was to detoxify
ammonia and convert it to the non- poisonous
urea. Studies have shown that urea accumulation
in fishes elevates solute concentration and body
fluid osmolality (Walsh et al., 2001; Okafor, 2007). It
also reduces vapour pressure of body fluids and
therefore minimizes evaporative loss of water
through the skin ( Janssens and Cohen, 1968;
Forster, 1970; Jones, 1980; Okafor, 2207; Oliekwe,
2010). It has been reported that urea plays the same
role in terrestrially adapted toads (Forster, 1970)
and in some land snails (Horne, 1971). All the
enzymes of the urea cycle have been detected in the
liver of P. aethiopicus of lake Victoria in Uganda
(Janssens and Cohen, 1966).
Such similar findings of increased urea
biosynthesis as well as urea accumulation have
been documented by Janssens and Cohen (1968) and
Funkhouser et al., (1972) during aestivation of the
East African lungfish, Protopterus aethiopicus and
the South American lungfish, Lepidosiren
paradoxa respectively. Chew et al., (2003) also gave
evidence of increased urea biosynthesis as well as
accumulation in the blood of the African lungfish,
Protopterus dolloi of Congo River Basin just after six
days of complete aerial exposure. Both Chew et al.,
(2004b ) and Ip et al., (2005) reported reduction in
ammonia production, but increased urea
biosynthesis in P.dolloi due to change of habitat.
Tay et al., (2006) also reported a reduced ammonia
excretion and increased urea excretion in the
climbing perch, Anabas testudineus when brought out
from water and exposed briefly to aerial life. Okafor
(2008b) reported tremendous increase in urea
levels during aestivation of the West African
lungfish, Protopterus annectens (O). Okafor (2010) also
reported increased urea biosynthesis in P. annectens
when exposed to brackish water (S=11.0‰) for a
few weeks. Hung et al., (2009) also gave evidence of
massive urea excretion in P.annectens when exposed
to terrestrial life.
 Urea Accumulation in Acid Water by the African Lungfish, Protopterus annectens (Owen)
The paper therefore suggests that in acid water,
there was a slight shift in the metabolic pathway of
P. annectens from ammonotelism in waters of
neutral pH to ureotelism in acid waters especially
in lower pH waters.
ACKNOWLEDGEMENT
The author wishes to thank Messers Clement
Asomugha and Uche Arukwe; both are Chief
Technologists in the Department of Biochemistry, of
Abia State University, Uturu - Nigeria as well as Dr.
Eddy Onuoha, another Chief Technologist in the
Department of Veterinary Microbiology and
Pathology of the University of Nigeria, Nsukka for
their technical assistance.
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—❋❋❋—
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Tay, Y.L., Loong, A.M., Hiong, K.C., Lee, S.J., Tng, Y.Y.,
Wee, N.L., Lee, S.M., Wong, W.P., Chew, S.F and
Wilson, J.M. 2006. Active ammonia transport and
excretory nitrogen metabolism in the climbing
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emersion or 10 minutes of forced exercise on land.
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Walsh, P.J., Grosell, M., Goss, G.G., Laurent, P., Bergman,
H.L., Bergman, A.N., Wilson, P., Alper S., Smith,
C.P., Kamunse, C. 2001. Physiological and
molecular characterization of urea transport by
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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 13-18
© Global Science Publications

INCIDENCE AND ANTIBIOTIC SENSITIVITY OF BACILLUS

CEREUS ISOLATED FROM READY TO EAT FOODS SOLD IN
SOME MARKETS IN PORTHARCOURT, RIVERS STATE, NIGERIA

O. K. AGWA, C.I. UZOIGWE AND E.C. WOKOMA

Department of Microbiology, University of Port Harcourt, Rivers State, Nigeria

Department of Crop and Soil Science, Faculty of Agriculture, University of Port Harcourt, Nigeria

(Received 20 August 2011; Accepted 30 September 2011)

Key words : Antibiotic sensitivity, Bacillus cereus isolates, Microbiological standards, Ready-to-eat indigenous

Abstract - Sixty-four food samples of “cooked rice”, “masa”, “agidi” and “epiti” purchased from local
markets in Obio-Akpor Local Government Area of Port Harcourt, Nigeria were examined
microbiologically. Plate count analysis on mannitol egg-yolk bromothymol blue polymyxin B (MYP) agar
revealed that “cooked rice” had the highest frequency of occurrence of Bacillus cereus isolates (29.51%),
“masa” (26.23%), “agidi” (22.95%) and “epiti” had the least frequency of 21.31%. All Bacillus cereus
isolates tested were found to be susceptible to rifampin (30µg), chloramphenicol (20µg), erythromycin
(30µg), ciprofloxacin (10µg), streptomycin (30µg), gentamycin (10µg) and lincocin (30µg) and 100%
resistance against norfloxapin (30ug), floxapen (30µg) and ampiclox (30µg). The study unveils the
presence of Bacillus cereus in food samples sold in Port Harcourt especially in ‘cooked rice” and “masa”
which may pose serious threat to the health of consumers and should not be ignored.

INTRODUCTION Dierick et al., 2005). Bacilli cause symptoms leading to

Present economic conditions have resulted in
situations, where food vending has become
increasingly important in most countries;
contributing significant income inflow for
households involved in selling these foods
(Mosupye and vonholy, 1999; FAO/WHO, 2003;
FAO/WHO, 2005 ). Hygiene and sanitation practices
obtained during preparation and marketing of
these foods provide ample opportunities for the
proliferation of these food with food-borne
pathogens (Desai and Varadaraj, 2009). Among the
food- borne pathogens, strains of Bacillus cereus are
of significance because of its ubiquitous nature and
ability to occur in a wide range of foods (Ehling-
schulz, et al., 2004a; Oguntoyinbo and Oni, 2004;
Reyes et al., 2007).
The unique properties of Bacillus cereus includes
heat resistant endospore forming ability, toxin
production in varieties of foods and the psychro-
trophic nature makes the organism a prime cause
of public health hazard (Griffith and Schraft, 2002;
illness, relatively mild lasting up to 24h, but could
be severe leading to hospitalisation and in some
cases death. The organism is found in a wide range
of habitat including air, water, dust and soil, from
where spores are introduced into cereal crops,
vegetables, animal hair, fresh water and sediments.
Raw plant foods especially rice, potatoes, beans,
peas, and spices are the most common sources of
Bacillus cereus (Eglezos et al., 2010). The occurrence of
the organism within the environment enables it to
enter the food chain through raw materials and is
a major problem in convenience foods and mass
catering (Beathe and William, 2000; Jensen et al.,
2005; Guinebretiere et al., 2006). Because of the role
of microorganisms in spreading diseases, the need
to assess the safety and quality of foods is very
important, in order to ensure safety of supply,
clean, wholesome and high quality delivery to the
public. When the food hygiene system fails, a
batch of food is contaminated with high level
microbes potential for food borne disease outbreak,
its distribution cannot be controlled, changes in

*Corresponding author - Obioma Agwa, E-mail : o_agwa@yahoo.com

14 AGWA
public health and deterioration and increases
susceptibility rate (Sahota et al., 2008).
In several studies that have been conducted on
the incidence of food-borne pathogens, emphasis
had been on Bacillus cereus (Yusuf et al., 1992; Umoh
and Odoba, 1999; Umar et al., 2006; Desai and
Varadaraj, 2009). The microbiological safety of foods
sold in the market is of major concern because of the
environment in which they are prepared, often in
places that may have poor sanitation, coupled with
use of containers which expose the food to
numerous potential contaminants such as heavy
metals and pesticides. Some of the food samples
have been tested for various microorga-nisms of
public health concern and Bacillus cereus was among
them (Tomlins et al., 2004).
This study was carried out to evaluate the
presence of Bacillus cereus in ready- to -foods
purchased from markets in Port Harcourt and
characterize the organism isolated in terms of
biochemical reaction and antibiotic susceptibility.
MATERIALS AND METHODS
Sampling site and collection
A total of sixty four food samples being sold in
various sales points in urban markets at Mile I,
Mile III, Oil mill, and Rumuokoro areas of Port
Harcourt were enumerated for the viable counts of
Bacillus cereus. The food samples comprised of
Sixteen samples each of “cooked rice”, “masa”,
“agidi” and “epiti”(cereal preparation) were
collected from the food vendors in sterile
disposable polyethylene bags brought to
laboratory for microbiological analysis. Analysis of
samples were performed within 60mins of
collection
Table 1. Ingredients of the food samples analysed
Samples Ingredients
Cooked rice Rice, meat, groundnut oil,
Seasonings(salt, onions, pepper,
tomato paste)
Masa Rice and raw milk
Agidi Powdered corn grain & seasonings
Epiti Powdered corn grain, plantain flour,
palmoil, seasonings.
Enumeration of Bacillus cereus
A homogeneous sample was prepared with 10g of
ET AL.
food sample, aseptically transferred into 90mL of
tryptone broth to give an initial 1:10 dilution.
Aliquots of 0.1mL from 10-5dilutions were surface
plated on sterile dried Mannitol egg-yolk
bromothymol blue polymyxin B (MYP) agar plates.
The plates were incubated at 37C for 24h examined,
then left for another 24h at room temperature and
re-examined. Sixteen presumptive colonies of
Bacillus cereus were randomly selected and counted
based on characteristic colony feature. The colonies
were purified on freshly prepared MYP agar plates.
After incubation, typical colonies of bluish green -
blue colonies with zones of egg yolk precipitate on
the medium were picked, streaked out to obtain
pure cultures and finally maintained on nutrient
agar slopes in the refrigerator (4C). The
presumptive isolates were identified by
morphological characteristics. The morphological
test includes appearance of cell, shape and
pigmentation. Further identification of the
organisms using biochemical characteristics are
Gram’s reaction, position of spores, motility, citrate
utilisation, oxidase, indoleproduction, urease,
voges-proskauer reaction, hydrolysis of starch,
nitrate reduction, gelatine hydrolysis, production
of gasandacid from glucose, sucrose, mannitol and
lactose (Cappuccino and Sherman, 2004).
Antibiotic sensitivity test
All Bacillus cereus isolates were tested for their
sensitivity to antibiotics by means of a disc
diffusion method (Bauer et al., 1966). The organisms
were investigated using antibiotics disc containing
rifampin (10µg), chloramphenicol (20µg),
erythromycin (30µg), ciprofloxacin (10µg),
streptomycin (30µg), gentamycin (10µg) ), floxapen
(30µg), ampiclox (30µg), norfloxapin (30ug),and
lincocin (30µg). The antibiotics disc were
spaciously placed on Mueller - Hinton agar plates
previously seeded with 6h-broth cultures of
Bacillus cereus isolates. The plates were incubated
at 37oC for18-24h. The different zones of inhibition
were measured to the nearest millimetre and
interpreted as sensitive, moderate sensitive and
resistant based on the interpretation table
recommended by the disc manufacturer (Oxoid,
1998).
RESULTS
The results of Bacillus cereus in some cereal based
ready to eat foods sold in Port Harcourt is depicted
 Incidence and Antibiotic Sensitivity of Bacillus Cereus Isolated from Ready to Eat Foods Sold in
in Fig. 1 and Table 2. Among the various markets
sampled, Oil mill market had the highest microbial
load of the organism, followed by rumuokoro, mile
3 and finally mile 1 market. A total of sixty-four
food samples “cooked rice”, “masa”, “agidi” and
“epiti” were assessed. The average count from the
food samples showed that about 7.2 x 105cfu/mL
were obtained from cooked rice, 6.4 x105cfu/mL
from masa, 5.6 x 105cfu/mL from agidi and 5.3 x
105cfu/mL from epiti. Sixteen presumptive colonies
of Bacillus cereus isolated revealed an irregular edge,
large, flat, rough bluish green to blue colonies with
zones of egg yolk precipitate. Antibiotic sensitivity
test of the isolates determined by the disc diffusion
method in accordance with the instructions of the
antibiotics disc manufacturer are shown in Table 3.
The isolates were highly resistant to ampiclox,
norfloxacin & floxapen, sensitive to erythromycin,
chloramphenicol, streptomycin rifampin and
ciprofloxacin and less sensitive to lincocin and
gentamycin.
Table 2. Cumulative plate count and total percentage
from various food samples
S/No Sample No. of Total % total
tested samples counts
(x106cfu/g)
1 CR 16 72 29.51
2 MS 16 64 26.23
3 AG 16 56 22.95
4 EP 16 52 21.31
Total 64 244 100
Keys: CR = cooked rice, MS = masa,
AG = agidi; EP = epiti
DISCUSSION
The public health significance of Bacillus cereus is of
high concern in view of the organism being
implicated in a number of food poisoning
outbreaks. The isolation of Bacillus cereus from all
the food samples could be explained by the
ubiquitous distribution of this organism and its
ability to form endospores (Kotiranta et al., 2000;
McKillip, 2000). A total of 64 samples (cooked rice,
masa, agidi and epiti) were analysed. From the
results obtained in the study, there were high
contamination levels of Bacillus cereus found in
cooked rice (7.2×105cfu/g) this is not surprising
because the organism is a normal flora of rice
followed by Masa (6.4×105cfu/g), a cereal
15
preparation from rice which harbour the
organism naturally with total percentage count of
26.23%. Masa was followed by agidi with 5.6 × 105
cfu/g and a total percentage count of 22.95% and
lastly epiti (5.3×105cfu/g) with total percentage
count of 21.31%. The variations in the value of
bacterial count percentages may be attributed to
numerous contaminations potential of the
organism, as most of the food samples were
prepared in places with poor sanitation practices,
poor storage and transport conditions. In most
cases, food vendors do not have adequate bathing
facilities, atimes starts their day without taking a
bath, therefore foods and ingredients are exposed to
repeated contamination from unwashed hands
and materials used for wrapping such as leaves,
newspaper and reusable polyethylene bags.
Purchasing ready-to-eat foods from markets
exposes one to a considerable risk of public health
due to poor hygiene practices (Mosupye and
Vonholy, 1999; Ehirim et al., 2001; FAO/WHO, 2003;
FAO/WHO, 2005). Agidi and epiti which had the
lowest percentage of the isolates could be attributed
to the method of processing the corn grain and
plantain (drying on moderately clean floor and
machine milling) (AOAC, 1995). The limited time &
temperature exposure during agidi preparation is
equally insufficient to destroy Bacillus cereus spores.
Bacillus cereus food poisoning is principally
associated with temperature abuse during the
storage of food. Temperature abuse can result in
spore germination and multiplication of the
vegetative cells leading to hazardous levels of
vegetative cells or toxins in the food at the time of
consumption (Granum, 2001). The extreme
resistance of the spores of this pathogen to heat,
desiccation, sanitizers and irradiation contributes
to their distribution, survival and persistence
within the entire food chain from raw agricultural
commodities to finished retailed products (Eglezos
et al., 2010)
The results of the antibiotic susceptibility of the
isolates are shown in Table 2. The antibiotic
susceptibility was determined by the disc agar
diffusion method in accordance with the instru-
ctions of the antibiotic disc manufacture (Oxoid).
All the Bacillus cereus isolates were susceptible to
Streptomycin (100%) Chloramphenicol (100%),
Rifampin (100%), Erythromycin (100%) and
Ciprofloxacin (100%) Gentamycin (100%) and less
sensitive to Lincocin (82%).
The findings of the present study correspond
16 AGWA ET AL.

Table 3. Antimicrobial sensitivities of Bacillus cereus isolates from food sample

Antibiotics (conc/µg) Food samples

Zones of inhibition in diameter mm status

CR AG EP MS

Rifampin (10µg) 32(S) 38(S) 32(S) 29(S)

Erythromycin (30µg) 28(S) 23(R) 26(S) 32(S)
Ampiclox (30µg) - - - -
Chloramphenicol (20µg) 40(S) 31(S) 31(S) 30(S)
Norfloxacin (30µg) - - - -
Streptomycin (30µg) 34(S) 40(S) 34(S) 26(S)
Gentamycin (10µg) 20(MS) 30(S) 24(S) 28(S)
Lincocin (30µg) 20(MS) 28(S) 30(S) 20(MS)
Floxapen (30µg) - - - -
Ciprofloxacin (10µg) 27(S) 23(S) 30(S) 27(S)
Bauer et al., (1966)

Keys: CR = Cooked rice ; AG = Agidi ; S = Sensitive ; EP = Epiti ; R = Resistant;

MS = Masa ; MS = Moderately Sensitive

Fig. 1 Distribution of the food samples within the markets analyzed

with those obtained by other researchers (Umar et
al., 2006; Whong and Kwaga, 2007). Previous works
have shown that antimicrobial susceptibility of
Bacillus cereus were highly susceptible to
streptomycin, chloramphenicol, erythromycin,
ciprofloxacin, and less susceptible to ampicillin,
ampiclox, cotrimazole, cloxacillin (Umar, et al.,
2006). Variations in the percentages may be due to
the differences in the concentrations of antimicro-
bial agents used, differences in the source of
isolates, drug resistance transfer and the overall
wide spread use of the antibiotics in the
environment. The development of drug resistance
may be due to the use of these drugs in medical and
veterinary practice to treat infections and misuse of
the drugs in the society, such practices can lead to
drug resistance strains.The antimicrobial pattern
of resistance of Bacillus cereus from foods is useful
in epidemiological studies but its effectiveness
decreases due to the negligence in utilisation policy
 Incidence and Antibiotic Sensitivity of Bacillus Cereus Isolated from Ready to Eat Foods Sold in
(Whong and Kwaga, 2007).
The following strategies should be undertaken to
control this pathogen: ensuring adequate
temperature (75oC) is reached during cooking of
food, hot foods should be kept hot (maintained at a
temperature greater or equal to 63 oC) and cold
foods should be kept cold (maintained at
temperature less than or equal to 5oC) to prevent
multiplication and toxin production. Training to
help food vendors to comply with regulations and
implement safe food handling practice; improve in
the equipments used to prepare and serve foods
and education campaign to increase consumer ’s
awareness about nutrition and safety of foods.
CONCLUSION
Food samples sold in markets are operating in less
than an acceptable and satisfactory environment;
efforts should be made to improve the safety of
street-vended foods. Illness associated with these
food products may be under reported, overlooked
and ignored, a few of the affected seek medical
attention owing to the mild nature and short
duration of symptoms. This must be taken
seriously as might lead to the release of toxins into
the body causing severe damage to the internal
organs and can eventually lead to death. Based on
the result, the effective antibiotic against Bacillus
cereus isolates are streptomycin, chloramphenicol,
rifampin, erythromycin, cipfloxacin and
gentamycin. Food-borne problems pose a serious
threat to human health, the economy of
individuals, families and nations. To ensure the
safety of food, Hazard Analysis and Critical
Control Point (HACCP) must be applied from the
primary producer to final consumption and its
implementation. Their control requires effort on the
part of the Government, food industry (vendors)
and consumers to meet the challenges of the future.
REFERENCES
AOAC 1995. Bacillus cereus in foods. Enumeration and
confirmation. Microbiological methods, AOAC
Official Method 980: 31. In: Cunniff, P. (Ed), Official
Methods of Analysis of AOAC International, 16 th ed.
vol. 1, AOAC International, Arhington VA, USA. Pp
52-54.
Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck,
M.1966.Antibiotic susceptibility testing by a
standardized single disk method. American Journal
of Clinical Pathology. 36:493-496.
17
Beattie, S.H. and Williams, A.C. 2000. Detection of
toxins. In :Encyclopedia of Food Microbiology (Vol. 1).
Edited by Robinson, R.K., Batt, C.A. and Ratel, P.D.
Academic Press, San Diego, USA. pp. 141-149.
Desai, S.V. and Varadaraj, M.C. 2009. Prevalence of
toxigenic traits in native food isolates of Bacillus
cereus in the city of Mysore, Southern India. Journal
of Microbiology and Antimicrobials. 1 (2) : 027-034.
Dierick, K., Vancoillie, E., Swiecicka, I., Megfroidt, G.,
Devlieger, H., Meulemans, A., Hoedemaekers, G.,
Fourie, L., Heyndrickx, M. and Mahillon, J. 2005.
Fatal family outbreak of Bacillus cereus associated
food poisoning. J. Clin. Microbiol. 43 : 4277- 4279
Eglezos, S., Huang, B., Dykes, E.A. and Fegan, N. 2010.
The prevalence and concentration of Bacillus
cereus in retail food products in Brisbane, Australia.
Food Borne Pathogens and Diseases. 7 (7) : 1-3.
Ehirim, J.E., Azubike, M.C., Ubbaonu, C.N., Anyanwu, E.
C., Ibe, K.M. and Ogbonna, M.O. 2001. Critical
control point of complementary food preparation
and handling in Eastern Nigeria. Bulletin of the
World Health Organisation. 79 (5) : 423-435.
Ehling-Schulz, M., Fricker, M. and Scherer, S. 2004.
Bacillus cereus, the causative agent of food borne
illness. Mol. Nutr. Food Res. 48 : 479-487
FAO/WHO. 2003. Assuring food safety and Quality:
Guideline for strengthening National food control
systems. Food and Nutrition, paper No. 76.
FAO/WHO.2005.Informal food distribution sector in
Africa (street foods): Importance and challenges.
Granum, P.E. 2001. Bacillus cereus: In: Food Microbiology.
Fundamentals and Frontiers (Eds. Doyle, M.P.,
Beuchat, L.R. and Montville, J.J.) pp. 373 - 381.
Griffiths, M.W. and Schraft, H. 2002. Bacillus cereus food
poisoning. In: Food borne Diseases, Cliver, D.O. and
Riemann, H.P. (Eds) Academic press, London. pp
261-270.
Guinebretiere, M-H., Fagerlund, A., Granum, P.E. and
Nguyen- The C. 2006. Rapid discrimination of cyt
K-1 and CytK-2 genes in Bacillus cereus strains by a
novel duplex PCR system. FEMS Microbiology
Letters. 259 : 74-80.
Jensen, G.B., Hansen, B.M., Eilenberg, J. and Mahillon, J.
2003. The hidden lifestyles of Bacillus cereus and
relatives. Minireview in Environmental Microbiology. 5
: 631-640.
Kotiranta, A., Lounatmaa, K. and Haapasalo, M. 2000,
Epidemiology and Pathogenicity of Bacillus cereus
infection. Microbes and Infections. 2 : 189-198.
McKillip, J.L. 2000. Prevalence and expression of
enterotoxins in Bacillus cereus and other Bacillus
spp. A literature review. Antonio Van Leewenhock. 77
: 393-399.
Mosupye, F.M. and Vonholy, A. 1999.Microbiological
quality and safety of ready-to-eat street-vended
foods in Johannesburg, South Africa. Journal of
Food Protection. 62 : 1278-1284.
18 AGWA
Oguntoyinbo, F.A. and Oni, O.M. 2004. Incidence and
characterization of Bacillus cereus isolated from
traditional fermented meals in Nigeria. J. Food
Prot. 67 : 2805-2808.
Reyes, J.E., Bastias, J.M., Gutierrez, M.R. and
Rodriguez, M.O. 2007. Prevalence of Bacillus cereus
in dried milk products used by Chilean school
feeding program. Food Microbiol. 24 : 1-6.
Sahota, P., Jairath, S., Pandove, G. and Krishan, M. 2008.
Emerging food borne pathogens- a review. Asian
Jr. of Microbiol. Biotech. Env. Science. 10 (4) : 921-926.
Tomlins, K. and Johnson, P.N. 2004. Developing food
safety strategies and procedures through
reduction of food hazards in street-vended foods
to improve food security for consumers, street-
food vendors and input supplies. Crop Post
—❋❋❋—
ET AL.
Harvest Programme (CPHP) Project R8270.
Funded by the DFID.
Umar, A.S., Yerima, M.B. and Uzal, U. 2006.
Antimicrobial sensitivities of Bacillus cereus isolated
from food samples sold in Bauchi metropolis to
selected antibiotics. Nigerian Journal of Microbiology.
20 (1) : 655-661
Umoh, V.J. and Odoba, M.B. 1999. Safety and quality
evaluation of street foods sold in Zaria, Nigeria.
Food Control. 10 : 9-14.
Whong, C.M.Z. and Kwaga, J.K.P. 2007.Antibiograms of
Bacillus cereus isolates from some Nigerian
foods. Nigerian Food Journals. 25 (1) : 178-183.
Yusuf, Z., Umoh, V.J. and Ahmad, A.A. 1992.
Occurrence and survival of enterotoxigenic
Bacillus cereus in some Nigerian flour based foods.
Food Control. 3 : 150-155.
Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 19-22
© Global Science Publications

EXTRACTION AND PURIFICATION OF NITROGENASE

REDUCTASE ENZYME (FE PROTEIN)
FROM RHIZOBIUM STRAIN
P. JAYALAKSHMI, P. SUVARNALATHA DEVI, *N.D. PRASANNA AND G. REVATHI

Department of Applied Microbiology, SPMVV, Tirupati 517502, A.P. , India.

(Received 10 July 2011; Accepted 30 August 2011)

Key words : Rhizobium, Nitrogenase, Fe protein, DEAE-Cellulose column.

Abstract - Biological nitrogen fixation involves the reduction of atmospheric dinitrogen (N2 ) into
ammonia (NH3) by a metalloenzyme nitrogenase. The nitrogenase reductase (Fe-protein) isolated from
Rhizobium strain was been purified and characterized. The nitrogenase Fe protein is a a 2 dimer
containing Fe 4S 4 cluster. The Fe-protein is similar to other Fe-proteins with respect to its molecular
weight. The Fe-protein was eluted with 400mM-Nacl on to a DEAE-cellulose column as a dark-brown
band at the top of the column. The molecular weight of the protein is about 60 K .Da. and the protein
concentration is about 14.2mg/mL.

INTRODUCTION homodimers with sequence identities =45% and

Rhizobium is an important soil bacterium which can
fix atmospheric nitrogen and increase soil fertility
(Boussingault, 1838). This is free-living soil
bacterium associated with legumes. 90% of natural
nitrogen fixation occurs through the agency of
certain microorganisms, all belong to a biological
group known as prokaryotes. All organisms which
reduce dinitrogen to ammonia do so with the aid of
an enzyme complex, nitrogenase. The nitrogenase
enzymes are irreversibly inactivated by oxygen,
and the process of nitrogen fixation uses a large
amount of energy (Dixon and Wheeler, 1986 and
Postgate, 1982).
The nitrogenase enzyme complex consists of two
components. Component 1 (also called nitrogenase
MoFe protein or the actual dinitrogenase reductase)
is a a2b2 tetramer in which the two non identical a
and ß subunits have similar molecular weights of
55,000 to 60,000. Component 2 (also called
nitrogenase Fe protein or dinitrogenase reductase)
is a dimer of two identical subunits, each having a
molecular weight of ca.35,000 (Martin Fuhrmann
and Hauke Hennecke, 1984). All Fe-proteins from
nitrogen-fixing bacteria (including eubacteria,
archeabacteria and cyanobacteria) are 60-70 KDa
contain an oxygen sensitive Fe 4S4 cluster that is
coordinated by invariant cysteine residues.
The Fe protein is the sole electron donor for the
MoFe protein that is thought to bind and reduce the
substrate. In the process of nitrogen fixation the Fe
protein accepts electrons from the one-electron
donors flavodoxin or ferridoxin and donates them
to the catalytically active MoFe protein after
formation of a protein-protein complex by the two
nitrogenase components. The (4Fe-4S) cluster is the
redox-active site involving in electron transfer to
FeMo-protein which cycles between reduced state
and oxidized state (Igarashi and Seefeldt, 2003).
The nitrogenase Fe protein has one binding site for
MgATP/Mg ADP. Fe subunit and binds two
nucleotides with positive cooperatively, with the
oxidized Fe 4S4 cluster state having a significantly
higher affinity than the one-electron reduced
cluster state. The exact role of MgATP hydrolysis in
nitrogenase function is still undefined, but it
requires both the Fe & MoFe protein and is
apparently associated with electron transfer in the
nitrogenase complex. Furthermore, the Fe protein
participates in the biosynthesis of FeMoCo and is
required for the insertion of this cofactor into MoFe
protein polypeptides during biosynthesis of MoFe

*Corresponding author - Jaya Lakshmi, E-mail : drpjayalakshmiphd@gmail.com; nd_prasanna@yahoo.co.in

20 JAYALAKSHMI ET AL.
protein. However, the iron-sulfur protein of Fe-
protein is not involved in these activities. Fe
protein may also have a regulatory function in the
biosynthesis of alternative nitrogenase.
MATERIALS AND METHODS
Collection and isolation of root nodule bacteria
Nodules of Groundnut were collected from S.V.
Agricultural College, Tirupati. Collected nodules
were washed with sterile water and then surface
sterilization was done using 70% ethanol and 0.1%
Hgcl 2 and repeatedly washed with sterile water.
After surface sterilization, nodules were crushed
and then resulting suspension streaked onto Yeast
Extract Mannitol Agar (YEMA) plates. After sub-
culturing, pure culture was obtained from a single
colony and preserved in 40% glycerol at -20oC for
experimental purpose and grown on Yeast Extract
Mannitol Agar medium (YEMA) at 30oC (Vincent,
1970). The cells were anaerobically centrifuged at
6000g for 10 mins. And were stored in liquid
nitrogen until used. All the preparations were done
in anaerobic condition in the presence of 1mM-
dithionite.
Extraction of Nitrogenase Reductase enzyme
Crude extract
Crude extracts were prepared by osmotic shock.
Frozen cell paste (50g) was thawed in 200mL of
glycerol buffer (4M-glycerol in 100mM-tris/acetate)
pH 8.1. The thawed cells were centrifuged at
10,000g for 15mins. And the glycerol buffer was
decanted. Next 10mL of glycerol buffer mixed with
20g of 4mM-diameter glass beads, 10mg of
ribonuclease and 20mg of lysozyme was added to
the pellet and thick slurry was made. After the
slurry has stood at 23oC for 15mins. 200mL of 0oC
breaking buffer (100mM-Tris/acetate pH 8.1, plus
2mM-dithiothretol) was added and the centrifuge
bottle was immediately capped and vigorously
shaken for 1m. The extract was centrifuged twice:
10mins. at 10,000g to remove unbroken cells and
90mins. at 45,000g to remove membrane particles.
The second centrifugation was with 40mL
centrifuge tubes closed with wired down serum
stoppers. The supernatant from the second
centrifugation is referred to as the crude extract
and the pellet as the chromatophore fraction.
Fe protein purification
The crude extract was removed from the centrifuge
tube with a syringe and was passed through a
DEAE-Cellulose column (2.5cm diameter X 15cm).
The negatively charged nitrogenase proteins were
bound tightly to the top of the column, and the
cytochromes and other proteins were washed
through. The column was washed sequentially
with 100mL portions of buffers (50mM-Tris/acetate,
pH 7.6 plus 2mM dithio-threitol) containing
100,200 or 400mM-Nacl. The 100mM-Nacl fraction
was discarded. The 200mM-Nacl wash eluted a
dark-brown band containing MoFe-protein and
this was frozen and stored at -20oC for later use.
The Fe-protein was in a fraction including
everything from the end of the MoFe-protein
fraction to the end of the dark-brown band eluted
with 400mM-Nacl. Immediately after the elution
the Fe-protein fraction was diluted with 1.5 vol. of
buffer and put on a DEAE-Cellulose column (1cm X
16cm) to concentrate it. The Fe-protein was eluted
from this column with 400mM-Nacl as a tight
band in 5-10mL volume. The concentrated Fe-
protein was desalted on a Sephadex G-25 column
(5cm X10cm) before being electrophoresed on a
preparative polyacrylamide gel column (3cm X
7cm). Solid sucrose was added to the concentrated,
desalted Fe-protein to give a 10% (w/w) solution,
and this was layered on the gel column under the
upper reservoir buffer. The 3cm-diameter gel had a
5cm-long section of 8.3% acrylamide separating gel
and a 2cm-long section of stacking gel. The
acrylamide/bisacrylamide ratio was 40:1. Both the
gel buffer and the reservoir buffer were 100mM-
Tris/borate pH 8.3. Before application of the protein,
the gel was pre-run for 3h in the presence of
dithionite at 60v (150pulses/s). The protein was run
into the gel slowly (overnight) at 40v (150 pulses/s,
6mA) and then was run through the separate gel at
150v (150 pulses/s, 20mA). The temperature was
kept low by putting the gel in a water-jacket or by
running the gel column in the cold room. Two
ferridoxins preceded the Fe-protein through the gel
and were collected and kept. The Fe-protein was
eluted with 100mM-Tris/acetate, pH 8.0 on to a
DEAE-Cellulose column (0.6cm X 4cm); this column
served as an on-line concentrator from which the
Fe-protein was eluted with 400mM-Nacl in Tris/
acetate, pH 7.7. The Fe-protein was stored in liquid
nitrogen until used.
 Extraction and Purification of Nitrogenase Reductase Enzyme (Fe Protein) from Rhizobium Strain 21

U.V.spectra
U.V.spectra of the Fe-protein from Rhizobium strain
was recorded with a Cary 14 spectrophotometer.
The proteins were precipitated twice in 1% HClO4
to remove iron-sulfur centers. After the second
precipitation, the proteins were resuspended in
1mL of 100mM-Tris borate; the final pH was about
9.0.
Molecular weights
SDS/polyacrylamide gels were run aerobically as
described by Laemmli (1950). The gels were stained
with Coomassie Brilliant Blue R-250 in water/acetic
acid/methanol (5:1:5, by vol.) and destained in the
same medium minus stain.

RESULTS AND DISCUSSION

The purification schemes for Rhizobium strain Fe-

protein was presented in Table 1. A DEAE-Cellulose
column holds both highly acidic nitrogenase
components. The preparative gel system was
essentially the same as that used by Shah and Brill
(1973) for the purification of A.vinelandii nitrogenase.
The lower yield of Rhizobium Fe-protein from the
preparative gel compared with that reported by
Shah and Brill (1973) for A.vinelandii Fe-protein
appears to result from an inherent low stability of
Rhizobium Fe-Protein. The degrees of purification for
Rhizobium Fe-protein from the crude extract were
19.0 fold respectively. The specific activity of the
enzyme was 5.2U/mg and the percentage of yield
was 40%.
Specific activity was measured by dividing the
total enzyme activity with total protein. Fold
purification was measured by dividing specific
activity with initial specific activity and the
1 2 M 3
Fig. 1 Separations of purified Rhizobium Fe-protein on
SDS/polyacrylamide gels.
Acrylamide gels (10%) with 0.1% SDS (acrylamide/
bisacrylamide ratio 3:7:0.3) were used. Gels were run
for 15m. at 100V to get the protein into the stacking gel
and then 250V for 1.5h. Lane 1 and 2 were referred to
as 10µg of crude extracts and M (molecular weight of
standards markers), Lane 3 purified protein.
percentage of yield was measured by dividing the
total enzyme activity with initial total enzyme
activity and multiplied with 100. The Fe-protein
from Rhizobium strain shows a band on SDS/
polyacrylamide gels. The molecular weight is about

Table 1. Purification of nitrogenase reductase enzyme from Rhizobial culture.

Fraction Total Conc.of Total Fold puri- Specific % yield

volume protein protein fication activity
(mg/mL)

Crude extract 100 10 1.0 - 0.30 100

1st DEAE-cellulose column 67 10.4 0.70 0.83 0.25 60
2nd DEAE-cellulose column 48 10.8 0.52 1.06 0.32 56
Sephadex G-25 column 32 12.2 0.39 1.2 0.38 50

Preparative polyacylamide 11 12.8 0.14 3.06 0.92 43

column
Purified column 4.5 14.2 0.021 19.0 5.2 40
22 JAYALAKSHMI ET AL.
60,000 (Fig. 1). The appearance of band was affected
neither by the SDS concentration used (0.1 or 1.0%)
nor by heating the SDS-treated protein at 100oC for
5mins. before applying it on the gel. We have
observed no change in the integrity of the band
during activation of Fe-protein.
The U.V. spectra of nitrogenase proteins has
been little investigated. Only Yates and Planque
(1975) have reported the spectrum of the Fe-protein
in the region below 330nm. The intense absorbance
by dithionite may have discouraged examination
in this region. It shows peaks at both 260nm and
280nm.
REFERENCES
Boussingault, J.B. 1838. Researches chimiques sur la
Vegetation, Entreprises Dans le But d’Examiner si
les Plantes Prennent de l’Azote a l’Atmosphere.
Crompt. Rendus. 6 : 102-112.
Dixon, R.O.D. and Wheeler, C.T. 1986. Nitrogen Fixation
in Plants. Blackie Chapman and Hall, New York. 15
- 25.
—❋❋❋—
Laemmli, U.K. 1970. Cleavage of structural proteins
during the assembly of the head of bacteriophage
T4. Nature. 227: 680-685.
Martin Fuhrmann and Hauke Hennecke 1984. Rhizobium
japonicum Nitrogenase Fe protein gene (nifH). J.
Bacteriol. 1005-1011.
Postgate, J.R. 1982. The Fundamentals of Nitrogen
Fixation. Cambridge University Press, Cambridge.
60-102.
Robert, Y., Igarashi and Lance C. Seefeldt, 2003.
Nitrogen fixation: The Mechanism of the Mo-
Dependent Nitrogenase. Critical Reviews in
Biochemistry and Molecular Biology. 38 (4) : 351-84.
Shah, V.K. and Brill, W.J. 1973. Mo-Dependent
Nitrogenase. Critical Reviews in Biochemistry and
Molecular Biology. Biochem. Biophys. Acta. 305 : 445
- 454.
Vincent, J.M. 1970. A Manual for the Practical study of the
Root-Nodule Bacteria. Burgess and Son Ltd. Great
Britain. 45.
Yates, M.G. and Planque, K. 1975. Nitrogenase from
Azotobacter chroococcum purification and properties
of the component proteins. Eur. J. Biochem. 60 : 467-
476.
Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 23-26
© Global Science Publications

A STUDY ON THE EFFECT OF DETERGENT SURYAGOLD ON

THE BIOCHEMICAL CONSTITUENTS OF THE FRESH WATER
FISH CIRRHINUS MRIGALA
J. VASANTHI*, R. KALAVATHI AND N. SARADHAMANI

Department of Zoology, Kongunadu Arts and Science college, Coimbatore, Tamilnadu, India

(Received 26 August 2011; Accepted 15 September 2011)

Key words : Detergent, Cirrhinus mrigala, Biochemical analysis.

Abstract - To get an idea about the nature of effect of detergent suryagold on the biochemical aspects
of aquatic organisms, fishes belonging to the species Cirrhinus mrigala were exposed to sublethal
concenteration of 3.5 mg/L (1/10th of LC50 value of 24 hrs ) for short term duration (24hrs, 48hrs and 72
hrs). Biochemical characteristics like glycogen, protein and cholesterol were estimated in liver, kidney,
gills and muscles. The glycogen and cholesterol content was found to increase in all tissues in different
hours of exposure but the protein content was found to decrease from the control. The results are
statistically analysed and most values were found to be non significant.

INTRODUCTION MATERIALS AND METHODS

Carbohydrates,proteins and lipids play a major
role as energy precursors for fishes under stress
conditions (Idler and Clemens, 1959 and Umminger,
1970).
Detergents constitute a key anthropogenic source
of nutrients to surface waters: historically, this
resulted in eutrophication of many freshwater
environments.Number of workers have studied
the effect of pollutants in physiology and
biochemistry of fishes (Jyothi and Narayan, 1997;
Kumar and Saradhamani 2004; Karthikeyan et al.
2007; Shanthi et al. 2009 and Sreenivasa and
Indirani, 2010). The effect of detergent Surya gold
on the fish has not been carried out. Therefore an
attempt has been made to study the possible
impact of detergent Surya gold on some
biochemical aspect of a freshwater fish Cirrhinus
mrigala.
The present work involved the study of -
1. Glycogen
2. Protein and
3. Cholesterol in the tissues like liver, kidney,
muscle and gills.
Cirrhinus mrigala is a freshwater carp fish found in
India, Pakistan, Bangladesh, Nepal and Burma. It
has a slender body, small head blunt snout and
thinner lips. Body silvery dark grey along the back.
Adult attains a maximum length of 90-100cm and a
weight of 1.4-2.8 kg.Fingerlings and adults both
feed on the huge amount of decaying organisms.
Bulk of samples of fishes Cirrhinus mrigala ranging
in weight from 4-5 g and measuring 4-6 cm in
length were procured from Aliyar fish farm. Fishes
were acclimatized to the laboratory conditions for
one month in large plastic tank (200 L). The fishes
were with fed adlibitium rice bran, wheat bran
and oil cakes.
Appropriate narrow range of concentration 10-
50mg was used to find the median lethal
concentration and the mortality was recorded for
every 24 hrs upto 72 hrs. It was found as 35 mg for
24 hrs using probit
Three groups of fishes were exposed to 3.5mg (1/
10 th of 24hrs LC 50 value) concentration of the
detergent ‘Suryagold’ for 24, 48 and 72 hrs
respectively. Another group was maintained as
control.

*Corresponding author - E-mail : vasanthigudalur_14@yahoo.com

24 VASANTHI ET AL.

Table 1. Changes in the Glycogen content (mg/g) in the liver, kidney, gills, Muscles of Cirrhinus mrigala on exposed to 3.5mg/L of detergent Surya gold on Short
At the end of the each exposure period, fishes
were sacrificed and tissues such as liver, gill,

193.05

106.70
41.81

55.31
muscle and kidney were dissected and removed.

%
The tissues(10 mg) were homogenized in 80%

Results are mean (± SD) of observations ; % = per cent increase / decrease over control ; * = Significant at 0.05 level ** = Significant at 0.01 level
methanol,centrifuged at 3500 rpm for 15 minutes

50.27*** ± 0.05
and the clear supernatant was used for the

39.27*** ± 0.05

40.70*** ± 0.05

59.33*** ± 0.05
Experiment
analysis of different parameters.
Total protein concentration was estimated by the

1.8604

4.0184

4.1803
method of Lowry et al. (1951). Quantitative
estimation of glycogen in the tissues was done
following the method as described by Kemp and
Kits (1945). Cholesterol was estimated based on

38.20 ± 0.05
28.70 ± 0.05

24.32 ± 0.05
13.40± 0.05
72 Hours
enzymatic method using cholesterol esterase,

Control

1.8376
cholesterol oxidase and peroxidase (Richmond,
1973).

RESULTS AND DISCUSSION

44.43

10.59

42.77

75.51
%
In the present investigation, the effect of a
detergent powder suryagold on biochemical
nature of glycogen, protein and cholesterol was

19.47*** ± 0.05

31.53*** ± 0.05

54.57*** ± 0.05

43.00*** ± 0.05
Experiment
studied in the different tissues (Liver, Kidney, Gills
and Muscles) of the freshwater fish, Cirrhinus

6.4710

1.0006

3.5689

7.1140
mrigala. The results were Tabulated (1-3) and
statistically analyzed.
Liver tissue was found to contain 24.57, 19.47,
39.27 mg/g, Kidney tissue was found to contain
13.48 ± 0.05

28.51 ± 0.05

37.88 ± 0.05

24.50 ± 0.05
36.07, 31.53, 40.7 mg/g, Gill tissue was found to
48 Hours

Control

contain 36.27, 54.57, 59.33 mg/g, Muscle tissue was

found to contain 51.40, 43.00, 50.27 mg/g of
glycogen in 3.5mg concentration of detergent Surya
gold in 24, 48 and 72 hours respectively.
82.94

109.53
24.80

-5.22

The control values were noted as 13.43, 28.90,

%

38.27, 24.53 mg/g in Liver, Kidney, Gills and Muscles

respectively. Increased blood sugar level that is
*** = Significant at 0.001 level ; NS = Non Significant
24.57*** ± 0.05

36.27*** ± 0.05

51.40*** ± 0.05
36.07*** ± 0.05

hyperglycaemic condition may be due to the

Experiment

conversion of stored glycogen into blood glucose

5.4105

3.1524

5.1939

1.5986

(glycogenolysis) by the inducement of adrenal

hormone namely glycocorticoides and
24 Hours

catecholamines (Radhakrishnaiah et al. 2005).

Liver recorded 15.45, 27.21, 10.80 mg/g, Kidney
recorded 4.21, 9.97, 2.55 mg/g, Gills recorded 4.23,
28.90 ± 0.05
13.43 ± 0.05

38.27 ± 0.05

24.53 ± 0.05

11.31, 5.03 mg/g, Muscle recorded 4.06, 5.37, 0.99 mg/

Control

g of protein in 3.5 mg concentration of detergent

Surya gold in 24, 48, 72 hrs exposures.
The control values noted as 34.41, 3.38, 4.35 and
7.33 mg/g in Liver, kidney, Gills and Muscles
term duration

respectively. The decrease in proteins could be due

to their breakdown for metabolic utilization and
Exposure

Muscles
Tissues

energy production in response to the toxic stress

Kidney
‘t’ test

‘t’ test

‘t’ test

‘t’ test
Liver

(Yellamma et al., 1995).

Gills
Table 2. Changes in the Protein content (mg/g)in the liver, kidney,gills, Muscles of Cirrhinus mrigala on exposed to 3.5mg/L of detergent Surya gold on Short
term duration

Exposure 24 Hours 48 Hours 72 Hours

Tissues Control Experiment % Control Experiment % Control Experiment %

Liver 34.41 ± 0.05 15.45*** ± 0.05 -55.10 34.00 ± 0.05 27.21*** ± 0.05 -19.97 34.45 ± 0.05 10.80*** ± 0.05 -68.65
‘t’ test 6.3808 3.9195 2.6637
Kidney 3.38 ± 0.05 4.21*** ± 0.05 24.55 3.30 ± 0.05 9.97*** ± 0.05 202.12 3.30 ± 0.05 2.55*** ± 0.05 -22.72
‘t’ test 1.7279 4.2092 2.5825
Gills 4.35 ± 0.05 4.23NS ± 0.05 -2.75 4.30 ± 0.05 11.31*** ± 0.05 163.02 4.40 ± 0.05 5.09*** ± 0.05 14.31
‘t’ test 0.0212 3.4502 9.4629
Muscles 7.33 ± 0.05 4.06*** ± 0.05 -44.61 7.30 ± 0.05 5.37*** ± 0.05 -26.43 7.36 ± 0.05 0.99*** ± 0.05 -86.54
‘t’ test 7.2807 5.9881 5.0599

Results are mean (± SD) of observations ;% = per cent increase / decrease over control ; * = Significant at 0.05 level ** = Significant at 0.01 level
*** = Significant at 0.001 level ; NS = Non Significant

Table 3. Changes in the Cholesterol content (mg/g)in the liver, kidney,gills, Muscles of Cirrhinus mrigala on exposed to 3.5mg/L of detergent Surya gold on
Short term duration

Exposure 24 Hours 48 Hours 72 Hours

Tissues Control Experiment % Control Experiment % Control Experiment %

Liver 26.27 ± 0.05 40.50*** ± 0.05 54.16 26.30 ± 0.05 70.40*** ± 0.05 167.68 26.20 ± 0.05 51.57*** ± 0.05 96.83
‘t’ test 2.0322 2.2032 2.0115
Kidney 49.80 ± 0.05 40.43*** ± 0.05 -18.81 46.4 ± 0.05 61.50*** ± 0.05 32.54 46.20 ± 0.05 48.30 NS ± 0.05 4.54
‘t’ test 0.0246 1.6028 4.2741
Gills 22.33 ± 0.05 49.47*** ± 0.05 121.54 22.36 ± 0.05 30.47*** ± 0.05 36.27 22.30 ± 0.05 51.50*** ± 0.05 130.94
‘t’ test 1.5359 1.9260 1.1462
Muscles 37.03 ± 0.05 51.53*** ± 0.05 39.15 37.06 ± 0.05 40.30*** ± 0.05 8.74 37.20 ± 0.05 61.40*** ± 0.05 65.05
‘t’ test 1.8850 7.5540 2.4296
A Study on the Effect of Detergent Suryagold on the Biochemical Constituents of

Results are mean (± SD) of observations; % = per cent increase / decrease over control ; * = Significant at 0.05 level
** = Significant at 0.01 level *** = Significant at 0.001 level ; NS = Non Significant
25
26 VASANTHI ET AL.
Liver was found to contain 40.50, 70.40, 51.57
mg/g,Kidney recorded 40.43, 61.50, 48.30 mg/g, Gill
tissue contain 49.47, 30.47, 51.50 mg/g, Muscle
recorded 51.53,40.30,61.40 mg/g of Cholesterol in
3.5 mg concentration of Surya gold in 24,48,72 hrs
exposures.
In control,the value of cholesterol was recorded
as 26.27, 46.43, 22.33 and 37.03 mg/g in Liver,
Kidney, Gills and Muscles respectively.Increase in
the triglyceride content might be due to increased
esterification reactions under stress condition.
CONCLUSION
To get an idea about the nature of effect of detergent
Surya gold on the biochemical aspects of aquatic
organisms, fishes belonging to the species Cirrhinus
mrigala were exposed to sublethal concentration of
3.5 mg/L (1/10th of LC50 value of 24 hrs) for short
term duration.Biochemical characteristics like
glycogen,protein and cholesterol were estimated in
Liver, Kidney, Gills and Muscles.
Glycogen showed maximum decrease in liver
tissue(193.05%) after exposed to 72 hrs and
minimum decrease was recorded in gills (-5.22%)
in 24 hrs exposure. Protein showed maximum
decrease in Kidney tissue (202.12%) after exposed to
48 hrs and minimum decrease was recorded in gills
(-2.75%) in 24 hrs exposure. Cholesterol showed
maximum decrease in Liver tissue (167.68%) after
exposed to 48 hrs and minimum decrease was
recorded in Kidney (4.54%) in 72 hrs exposure. It
can be concluded that the toxicity of the detergent
may lead to severe effect of aquatic organisms.
REFERENCES
Finney, D.J. 1971. Probit Analysis, 3rd edition, (London :
Cambridge University Press), London. Pg. 20.
Idler, D.R. and Clemens, W.A. 1959.The energy
expenditures of fresh water river Sockeye Solmon
during spawning migration to chikes and stuar
—❋❋❋—
lakes. Inct. Pacific Salmon Fish. Comm. Prog. Jackson
Printing, New westminister B.C., 80.
Jyothi, B. and Narayan, G. 1997. Effect of phorate on
certain protein profiles of serum in freshwater
fish, Clarias batrachus (Linn.) J. Environ. Biol. 18 (2) :
137-140.
Karthikeyan, S., Palaniappan, P.R. and Sabhanayakam,
S. 2007. Influence of pH and water hardness upon
nickel accumulation in edible fish Cirrhinus mrigala
(Ham). J. Environ. Biol. 28 (2) : 489-492.
Kemp, A. and A.J.M. Kits Vaheijnigen, 1945. A
calorimetric micro-method for the determination
of glycogen in tissues. J. Biochem. 56 (4) : 646-648.
Kumar, K. and N.Saradhamani, N. 2004. A study on the
effect of a pesticide, avaunt on protein changes of
the freshwater fish Cirrhinus mrigala. 3 (3) : 225-259.
Lowry, O.H., Rose Brough, N.J., Farr, A.L. and Randall ,
R.J. 1951. Protein measurements with the folin
phenol reagent. J. Biol. Chem. 193 : 265-275.
Radhakrishnaiah, K., Venkatramana, P., Suresh, A. and
Sivaramakrishna, B. 2005.Effect of lethal and
sublethal concentration of copper on the
glycolysis in liver and muscle of freshwater
teleost Labeo rohita (Ham). J. Environ. Biol. 139 (1) :
63-68.
Richmond, W. 1973. Preparation and properties of a
cholesterol oxidase from Nocardia sp. and its
application to the enzymatic assay of total
cholesterol in serum. Clin. Chem. 19 : 1350-1356.
Shanthi, K., Kiran Joseph and Manimegalai, M. 2009.
Studies on biochemical changes in the liver and
kidney due to sterling biotech limited industry
effluent in freshwater fish, Labeo rohita (Hamilton).
Indian J. Environ. & Ecoplan. 16 (1) : 145-150.
Sreenivasa, V. and Indirani, R. 2010. Impact of
Dimethoate on biochemical constituents in the
fish, Oreochromis mossambicus. J. Ecotoxicol. Environ.
Monit. 20 (2) : 151-156.
Umminger, B.L. 1970. Physiological studies on super
cooled hill fish Fundulus heteroclitus. 111;
carbohydrate metabolism and survival at
subzero temperature. J. Exp. Zool. 173 : 159-174.
Yellamma, K.; Ayyanna, K. and Reddy, T.V. 1995.
Modulation in protein metabolism of Oziotelphus
senen during cypemethrin intoxication. Chem.
Ecol. 11 : 113-116.
Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 39-47
© Global Science Publications

BIOCHEMICAL AND HISTOLOGICAL STUDIES ON THE

DIABETIC MANAGEMENT POTENTIAL OF PTEROCARPUS
MARSUPIUM IN THE MSG OBESE MOUSE
PRISCILLA SURESH AND C.D. LETHI*

Department of Zoology, Bishop Heber College, Trichy, India

* Department of Zoology, Holy Cross College, Trichy, India

(Received 17 August 2011; Accepted 19 September 2011)

Key words : MSG, Homeostasis, Hyperglycemia, Pancreatic histology.

Abstract - Obesity is a global public health threat and one of the main risk factors for Type 2diabetes,
which accounts for over 90% of all cases of Diabetes. Obesity related diabetes or adult onset diabetes
is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency
and hyperglycemia. Accumulation of fatty acids or fatty acid derivatives in muscle and liver produce
insulin resistance. Monosodium glutamate is a food additive commonly known as MSG, Ajinomotto, is
a sodium salt of glutamic acid. Monosodium Glutamate (MSG) 4mg/g body weight was administered
subcutaneously to the neonatal mice. Each animal received eight injections on every alternate day
from the 5 th day of birth onwards. The mouse was tested for obesity and diabetes on the 60th day. In
order to manage type 2 diabetes the Pterocarpus marsupium which is used as hypoglycemic plant in
folklore medicine was chosen. The obese mouse was force-fed with Pterocarpus crude extract and
Pterocarpus distillate separately every day for 60 days. Lee index of the mouse, fasting blood sugar
level, oral glucose tolerance test and pancreatic histology were studied. Administration of MSG has
shown impact on Lee index, induced hyperglycemia and the mouse could not show glucose
homeostasis. The most marked morphological change in obese mouse was an increase in size of islets
and mean islet area was significantly greater in obese mouse compared to the control. P.marsupium
treatment to the MSG obese mouse showed hypoglycemia and glucose homeostasis. Pancreatic
sections in the Pterocarpus marsupium crude and Pterocarpus marsupium distillate treated obese mouse
showed the formation of new islets by neogenesis from pancreatic ducts. In the MSG obese mouse,
the effect of the treatment was more significant in Pterocarpus marsupium crude extract than distillate.
Pterocarpus marsupium has potential to manage Type 2 diabetes.

INTRODUCTION disequilibrium between energy intake and

Obesity is a risk factor for type 2 diabetes in any
population. Type 2 diabetes is characterized by
disorders of insulin action and insulin secretion,
either of which may be predominant feature. Most
patients with type 2 diabetes are obese, when they
develop diabetes and obesity aggravates the
insulin resistance. The risk of developing type 2
diabetes increases with age, obesity and physical
inactivity. Obesity and overweight are chronic
conditions characterized by excess body fat that is
quantified by the elevation in body weight of
patients (Seidell and Flegel, 1997). In general, it is
accepted that overweight and obesity result from
*Corresponding author - E-mail : priscisf@gmail.com
expenditure (Woods et al.,1998) and these conditions
have large impact on several metabolic and chronic
ailments including heart disease, cancer, arthritis,
hypertension, hyperlipidemia and type 2 diabetes
associated with insulin resistance (Wickelgren,
1998; Munoz et al., 2004). Much has been learned in
the past decade regarding the regulation of obesity
as it relates to the molecular regulation of appetite
that affects energy homeostasis,particularly as
positive energy balance upsets lipid and glucose
metabolism. (Schoonjans et al.,1996).
MSG is reported to induce obesity in adult
animals, (Betran, et al., 1992). Monosodium
glutamate is commonly known as MSG, ajinomoto,
40 PRISCILLA AND LETHI
vetsin, (or) accent is a sodium salt of glutamic aid.
MSG is a food additive and it is commonly
marketed as a “flavor enhancer ”. Many studies
have shown that over feeding has been associated
with metabolic complications such as insulin
resistance, hypertension, diabetes, and is cause of
heart disease, particularly when the excess
deposition in the deep abdominal area (Kisseban,
1991).
Different medicinal systems are using the active
plant constituents which discovered as natural
hypoglycemic medicine, came from the virtue of
traditional knowledge. Herbal drugs are
considered free from side effects than synthetic one.
They are less toxic, relatively cheap and popular
(Momin, 1987). In India, medicinal plants have been
used as natural medicine. Recently the plants and
herbs are being used as decoctions or in other
extracted forms for their blood sugar lowering
potential. There are some useful reviews on Indian
medicinal plants having blood sugar lowering
potentials (Mukherjee et al., 1981; Grover et al., 2002;
Saxena et al., 2004; Mukherjee et al., 2006).
P. marsupium is a moderate to large deciduous tree
that belongs to the family papilionaceae. It is
commonly distributed in the hilly regions through
out the Deccan peninsula in India. The anti diabetic
effect of P.marsupium is exploited in the traditional
medical practices in India including folklore. Water
stored over night in tumbler made up of the heart
wood of P.marsupium is believed to have anti diabetic
effects (Kedar and Chakravarthy,1981) are used in
the treatment of diabetes by Ayurvedic (a
traditional Indian medicine system) physicians in
India. As a result of its effectiveness are various
aspects of hypoglycemic/anti diabetic effect of
P.marsupium during the last five decades.
Plants grow throughout India and use as
hypoglycemic plant in folklore medicine (Mukherjee
et al., 1996). Aqueous bark extract lowered blood
sugar and improved glucose tolerance of diabetes
with no side effects observed (Pandey et al., 1975).
The epicatechin from bark increased the CAMP
content of the pancreatic islets associated with
increased insulin release, conversion of pro insulin
to insulin and cathepsin B activity in rats (Ahmed et
al., 1991).
Reports are also available on the clinical trials
made in diabetic patients using the aqueous on the
clinical of the heart wood of P. marsupium which
proved successful in controlling hyperglycemia in
them (Sepana and Bose, 1956; Pandey and Sharma,
1975; Rajasekharan and Tuli, 1976; Ojna et al., 1978).
Nearly 10 patients were given the water stored in
a heart wood container of this plant for a period of
1 month blood glucose levels decreased from the
second week of treatment and were maintained at a
normal level with the treatment was withdrawn
(Kedar and Chakravarthy, 1981). In another trial 93
newly diagnosed/untreated Type II diabetic
patients were administered with P. marsupium
extract for a period of 4 weeks. In the treated
patients both fasting and postprandial blood
glucose levels decreased significantly (Indian
council of medical Research, 1998).
Although there is a lot of information on the anti
diabetic potential of P.marsupium with reference to
type I diabetes, there is no study on its effect on
MSG obese and obesity-induced diabetic animals.
The present study was aimed to evaluate the effects
of P. marsupium in MSG induced obese mouse with
type II diabetes.
MATERIALS AND METHODS
Experimental animal
The inbred strain of albino mouse, Mus musculus
was used for this study. The animals which were
primarily obtained from King Institute, Guindy,
Chennai were made to breed in the animal house
attached to the Department of Zoology, Holy Cross
College, Tiruchirappalli. They were fed on
standardized rat feed. The newly bred 5 days old
mice pups were selected for the study.
Induction of obesity-based diabetes
Diabetes was induced in the mouse with the
subcutaneous injection of Monosodium Glutamate
(MSG) 4mg/g of body weight. The required amount
of salt was weighed, dissolved in 0.5mL saline and
was injected subcutaneously to the neonatal mice.
Each animal received a total of eight injections, on
every alternate day from the 5 th day of birth
onwards. The mouse was tested for obesity and
diabetes on the 60th day after the administration of
the MSG by measuring the blood sugar level.
Experimental Design
The parameters were studied on the 0, 30th and 60th
day of the MSG obese mice. The experimental set up
has included four groups.
- Group I represented the normal control which
received no MSG injection and treatment of
P.marsupium extract.
 Biochemical and Histological Studies on the Diabetic Management Potential of
- Group II represented obese - induced control,
which received MSG (4mg/g).
- Group III received a 4mg/g MSG and the obese
mouse was force-fed with aqueous extract of
P. marsupium crude extract every day for 60 days.
- Group IV received a 4mg/g MSG and the obese
mouse was force-fed with aqueous extract of
P.marsupium distillate (Crude extract was distilled
seven times) every day for 60 days.
The parameters traced were Lee index, fasting
blood sugar, oral glucose tolerance test and
pancreatic histology.
Preparation of extract
10gm powder of P.marsupium heartwood was
prepared by dissolving in 100mL of distilled water,
left it for overnight, boiled and filtered. To prepare
distilled extract, the crude extract was distilled
seven times. Oral administration of 0.25 mL of the
crude extract and distillate were given separately
to the MSG induced obese mice.
Measurements and estimations
Lee Index
The Lee Index was calculated by dividing the cube
root of the body weight by nose to anus length and
multiplying by 1000. Lee Index was calculated on
30th, 60th, 90th and 120th days of the experiment.
Blood Sugar level
Blood sugar was estimated using Glucose oxidase
method (GOD) (Trinder, 1969). Serum was
separated from the blood sample by centrifugation
at 2000 rpm. 10µL of serum was mixed with 1 mL
of GOD reagent and incubated for 10 min. at room
temperature. The optical density was measured at
505 nm using a digital colorimeter.
Oral Glucose Tolerance Test (OGTT)
After collecting the blood from fasted animal, they
were force-fed with 1g/gm body weight of glucose
using baby oral feeding tube. Blood samples were
collected every 30min for one and half hours for
sugar estimation.
Histological sections
For light microscopic studies, pancreas was fixed
in Gilson’s fixative (Gray, 1964) for 24h. The tissue
was washed well, processed through series of
alcohol for dehydration, cleared in xylol and finally
embedded in paraffin wax. Sections of 5µ thickness
were cut using rotary microtome (Weswox optic)
41
and stained with Ehrlich’s haematoxylin and
counterstained with 0.5% aqueous phloxine
(Gomori, 1941). The sections were mounted in DPX
and were studied and photographed using
photomicrography unit.
RESULTS
The results of this study revealed that the neonatal
administration of monosodium glutamate (MSG)
induced hyperglycemia in the albino mouse. The
animals were screened for the diabetic conditions
and the effect of Pterocarpus marsupium on the MSG
obese mouse studying lee index, blood sugar level,
oral glucose tolerance test and pancreatic histology.
Lee index
Table 1 and Figure 1 illustrate the data on the lee
index of the control, MSG administered, Pterocarpus
crude and Pterocarpus distillate. In the control
animal the lee index were 292.1 ± 0.28, 326.3 ± 4.5,
326.8 ± 7.2, 327.5 + 2.3 on 30th, 60th, 90th day and 120th
day respectively. Administration of 4 mg/g MSG
induced an obese condition. The corresponding
values in the obese animals were 332.39 ± 6.17,
333.5 ± 12.1, 336.9 ± 14.7 and 337.5 ± 4.5. Lee index
in the P. marsupium crude treated MSG obese mouse
was observed to be 332.3 ± 6.17 on 30th day, 324.9 ±
6.7 on 60th day, 323.3 ± 8.48 on 90th day and 322.7±
5.4 on 120th day and in P. marsupium distillate treated
MSG obese mouse the Lee index were 332.3± 6.17,
328.6 ± 4.9, 326.9 ± 13.1 and 324.2 ± 4.8
Administration has shown noticeable impact on
the lee index.
Fasting blood sugar level
The data on the fasting blood sugar level in control,
MSG administered and P.marsupium crude and
distillate treated animals are recorded in Table 2
Figure 2. Administration of MSG administered
obese condition with hyperglycemia as 151.2 ± 144
mg/dl, 177.2 ± 41.24 mg/dl, 171 ± 21.83mg/dl
respectively on the 0, 30th and 60th days. The
corresponding values for the P. marsupium crude
treated were 170± 13.63, 106± 14.54, 80± 11.60 and
P.marsupium distillate treated were 167.5 ± 25.09mg/
dl, 125 ± 31.22mg/dl and 116 ± 18.018mg/dl. The
fasting blood glucose levels in control mouse were
70.5±3.70mg / dl on 0 day, 88±11.05mg /dl on 30th
day and 81±10.23mg /dl on 60 th day. Pterocarpus
marsupium treatment in the MSG administered
mouse did show the impact on the blood sugar.
42 PRISCILLA AND LETHI

Table 1. Lee index calculated in control, MSG administered, Pterocarpus crude and Pterocarpus distillate treated albino mouse, M. musculus on 30th, 60th, 90th and

326.9±13.1
332.4±6.17
Table 2. Fasting blood sugar (mg/dl) in control, 4mg/g

328.6±4.9

324.2±4.8
MSG administered and Pterocarpus crude and distillate

index
treated albino mouse, Mus musculus on 0, 30th and 60th

Lee
day of the experiment.

8.04±0.22
Pterocarpus Distillate

Days Control Msg Admi- Pterocarpus Pterocarpus

9.4±0.80
9.0±0.40

9.6±0.48
(cm) nistered Crude distillate
L

0day 70.5±3.70 151.2±10.14 170±13.63 167.5±25.09

19.12±1.82
26.07±4.10
30th day 88±11.05 177.2±41.24 106±14.54 125±31.22

28.9±3.79
30.5±3.5
60th day 81±10.23 171±21.83 80±11.60 116±18.02
Wt
(g)

Oral Glucose Tolerance Test

332.4±6.17

323.3±8.48

The results of the oral glucose tolerance test are

324.9±6.7

322.7±5.4

recorded in the Table 3 and Figure 3a, 3b, 3c. The

index
Lee

data revealed that the control mouse on 0 day was

able to show glucose homeostasis within 90
minutes of glucose administration. The fasting
8.04±0.22

10.3±0.35
9.8±0.28
9.3±0.5

blood glucose level noted was 91.50± 2.12mg/dl. On

Pterocarpus Crude

(cm)

administration of glucose, the levels were

L

143±2.82mg/dl in 30 minutes,162.5±24.78mg/dl in
60minutes and 123.5±0.70mg/dl in 90minutes. The
19.12±1.82
27.25±2.8

MSG administered mouse could not show glucose

32.1±0.4
36.2±1.9

homeostasis in 90 minutes. The values recorded

Wt
(g)

were 140.3±14.29mg/dl, 258± 16.02 mg/dl, 173±

14.73mg/dl,155±18.08 mg/dl respectively at
336.9±14.69
333.5±12.01
332.4±6.17

0,30,60,90 minutes. The data of the oral glucose

337.5±4.5

tolerance test recorded on 30th day and 60th day

index
Lee

revealed the control mouse was able to show

glucose homeostasis within 90 minutes of glucose
administration. Similarly the MSG administered
292.1±0.28 19.12±1.821 8.04±0.21

9.6±0.42
9.0±0.58
Msg Administered

mouse could not show glucose homeostasis in 90

(cm)

10±0

minutes. The values recorded on 60 th day were

L

140±14.29 mg/dl,258±16.02mg/dl,173±14.73mg/dl
26.85±2.51

and 164±4.94 mg/dl respectively at 0,30,60,90

33.8±2.70
38.5±1.6

minutes.
In Pterocarpus crude extract treated MSG obese
Wt
(g)

mouse the corresponding values were 84±1.41mg/

dl, 171±2.12mg/dl, 219±2.12mg/dl and 115±4.24mg/
327.5±2.3
326.3±4.5
326.8±7.2

dl and in Pterocarpus distillate treated MSG obese

index
Lee

mouse the values were 123±4.63mg/dl, 209±

5.87mg/dl, 155±4.56mg/dl,122±4.6mg/dl. After
treatment, the mouse able to show glucose
8.15±0.53 6.8±0.28
8.0±0.38
9.2±0.23
9.6±0.1

homeostasis.
120th day of the experiment.

(cm)
L

Pancreatic Histology
Control

27±0.26

Figure panel 4A and 4 B presents the sections of

31±1.6
17±2.5

30th day pancreatic islets from the control and

Wt
(g)

obese mouse. Light microscopic analysis of these

pancreatic islets showed typical profile images of
120th day
30th day
60 day
90th day

circular, oval or elongated shapes surrounded by

Days

exocrine pancreatic tissue. A thin connective sheath

th

surrounded each islet. Numerous blood capillaries

 Biochemical and Histological Studies on the Diabetic Management Potential of 43

Table 3. Blood sugar level (mg/dl) obtained through Oral Glucose Tolerance Test (OGTT) in control, 4mg/g MSG
administered, Pterocarpus crude and distillate treated albino mouse, M.musculus on 0, 30 th & 60 th day of the
experiment. The glucose levels were measured at the 0,30th, 60th & 90th minutes after the administration of glucose.

Day Group Fasting 30 min 60 min 90 min

0 Control 91.50±2.12 143±2.82 162.5±24.78 123.5±0.70

MSG 140.3±14.29 258±16.02 173±14.73 155±18.08
30th Control 91.0±1.41 136±5.65 166.5±8.48 10±14.14
MSG 140±5.65 166±36.76 179±5.65 146±16.6
Pterocarpus Crude 125±1.65 168±1.56 126±2.34 11±3.67
Pterocarpus Distillate 131.5±12.02 202±43.8 238.5±20.50 155.5±2.12
60th Control 91.50±2.12 143±2.82 162.5±24.78 115±3.53
MSG 140±14.29 258±16.02 173±14.73 164±4.94
Pterocarpus Crude 84±1.41 171±2.12 219±2.12 15±4.24
Pterocarpus Distillate 123±4.63 209±5.87 155±4.56 122±4.6

Table 4. Area of islets (mm2 ) in the pancreatic sections
of Control, MSG administered, P.marsupium crude and
distillate treated albino mouse M.musculus
Control MSG Pterocarpus Pterocarpus
administered Crude Distillate
treated treated
0.121 0.192 0.119 0.143
± 0.018 ±0.017 ±0.019 ±0.007
permeated the entire islet. Islets from MSG obese
mouse showed marked changes compared with the
control animals. The most marked morphological
change in obese mouse was an increase in size of
islets compared with the control. Mean islet area in
mm 2 /tissue section/mouse, was significantly
greater in obese mouse 0.192± 0.017mm2 compared
to the control 0.121± 0.018 mm 2. Histomorpho-
logical and morphometric examination of the
stained pancreatic sections in the Pterocarpus
marsupium crude and Pterocarpus marsupium distillate
treated obese mouse (Fig.4C and 4D) showed the
formation of new islets by neogenesis from
pancreatic ducts and the mean islet area of treated
obese mouse was similar to that of the control
0.119± 0.019mm2 and 0.143± 0.007mm2.
DISCUSSION
The results obtained from the study of the neonatal
administration of MSG confirms its potential to
induce obesity and the obesity-mediated
hyperglycemia. The findings also revealed the effect
of MSG on the increase in blood sugar. Keeping in
view of the normal blood sugar level in mammals
(80-120mg %) the MSG administration is found to
exert a marginal hyperglycemic impact in mouse at
a dosage of 4 mg/g. The finding that the MSG
induces obesity is in confirmity with the earlier
research reports by Jezova et al. (1998); Matsuki et
al. (2003); Hirata et al. (1997) and Harris et al. (2001)
and Olney (1969). In streptozotocin induced
diabetes, the animal looses weight and consumes
more feed and water obviously the diabetes
induced in this case is more in the direction of type
2 diabetes.
However this speculation needs further
confirmation with experiments on insulin
resistance. Nevertheless, this speculation can be
supported by the report of Hirata et al. (1997)
where monosodium glutamate obese rats
developed glucose intolerance and insulin
resistance to peripheral glucose uptake.
Lee index used as a standard to measure the
obesity (or) adiposity is measured in control, MSG
administered, P. marsupium crude and P. marsupium
distillate. The observation made on the body
weight reveals that there is an increase in the body
weight of MSG administered mouse, revealing the
occurrence of obesity after nearly two months old
MSG administered mouse. Reports are already
available where MSG is used to induce obesity.
Maletinska et al. (2006) administered MSG to
induce obesity in mouse. According to them the
increase in body weight is due to accumulation of
fatty acids derivatives on the muscle.
A comparison of the obese conditions in the
streptozotocin induced and MSG-induced diabetic
conditions shows that the physiological and
histological manifestations are different in both the
44 PRISCILLA AND LETHI

Fig. 1 Lee Index in control, MSG administered, Pterocarpus crude and distillate treated
Albino mouse, Mus musculus on 30th, 60th, and 90th day of the experiment.

Fig. 2 The fasting blood sugar level (mg/dl) in control, MSG administered and P. marsupium crude and
distillate treated Albino mouse, M.musculus on 0, 30th and 60th day of the experiment.

conditions, although there is occurrence of
hyperglycemia in them. The damage to the
pancreatic endocrine tissue is minimal in MSG
administered mouse. Work has been carried out in
our laboratory in type I diabetes using
streptozotocin, which revealed a drastic loss of
islets and b cells (Farzana, 2005). Obesity is
accompanied by an increase in beta cell mass,
increased insulin secretory capacity and
maintenance of normoglycemia by Nils Bilestrup
(2004). Bock et al. (2003) using stereological
methodology, indicated that an increase in islet
mass in obese mice was because of islet
enlargement with no change in islet number
compared with control mice.
Obesity in humans and rodents have been
associated with alterations in pancreatic islet
morphology. One consistent effect seen in obese
humans and rodents is an increase in the number of
islets and enlargement of islets because of
hyperplasia or hypertrophy of islet b cells (Ogilvie,
1933; Wissler et al., 1949; Bonner-weir, 2000; Hong et
al., 2002). Concomitant increase in insulin content
has been described in islets of obese animals
(Malaisse et al., 1968; Genuth, 1969).
MSG obese mouse treated with Pterocarpus
marsupium crude and distillate separately inhibited
pancreatic islets cell death, particularly through its
potent anti-inflammation potential. Once absorbed
via the digestive tract, P.marsupium can enter
pancreatic islet b cells and exert its anti-apoptotic
effect possibly using its potent anti-inflammatory
and antioxidant activities (Joshi et al., 2004). Dor et
al., (2004) have suggested that new islets can form
by proliferation from existing differentiated islet
cells or by neogenesis from pancreatic ducts
 Biochemical and Histological Studies on the Diabetic Management Potential of 45

Fig. 3a Blood sugar level (mg/dl) using oral glucose tolerance test in control, MSG administered,
Pterocarpus crude and distillate treated Albino mouse, M.musculus at 0 (fasting),
30, 60 and 90min on 0 day of the experiment.

Fig. 3b Blood sugar level (mg/dl) using oral glucose tolerance test in control, MSG administered,
Pterocarpus crude and distillate treated albino mouse, M.musculus at 0 (fasting),
30, 60 and 90min on 30th day of the experiment.

Fig: 3c Blood sugar level (mg/dl) using oral glucose tolerance test in control, MSG administered, Pterocarpus crude
and distillate treated albino mouse, M.musculus at 0 (fasting),30,60 and 90min on 60th day of the experiment.

(Bonner-Weir,2000)
Pterocarpus marsupium has potential to
manage Type 2 diabetes. Further studies are
required to explore the physiological implications
of these findings on the development of diabetes
and its management by herbal extract.
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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 49-53
© Global Science Publications

EFFICACY OF LACTOBACILLUS PLANTARUM TO INHIBIT

THE GROWTH OF AEROBIC BACTREIAL
BURN WOUND PATHOGENS
NEMADE SNEHAL, MISHRA SHWETA AND M. MUSADDIQ

P.G. Department of Microbiology, Shri Shivaji College, Akola 444 001,(M.S.) India.

(Received 20 July 2011; Accepted 15 September 2011)

Key words : Burn wound, Bacteria, Lactobacillus plantarum, Bacteriocin,

Abstract - Burns are amongst the most devastating of all injuries with out comes spanning the spectrum
from physical impairment and disabilities to emotional and mental consequences Infection in burn
patient is leading cause of morbidity and mortality and continue to be one of the most challenging
concern for the burn team. Alongwith host factor contamination by endogenous and exogenous
sources increases the risk. Considering the changing epidemiology, high rate of infection, the problem
of drug resistance in common pathogenic microorganisms, the present study was planned to
determine the bacterial profile of burn wound with its antibiogram study. These days bacterial
resistance has become a major cause for the management of burn wound infections. Use of different
combinations of antibiotics has shown the possible synergistic effect in the recovery of severe drug
resistant infections. Hence, it has led many investigators to search for new drug therapy. However,
indiscriminate using of antibiotics has induced abnormalities on the resident normal flora of gastro-
intestinal tract and led a major problem at digestibility level in the children. Because of this several
workers has reported the effect of antibiotics with other bacteriostatic agents. Hence, with the view to
minimize the indiscriminate use of antibiotics and to incorporate the bacteriostatic agent in the therapy
for the management of burn wound infections, the present investigation has been carried out to
evaluate the inhibitory potential of Lactobacillus plantarum against different burn wound associated
bacterial isolates. The results revealed the utility of L. plantarum and its derivatives to combat aerobic
bacterial burn wound pathogens.

INTRODUCTION disposing factors (Morsi, 1990).

Burn is a preventable tragedy, which is unfortu-
nately common in India. Incidence of burn injury
has declined steadily, over the past several decades
in rest of the world but, it is still on higher side in
our country because of illiteracy prevalent in rural
India and its social fabric where the practice of
bride burning is still reported in newspapers
almost everyday (Dogra, 2004).
Diagnosis of burn infection involve several risk
factors like extent of burn, depth of burn, age of
patient, pre-existing disease, wound dryness,
acidosis, etc. and microbial factors like density,
motility, metabolic products, toxins, antimicrobial
resistance, etc. Pre-existing immunological
deficiencies and metabolic abnormalities are pre-
*Corresponding author - E-mail : dr.m.musaddiq@rediffmail.com; shaikhasimiqbal1@gmail.com
Infection remain the leading cause of death
among patients who are hospitalized for burns.
The risk of infection is directly related to the extent
of burn and to the impaired resistance resulting
from skin disruption. Burn wound can be classified
as wound cellulitis which involve unburnt skin at
the margin of burn or as in invasive wound
infection which is characterized bymicrobial
invasion of biable tissue beneath the eschar.
Excessive antimicrobial use has depleted bacterial
infection but invasive fungal infection has risen
significantly. Overall mortality rates from burn
wound sepsis remains high (Gibran, 2000).
Burn injury cause mechanical disruption of skin
which allows invasion by environmental microbes
into deeper tissues. Avascularity of eschar also
50 SNEHAL
supports easy proliferation and penetration of
microbes. Burn wound infection is sterile
immediately following injury, however it is
repopulated quickly with Gram positive bacteria
[eg. Staphylococcus aureus, Enterococcus faecalis,
Clostridium perferingens, etc.] from environment and
hair follicles, during first 48 hrs. and are replaced
by Gram negative organisms [eg. E. coli,
Pseudomonas aeruginosa, K. pneumoniae, etc.] after 5-7
days. Due to several pathogenicity markers and
antibiotic resistance ability hey proliferate deep
into sub-eschar space. Invasive wound infections
occur due to inadequate host defense (Akayleh,
1999).
Despite advances in the use of topical and
parenteral antimicrobial therapy and the practice
of early tangencial excision, bacterial infection
remains a major problem in the management of
burn victim today. The goal of burn wound
management is to reduce the onset and density of
bacterial contamination which usually occurs by
the second week after injury, and thereby to
prevent invasive wound infection (Lawrence 1994).
The choice of antibiotics was limited in curing such
infections. High antimicrobial resistance was
probably promoted due to selective pressure
exerted on bacteria due to numerous reasons like
non-adherence to hospital antibiotic policy,
excessive and indiscriminate use of broad-
spectrum antibiotics etc. These multi drug resistant
strains establish themselves in the hospital
environment in areas like sinks, taps, railing,
mattress, toilets and thereby spread from one
patient to another. The resistance of organisms to
antibiotics poses a challenge to burn care because it
reduces effectiveness of treatment and may increase
morbidity and mortality.
Hence attention was switched on to search for
new and better alternative remedies to treat
multiple drug resistant pathogens.
Strains of several Lactobacillus sps have proven to
exert a range of health promoting activities such as
immunomodulation enhancement of resistance
against pathogens reduction of blood cholesterol
levels and others (Gill and Rutherferd 2001).
Amongst these is Lactobacillus plantarum which is
able to produce extracellularly released
proteinaceous antimicrobial subs. Including some
primary metabolites like lactic acid, acetic acids,
ethanol, and carbon dioxide as well as the
production of other antimicrobial compounds, such
as formic and benzoic acids, hydrogen peroxide,
ET AL.
diacetyl, acetoin and bacteriocins (Piard et al., 1991).
Several investigators (Valdez et al., 2005,
Horcharoen and Sookkhee, 2007) have analysed its
inhibitory action with respect to P. aeruginosa
infections and had concluded them as potential
therapeutic agents for its local treatment. However,
little information is available regarding it.
Considering the mortality rate in burns
associated with septicaemia, the present piece of
work aims to determine the bacterial colonizers
associated with burn wound along with their
sensitivity/resistance pattern studies. The study
also attempts to suggest an alternative remedy to
fight with these organisms utilizing Lactobacillus
plantarum and its derivatives.
MATERIALS AND METHODS
Burn wound biopsy and superficial wound swabs
were collected in sterile trypticase soya broth.
Sample were inoculated on MacConkey Agar and
Blood Agar and incubated. Based on observations
made samples were inoculated on respective
selective media and were further identified by
standard conventional method. The identified
isolates were subjected to antibiotic sensitivity test
by using Kirby Bauer method (1966). Antibiotics
used were Amoxycillin (10 mcg), Chloramphenicol
(50 mcg), Kanamycin (10 mcg), tetracycline (30
mcg), Nalidixic acid (30 mcg), Penicillin (10 mcg),
Co-trimoxazole (25 mcg), and Norfloxacin (10 mcg).
Lactobacillus plantarum strains were isolated from
various samples viz milk, curd, fresh fish, frozen
fish. MRS agar and broth were used for
enumeration and culture of Lactobacillus plantarum.
Samples were homogenized, serially diluted and
pour plated on MRS plates; and incubated in 5%.
CO2 at 37°C for 48-72 hours. Well isolated colonies
with typical characteristic were picked and
exposed on the basis of morphological, cultural,
physiological and biochemical characteristics (Nair
et al.,2004-2005). Antagonistic activity was detected
by using Tryptic Soy Broth containing only 0.2%
glucose and 0.75% agar. Lactobacillus plantarum
cultures were grown in MRS broth while another
set was prepared by using media optimized for
bacteriocin production. Antimicrobial sensitivity
pattern was studied by the Agar Well Diffusion
technique. Results were recorded as per zone size
in mm. Effect of several physical and chemical
parameters was also analysed on the inhibitory
potential by standard methods.
 Efficacy of Lactobacillus plantarum to Inhibit the Growth of Aerobic Bactreial 51

RESULTS AND DISCUSSION Table 5. Assessment of Lactobacillus plantarum to inhibit

bacterial burn wound pathogens
Table 1. Distribution of burns by age Name of Bacteriocin Producing L.Plantarum
Organism
Age Groups (Year) Percentage
L. plantarum (Cell-Free L. plantarum
<=7 3.5 % supernatant in (whole
8 – 35 54 % medium optimized broth)
36 – 65 33 . 4 % for bacteriocin
> = 67 9 .1 % production)

S. aureus 36/53 (68%) 33/53 (62%)

P. aeruginosa 26/44 (59%) 24/44 (65%)
Table 2. Aetiological distribution of Burns.
P. vulgaris 17/36 (47%) 22/36 (61%)
Aetiology Percentage E. coli 12/29 (40%) 14/29 (48%)
K. pneumoniae 13/24 (54%) 9/24 (62%)
Chemical Burns 50.8% A. baumanii 6/16 (38%) 5/16 (31%)
Scald Burns 43.56%
Dry Heat 4.45%
Electrical Burns 3.03% Table 6. The antimicrobial activity of selected L.
plantarum isolates. Data were shown in the average
diameter of inhibition zone (mm)

Test Organism Mean Diameter of Clear

Table 3. Types of Microorganism Isolated from Burn
Zone In L. Plantarum (Mm)
wound infection
S. aureus 25.00
Name of Microorganism Percentage
E. coli 17.50
Pseudomonas 50.74% P. aeruginosa 16.00
S. aureus 42.6% A. baumanii 15.50
Klebsiella 44.7% K. pneumoniae 14.50
E.coli 29.5% P. vulgaris 13.50
Proteus 26.2%
Enterococcus 17.9% Present investigation indicates that amongst the
Enterobacter 14.2% burn patients analyzed, 59.9% had second degree
Acinetobacter 14 %
burns, 13.8% had third degree burns and the
Candida 11.3%
reminder had first degree burn. Etiology and
Aspergillus 7.4%
Clostridium 3.4% degree of burn was recorded prior sampling and
are shown in Table 1 and 2. respectively. People
between the age group of 8 to 35 were found more

Table 4. Antibacterial Resistance Pattern (%) exihibited by obtained isolates

Name of Organism Name of Antibiotics

Ch Na Co Nx K P Am C

S. aureus 1 51 53 50 50 78 25 3
E. faecalis S 31 S 27 31 79 23 S
Enterobacter aerogens S S 2 32 S 62 19 S
E. coli 32 51 69 25 80 75 25 42
P. vulgaris S 53 53 50 51 52 50 S
P. mirabilis S 56 54 48 42 49 46 S
Pseudomonas aeruginosa 18 78 71 S 34 52 R S
Klebsiella pneumoniae 41 52 54 15 85 68 16 S
Acinetobacter sps. 29 R 39 R R 56 24 43

Ch - Chloramphenicol, Na - Nalidixic acid Co-Co-Trimoxazole, Nx - Norfloxacin, K - Kanamycin,

P - Penicillin, Am - Amoxycilin, C - Colistin
52 SNEHAL
prone to burn incidences.
Bacteriological analysis of surface swab and
wound biopsy was made by conventional
biochemical methods according to standard
microbiological techniques. Table 3 shows the types
of bacteria isolated from burn wound along with
the percentage incidence of occurrence. Various
types of bacteria were isolated from burn wound
culture and the picture clearly indicates
Pseudomonas as the commonest bacteria, accounting
for 50.74% cases followed by S. aureus (44.7%),
Klebsiella (42.6%). Only two percent of wound
swabs were found to be sterile.
Our results are in accordance with the findings
of Mehta et al., (2007) who studied on the bacterial
isolates obtained from burn wound for 8 long years
and supported the existence of Pseudomonas as the
most eminent colonizer of wound.
Results revealed that all the isolates analysed
were sensitive to Chloramphenicol and Colistin.
Chloramphenicol inhibited considerably all the
strains of Staphylococcus aureus, Enterococcus faecalis
and all Proteus species while few strains of E. coli (32
%), Pseudomonas aerugionsa (18%), K. pneumoniae (41%)
were found resistant on it. Co-trimoxazole was
found satisfactory in inhibiting strains of
Enterococcus faecalis, Enterobacter aerogens while the
remaining isolates exhibited variable percentage of
resistance towards it.
Amoxycilin was found to have considerable
inhibition against all obtained isolates with
variable percentage viz. S. aureus (25%), E. faecalis
(23%), E. coli (25%), P. vulgaris (50%), P. mirabilis (46%),
Klebsiella (16%), Acinetobacter (24%).
Kanamycin and Nalidixic acid resistance was
revealed by all the obtained isolates with variable
percentage viz. S. aureus (50%) isolates were
resistant to Kanamycin while some (51%) isolates
exihibited resistance towards Nalidixic acid. Few
strains of E. faecalis (31 %), E. coli (80%), P. vulgaris
(51%), P. mirabilis (42%), Pseudomonas (34%), Klebsiella
(85%), were found resistant to Kanamycin and least
inhibition was recorded in presence of Penicillin-G.
All bacterial isolates exihibited variable percentage
of resistance towards it.
It is worth to note that maximum organisms
exhibited multiple drug resistance specially
Pseudomonas aeruginosa and Staphylococcus aureus.
Considering the multiple drug resistance
characteristics, immonocompromise status of
patients, side effects caused due to the over
exposure of antibiotics, attention was switch on to
ET AL.
formulate or search an alternative remedy. L.
plantarum strains were isolated from various
sources, isolated and identified. Isolates able to
ferment Arabinose, Mannitol, Fructose, Raffinose,
Ribose, Lactose, Sorbitol, Maltose, Sucrose.were
confirm identified as Lactobacillus plantarum. The
confirmed isolates were further analyzed for
presence or absence of inhibitory potential. Whole
broth of Lactobacillus plantarum as well as cell free
supernatant prepared from medium optimized for
bacteriocin production was utilized for analysis of
inhibitory potential.
Organisms found pre-dominantly associated
with burn wound infection and exhibiting
multidrug resistance were used for further study to
analyze inhibitory action exhibited on them by
Lactobacillus plantarum isolates.
The culture supernatant obtained from 50
lactobacillus isolates were tested for antibacterial
activity against same group of Lactobacilli. Among
them 10 strains were observed to have inhibitory
potential. These isolates were used for further
study.
From the results it was revealed that, cell free
supernatants in medium optimized for bacteriocin
production from L. plantarum inhibited S. aureus
(68%) and P. areuginosa (59%) significantly. The
antimicrobial proteins from the selected cell free
supernatant was capable of inhibiting
considerably P. vulgaris (47%) and E. coli (40%). The
degree of sensitivity was least amongst the clinical
isolates of A. baumanii (38%).
When the same sample were tested for the
activity-spectrum by using whole broth of L.
plantarum, K. pneumoniae (36%) was found to be
highly sensitive in whole broth as compared to in
cell free supernatant in medium optimized for
bacteriocin production (54% in cell free supernatant
and 62% in whole broth). Other isolates analyzed
were found to have least inhibition against L.
plantarum in whole broth than cell free supernatant
in medium optimize for bacteriocin production
except K. pneumonea like S. aureus (62%) and P.
aeruginosa (65%) followed by P. vulgaris (61%), E. coli
(48%) and A. baumanii (31%).
The inhibitory action exhibited by L. plantarum
may be due to accumulation of main primary
metabolites (lactic and acetic acids, ethanol and
CO 2) as well as to the production of other
antimicrobial compounds, such as formic and
benzoic acids, hydrogen peroxide, diacetion and
bacteriocins, in case of whole broth. But, in medium
 Efficacy of Lactobacillus plantarum to Inhibit the Growth of Aerobic Bactreial
optimized for bacteriocin production, the
conditions and formulation are so adjusted so as to
suit bacteriocin production suppressing the other
components. Thus it may be inferred that
bacteriocins from L. plantarum can be used to check
burn infections pathogens.
Similar conclusions have been derived by
Horcharoen and Sookkheee (2007). They studied
antimicrobial activity of Lactobacillus against
urovaginal pathogens. As per their conclusions L.
plantarum may be excellent candidate for eventual
use as probiotics to restore the balances of normal
microbiota in urovaginal ecosystem as well to
prevent uropathogenic bacterial infections.
Valdez et al. (2005) also studied interference of
Pseudomonal growth by L. plantarum whole cell and
cell free supernatant. They isolated Pseudomonas
majorly from burn wound and evaluated in-vitro as
well as in-vivo. The inhibitory potential exhibited
by L. plantarum strains on Pseudomonas aeruginosa
infections. According to their inferences L. plantarum
and or its by-product are potential therapeutic
agents for local treatment of P. aeruginosa burn
infections.
Findings from the investigation "Efficacy of
Lactobacillus plantarum to inhibit the growth of
aerobic bacterial burn wound pathogens" reflects
effective use of L. plantarum or its derivatives to cure
burn infectious pathogens and might help in
reduction of mortality rate, in burn care unit.
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Al Akayleh, A.T. 1999. Invasive burn wound infection.
Annals of Burn and Fire Disasters. 12 : 1-5.
Anantnarayan and K.J. Panikar: Text book of
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Bauer, A.W., Kirby, W.M. and Sheories J.C. 1996.
Antibiotic susceptibility testing by standardised
single disc method. An J. Clin Pathol. 48 : 493 - 497.
Bergey, 1989. Manual of Systematic Bacteriology. 4.
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Horcharoen, K. and Kookkhees 2005. Antimicrobial
activity of Lactobacillus against the urovaginal
pathogenic bacteria. Department of microbiology,
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Valdez, J. C. , Peral, M. C., Rachid, M., Santana, M. and
Perdigon, G. 2005. Interference of Lactobacillus
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grams : A eight years study. Indian J. Plast. Surg. 40
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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 63-68
© Global Science Publications

INSIGHT OF ANAEMIA WITH SOCIO DEMOGRAPHIC

ACQUAINTANCES AMONG PREGNANT WOMEN
IN SOUTH INDIA

HEPSIBAH PALIVELA 1, M.S. NEEHARIKA 2,V. SAI KUMAR 3 AND M. ANUSHA2

1
Department of Pharmacy, Al-Ameer College of Pharmacy, Gudilova,
Anandapuram, Visakhapatnam, India
2
Department of Pharmacy, A.Q.J.College of Pharmacy, Gudilova,
Anandapuram, Visakhapatnam, India

(Received 13 August 2011; Accepted 15 September 2011)

Key words : Anaemia, Malnutrition, Pregnant women, Sociodemographic factors, Poverty.

Abstract - This research investigates the anaemic cases encountered among pregnant women in
Tirupati, Chittoor district, Andhra Pradesh. In India the impact of anaemia recorded alarming mortality
rates among the selected population. A cross-sectional study was conducted in the Govt. Maternity
Hospital of Tirupati for a period of 9 months to acknowledge the impact of anaemia at all the trimesters
of the pregnant women.A total of 550 pregnant women were registered. Study variables such as
haemoglobin content (Tallquist's method), age, weight, socio demographic factors like occupation,
monthly income, education and clinical factors like birth interval and number of abortions were
considered. A high prevalence of anaemia (91.09%) among the enrolled patients was observed. A major
proportion of them (48.00%) had moderate anaemia. Despite the measures taken to control anaemia in
pregnancy, the severity of nutritional anaemia continues to remain a public health issue of greater
magnitude, suggesting that these measures have been largely ineffective. Hence, health promotions
to improve the diet and iron supplement for women are required to redress nutritional deficits.

INTRODUCTION Apparently, the present investigation was

The impact of anaemia is on the higher side and
stands as a major public health issue to be dealt. In
India the vital cause of high mortality rates
accounting up to 20% is due to the maternal deaths.
(Govt of India, Health Information of India, 1995).
According to WHO, in developing countries the
prevalence in pregnant women is 56% ranging
from 35 to 100% among distributed regions in the
world (WHO, 1992). Descriptive and methodo-
logical studies were reported on the prevalence of
anaemia in distributed regions of India (Luwang et
al. 1980).
The consequences of anaemia among women
undergo the increased risk of low birth weight,
perinatal and neonatal mortality and also the
maternal morbidity and mortality (Roy et al. 1992).
carried out to find out the prevalence of anaemia
with socio demographic factors in pregnant
women.
The high prevalence of anaemia among women
in India is a burden for them and their families that
implies the economic development & productivity
of the country (Vijayaraghavan et al. 1990).
The results reflect the effect of poverty on
women’s nutrition and anaemia status, regardless
of whether they live in rural or urban areas
(Bentley and Griffiths, 2003).
METHODOLOGY
The research was conducted in the Govt.
Maternity Hospital of Tirupati for a period of 19
months encountering five hundred and fifty

*Corresponding author - Dr. Hepsibah Palivela, Associate Professor, Gudilova, Anandapuram, Visakhapatanm
E-mail : premhkp@yahoo.com
64 PALIVELA ET AL.
patients registered in out-patient unit and
maternity wards. These patients were selected
from the hospital as they can endow the adequate
number of cases for the study. The women with
multiple pregnancies and bleeding disorders were
not accomplished in this data for evaluation.
A questionnaire was developed to assess the
awareness and practices of pregnant ladies with
the assistance of doctors. The affected patients were
enrolled based on their haemoglobin content
(Tallquist’s method) (Human Physiology Vol-I by
C.C. Chatterjee).
These subjects were interviewed using a pre-
tested schedule. The severity of anaemia was
classified as per WHO criteria (WHO, Geneva, 1989).
The study population encountered fit in the class
below poverty line presenting the blow of anaemia.
Chi-square test was employed to acknowledge
the significance of the characteristic factors
considered in the research study.
RESULTS AND DISCUSSION
Of the 550 pregnant women enrolled in this study,
49 were recorded as normal with haemoglobin
content of >13 gm/dL. Anaemia was recorded in
501 (91.09%) of the enrolled women categorizing
them into mild (with Hb content of 11-13gm/dL),
moderate (9-11gm/dL) and severely anaemic
(<9gm/dL), of which higher number of moderately
anaemic patients were recorded.
Our proposition on the relationship of age to
anaemia was partially supported. Women in the
20-25 yrs age group constituted the highest
percentage of anaemic cases (36.55%) followed by
25-30 yrs age group (17.64%) suggesting the fact
that the women below the age of 30 yrs are more
prone to be anaemic.
Although overweight women had higher
prevalence of any degree of anaemia compared to
normal-weight and thin women, high prevalence
was observed across all groups. The percentage of
women observed to be anaemic was high (33.27%)
for women of weight group 55-65 kg, compared to
the other weight groups. For the same weight
group, the prevalence of anaemia ranged from
10.36% for mild anaemic, 14.73% for moderately
anaemic to 8.18% for severely anaemic pregnant
women suggesting dietary deficiency or other
problems among women who have no apparent
resource constraints (Griffiths & Bentley, 2001).
Other problems may include hook worm or
malaria infection, acute infections, micronutrient
deficiency that interferes with iron metabolism, or
poor dietary patterns that compromise adequate
iron intake. Among over-weight and higher-income
women, with moderate to severe anaemia, we
construe that diet may play an important role but
we are unable to assess its role with the current
data.
Our proposition related to sociodemographic
factors namely occupation, monthly income,
education and anaemia were partially supported.
We had expected to find the highest prevalence of
anaemia among those women who are illiterate
and poorest based on the standard of living index.
However, the poorest illiterate and educated
women both had the greatest risk of anaemia and
had similar probabilities of being anaemic. Women
from low standard of living group (farmers-37.64%)
were observed to have the highest odds of being
mildly (8.73%), moderately (17.64%) or severely
anaemic (11.27%) when compared to coolies
(30.00%) and maids (23.45%) belonging to the same
standard of living. A higher prevalence (46.55%) of
anaemia was recorded among 256 of the enrolled
subjects with a monthly income of < Rs.1500.
Illiteracy among patients was high with 185
(33.64%), 143 (26.00%) primary, 121 (22.00%) higher
grade and 52 (9.46%) at college level.
When first pregnancy was delayed up to 18
years or later, the pregnant women were more
often normal or had mild anaemia. However, these
trends were statistically not significant (Table 2).
A high prevalence of anaemia (91.09%) among
pregnant women was observed. Majority (48.00%)
had moderate anaemia (Table 1).
It is obligatory to improve women’s overall
nutrition status and their access to resources
(income) which will have the greatest impact on
reducing anaemia in India (World Bank, 1993).
Integrated programs for hook worm eradication,
malaria prophylaxis, or that address other
micronutrient deficiencies would also be important
for reducing the affliction of anemia (Stoltzfus,
1997; Gillespie & Johnston, 1998).
CONCLUSION
In conclusion, the high prevalence of anemia
among women in India is a millstone for them, for
their families, and for the pecuniary progress and
productivity of the country. Iron supplementation
programs, for a variety of reasons, have not been
Table 1.

0 0.00 0.00 0.00 24 4.36 11.60 278.40 61 11.09 10.50 640.50 27 4.91 8.50 229.50 112 20.36 10.25
8 1.45 13.50 108.00 49 8.91 11.20 548.80 99 18.00 9.81 971.19 53 9.64 8.20 434.60 201 36.55 9.72
12 2.18 13.20 158.40 30 5.45 11.31 339.30 46 8.36 9.92 456.32 21 3.82 8.34 175.14 97 17.64 10.01
23 4.18 13.41 308.43 23 4.18 11.72 269.56 41 7.45 10.17 416.97 16 2.91 8.61 137.76 80 14.55 10.30
6 1.09 13.52 81.12 3 0.55 11.87 35.61 6 1.09 10.42 62.52 2 0.36 8.40 16.80 11 2.00 10.45
49 8.91 13.41 13.39 129 23.45 11.54 11.41 253 46.00 10.16 10.07 119 21.64 8.41 8.35 501 91.09 10.15
4 0.73 13.21 52.84 25 4.55 11.41 285.25 34 6.18 10.31 350.54 16 2.91 8.31 132.96 75 13.64 10.25
12 2.18 13.43 161.16 35 6.36 11.16 390.60 60 10.91 10.22 613.20 34 6.18 8.64 293.76 129 23.45 10.06
16 2.91 13.67 218.72 57 10.36 11.51 656.07 81 14.73 10.48 848.88 45 8.18 8.27 372.15 183 33.27 10.26
12 2.18 13.52 162.24 19 3.45 11.68 221.92 44 8.00 10.82 476.08 23 4.18 8.49 195.27 86 15.64 10.39
5 0.91 13.62 68.10 9 1.64 11.59 104.31 11 2.00 9.91 109.01 8 1.45 8.37 66.96 28 5.09 10.01
49 8.91 13.49 13.53 145 26.36 11.47 11.44 230 41.82 10.35 10.42 126 22.91 8.42 8.42 501 91.09 10.19
15 2.73 13.56 203.40 45 8.18 11.35 510.75 81 14.73 10.65 862.65 39 7.09 8.51 331.89 165 30.00 10.34
14 2.55 13.84 193.76 39 7.09 11.51 448.89 59 10.73 10.41 614.19 31 5.64 8.32 257.92 129 23.45 10.24
20 3.64 13.29 265.80 48 8.73 11.81 566.88 97 17.64 10.25 994.25 62 11.27 8.41 521.42 207 37.64 10.06
49 8.91 13.56 13.53 132 24.00 11.56 11.56 237 43.09 10.44 10.43 132 24.00 8.41 8.42 501 91.09 10.21
6 1.09 13.49 80.94 63 11.45 11.12 700.56 132 24.00 10.21 1347.72 61 11.09 8.29 505.69 256 46.55 9.98
18 3.27 13.28 239.04 53 9.64 11.67 618.51 67 12.18 9.81 657.27 21 3.82 8.66 181.86 141 25.64 10.34
25 4.55 13.64 341.00 20 3.64 11.41 228.20 65 11.82 10.42 677.30 19 3.45 8.31 157.89 104 18.91 10.22
49 8.91 13.47 13.49 136 24.73 11.40 11.38 264 48.00 10.15 10.16 101 18.36 8.42 8.37 501 91.09 10.18
10 1.82 13.11 131.10 47 8.55 11.81 555.07 97 17.64 9.59 930.23 41 7.45 8.44 346.04 185 33.64 9.90
12 2.18 13.81 165.72 33 6.00 11.45 377.85 81 14.73 10.25 830.25 29 5.27 8.39 243.31 143 26.00 10.15
20 3.64 13.52 270.40 32 5.82 11.21 358.72 62 11.27 10.41 645.42 27 4.91 8.61 232.47 121 22.00 10.22
7 1.27 13.52 94.64 16 2.91 11.35 181.60 24 4.36 10.35 248.40 12 2.18 8.52 102.24 52 9.45 10.24
49 8.91 13.49 13.51 128 23.27 11.46 11.51 264 48.00 10.15 10.05 109 19.82 8.49 8.48 501 91.09 10.13
37 6.73 13.17 487.29 114 20.73 11.56 1317.84 192 34.91 9.64 1850.88 114 20.73 8.74 996.36 420 76.36 9.92
12 2.18 13.21 158.52 10 1.82 11.32 113.20 52 9.45 10.88 565.76 5 0.91 8.31 41.55 67 12.18 10.75
0 0.00 13.42 0.00 5 0.91 11.79 58.95 9 1.64 10.95 98.55 0 0.00 8.62 0.00 14 2.55 11.25
49 8.91 13.27 13.18 129 23.45 11.56 11.55 253 46.00 10.49 9.94 119 21.64 8.56 8.72 501 91.09 10.64
22 4.00 13.22 290.84 26 4.73 11.66 303.16 101 18.36 9.79 988.79 17 3.09 8.77 149.09 144 26.18 10.01
8 1.45 13.83 110.64 58 10.55 11.85 687.30 120 21.82 10.53 1263.60 47 8.55 8.31 390.57 225 40.91 10.41
Insight of Anaemia with Socio Demographic Acquaintances among Pregnant Women

19 3.45 13.72 260.68 17 3.09 11.43 194.31 73 13.27 10.12 738.76 42 7.64 8.92 374.64 132 24.00 9.91
49 8.91 13.59 13.51 101 18.36 11.65 11.73 294 53.45 10.15 10.17 106 19.27 8.67 8.63 501 91.09 10.11
65
 66
Table 2.

Characteristic Anaemia Normal

Mild (11-13gm/Dl) Moderate(9-11gm/Dl) Severe (<9gm/Dl) Total ( >13gm/Dl) PValue

No. % Hb %±Sd No. % Hb %±Sd No. % Hb %±Sd No. % Hb %±Sd No. % Hb %±Sd
AGE (yrs)

15-20 24 4.36 11.6±0.2 61 11.09 10.5±0.67 27 4.91 8.5±0.21 112 20.36 10.25 0 0.00 0.00
20-25 49 8.91 11.2±0.42 99 18.00 9.81±0.26 53 9.64 8.2±0.46 201 36.55 9.72 8 1.45 13.5±0.29
25-30 30 5.45 11.31±0.26 46 8.36 9.92±0.41 21 3.82 8.34±0.39 97 17.64 10.01 12 2.18 13.2±0.64 <0.05
35-40 23 4.18 11.72±0.41 41 7.45 10.17±0.62 16 2.91 8.61±0.81 80 14.55 10.30 23 4.18 13.41±0.33
45-50 3 0.55 11.87±0.61 6 1.09 10.42±0.32 2 0.36 8.4±0.17 11 2.00 10.45 6 1.09 13.52±0.19
ALL 129 23.45 11.54±0.38 253 46.00 10.16±0.46 119 21.64 8.41±0.41 501 91.09 10.15 49 8.91 13.41±0.36

WEIGHT (kg)

35-45 25 4.55 11.41±0.21 34 6.18 10.31±0.29 16 2.91 8.31±0.32 75 13.64 10.25 4 0.73 13.21±0.41
45-55 35 6.36 11.16±0.43 60 10.91 10.22±0.62 34 6.18 8.64±0.21 129 23.45 10.06 12 2.18 13.43±0.29
55-65 57 10.36 11.51±0.29 81 14.73 10.48±0.17 45 8.18 8.27±0.34 183 33.27 10.26 16 2.91 13.67±0.52 <0.05
65-75 19 3.45 11.68±0.36 44 8.00 10.82±0.32 23 4.18 8.49±0.51 86 15.64 10.39 12 2.18 13.52±0.81
75-85 9 1.64 11.59±0.34 11 2.00 9.91±0.56 8 1.45 8.37±0.42 28 5.09 10.01 5 0.91 13.62±0.19
ALL 145 26.36 11.47±0.33 230 41.82 10.35±0.39 126 22.91 8.41±0.36 501 91.09 10.19 49 8.91 13.49±0.44

OCCUPATION
PALIVELA ET AL.

COOLIE 45 8.18 11.35±0.29 81 14.73 10.65±0.27 39 7.09 8.51±0.61 165 30.00 10.34 15 2.73 13.56±0.42
MAIDS 39 7.09 11.51±0.31 59 10.73 10.41±0.81 31 5.64 8.32±0.51 129 23.45 10.24 14 2.55 13.84±0.28 <0.05
FARMERS 48 8.73 11.81±0.18 97 17.64 10.25±0.19 62 11.27 8.41±0.29 207 37.64 10.06 20 3.64 13.29±0.51
ALL 132 24.00 11.56±0.26 237 43.09 10.44±0.42 132 24.00 8.41±.047 501 91.09 10.21 49 8.91 13.56±0.40

MONTHLY INCOME
<1500 63 11.45 11.12±0.67 132 24.00 10.21±0.31 61 11.09 8.29±0.56 256 46.55 9.98 6 1.09 13.49±0.17
1501-2500 53 9.64 11.67±0.29 67 12.18 9.81±0.62 21 3.82 8.66±0.28 141 25.64 10.34 18 3.27 13.28±0.61 <0.05
2501-4000 20 3.64 11.41±0.43 65 11.82 10.42±0.29 19 3.45 8.31±0.41 104 18.91 10.22 25 4.55 13.64±0.24
ALL 136 24.73 11.4±0.46 264 48.00 10.15±0.41 101 18.36 8.42±0.42 501 91.09 10.18 49 8.91 13.47±0.34

EDUCATION
ILLITERATES 47 8.55 11.81±0.36 97 17.64 9.59±0.18 41 7.45 8.44±0.35 185 33.64 9.90 10 1.82 13.11±0.29
PRIMARY 33 6.00 11.45±0.41 81 14.73 10.25±0.29 29 5.27 8.39±0.26 143 26.00 10.15 12 2.18 13.81±0.41
HIGH SCHOOL 32 5.82 11.21±0.38 62 11.27 10.41±0.61 27 4.91 8.61±0.31 121 22.00 10.22 20 3.64 13.52±0.17 <0.05
COLLEGE 16 2.91 11.35±0.19 24 4.36 10.35±0.51 12 2.18 8.52±0.19 52 9.45 10.24 7 1.27 13.52±0.38
LEVEL
ALL 128 23.27 11.45±0.34 264 48.00 10.15±0.4 109 19.82 8.49±0.28 501 91.09 10.13 49 8.91 13.49±0.31
37 6.73 13.17 114 20.73 11.56 192 34.91 9.64 114 20.73 8.74 420 76.36 9.92
12 2.18 13.21 10 1.82 11.32 52 9.45 10.88 5 0.91 8.31 67 12.18 10.75
0 0.00 13.42 5 0.91 11.79 9 1.64 10.95 0 0.00 8.62 14 2.55 11.25
49 8.91 13.27 129 23.45 11.56 253 46.00 10.49 119 21.64 8.56 501 91.09 10.64

22 4.00 13.22 26 4.73 11.66 101 18.36 9.79 17 3.09 8.77 144 26.18 10.01
8 1.45 13.83 58 10.55 11.85 120 21.82 10.53 47 8.55 8.31 225 40.91 10.41
19 3.45 13.72 17 3.09 11.43 73 13.27 10.12 42 7.64 8.92 132 24.00 9.91
49 8.91 13.59 101 18.36 11.65 294 53.45 10.15 106 19.27 8.67 501 91.09 10.11

Characteristic Anaemia Normal

Mild (11-13gm/Dl) Moderate(9-11gm/Dl) Severe (<9gm/Dl) Total ( >13gm/Dl) P Value

No. % Hb %±Sd No. % Hb %±Sd No. % Hb %±Sd No. % Hb %±Sd No. % Hb %±Sd

No. of Abortions

0 114 20.73 11.56±0.22 192 34.91 9.64±0.36 114 20.73 8.74±0.29 420 76.36 9.92±0.47 37 6.73 13.17±0.25
1 10 1.82 11.32±0.51 52 9.45 10.88±0.29 5 0.91 8.31±0.66 67 12.18 10.75±0.43 12 2.18 13.21±0.69
2 5 0.91 11.79±0.82 9 1.64 10.95±0.73 0 0.00 8.62±0.78 14 2.55 11.25±0.65 0 0.00 13.42±0.58 <0.05
ALL 129 23.45 11.55±0.41 253 46.00 10.49±0.55 119 21.64 8.56±0.38 501 91.09 10.64±0.58 49 8.91 13.26±0.54

BIRTH INTERVAL (in months)

<18 26 4.73 11.66±0.12 101 18.36 9.79±0.49 17 3.09 8.77±0.28 144 26.18 10.01±0.94 22 4.00 13.22±0.84
18-35 58 10.55 11.85±0.48 120 21.82 10.53±0.18 47 8.55 8.31±0.64 225 40.91 10.41±0.42 8 1.45 13.83±0.21 <0.05
>36 17 3.09 11.43±0.25 73 13.27 10.12±0.22 42 7.64 8.92±0.21 132 24.00 9.91±0.34 19 3.45 13.72±0.74
ALL 101 18.36 11.65±0.83 294 53.45 10.12±0.47 106 19.27 8.67±0.48 501 91.09 10.11±0.88 49 8.91 13.59±0.33
Insight of Anaemia with Socio Demographic Acquaintances among Pregnant Women
67
68
effective in reducing anemia prevalence (Galloway
& McGuire, 1991; Vijayaraghaven et al., 1990) and
operational research on how best to improve
existing iron supplementation programs is needed
(Yip, 1994). New and innovative strategies are
needed, primarily those that improve the overall
health and nutrition status of adolescent girls
before they enter their reproductive years (Kurz &
Johnson-Welch, 1994; Kanani & Poorjara, 2000;
Creed-Kanashiro et al., 2000). This will necessitate
customised programs that target women in all
socioeconomic groups so as to promptly enhance
the growth of marginalised segment of the Indian
population.
ACKNOWLEDGEMENT
We would like to thank the respondents in the
study for their willingness to answer our intricate
queries. We are grateful to the hospital authorities
for their continuous support and the assistance of
Ms. B. Jyothirmaye and Ms. S. Jayanthi in the
fulfillment of the investigation.
REFERENCES
Bentley, M.E. and Griffiths, P.L. 2003. The burden of
anaemia among women in India. Ind. J. Cli. Nutr.
57: 52-60.
Creed-Kanashiro, H.M., Uribe, T.G., Bartolini, R.M.,
Fukumoto, M.N., Lopez, T.T., Zavaleta, N.M. and
Bentley, M.E. 2000. Improving dietary intake to
prevent anemia in adolescent girls through
community kitchens in a periurban population of
Lima, Peru. J. Nutr. 130 : 459S-261S.
Govt of India. Health Information of India, 1995, DGHS,
Nirmal Bhawan, New Delhi.
Griffiths, P.L. and Bentley, M.E. 2001. The nutrition
transition is underway in India. J. Nutr. 131 : 2692-
2700.
PALIVELA ET AL.
Gillespie, S. and Johnston, J. 1998. Expert Consultation
on Anemia Determinants and Interventions,
Ottawa: The Micronutrient Initiative.
Galloway, R. and McGuire, J. 1994. Determinants of
compliance with iron supplementation: supplies,
side effect, or psychology?. Soc. Sci. Med. 39 : 381-
390.
Kurz, K. and Johnson-Welch, C. 1994. The Nutrition and
lives of adolescents in developing countries:
Findings from the Nutrition of Adolescent Girls
Research Program, Washington, DC:
International Center for Research on Women.
Kanani, S.J. and Poojara, R.H. 2000. Supplementation
with iron and folic acid enhances growth in
adolescent Indian girls. J. Nutr. 130 : 452S - 455S.
Luwang, N.C., Gupta, V.M. and Khanna, S. 1980.
Anaemia in pregnancy in rural community of
Varanasi. Ind J Prev. Soc. Med. 11 : 83-88.
Preventing and controlling iron deficiency anaemia
through primary health care, WHO, Geneva, 1989.
Roy, S. and Chakravorty, P.S. 1992. Maternal and
perinatal outcome in severe anaemia. J Obstet
Gynae Ind. 42 : 743-750.
Stoltzfus, R.J. 1997. Rethinking anemia surveillance.
Lancet. 349 : 1764 -1766.
Vijayaraghavan, K., Brahmam, G.N.V., Nair, K.M.,
Akbar, D. and Praalhad Rao, N. 1990. Evaluation of
National Nutritional Anemia Prophylaxis
Programme. Ind. J. Pediatr. 57 : 183-190.
Vijayaraghavan, K., Brahmam, G.N.V., Nair, K.M.,
Akbar, D. and Praalhad Rao, N. 1990. Evaluation of
National Nutritional Prophylaxis Programme. Ind.
J. Pediatr. 57 : 183-190.
World Health Organization. The prevalence of anaemia
in women: A tabulation of available information,
2nd Ed., Geneva: WHO, 1992.
World Bank 1993. Invest in Health, World Development
Report. pp195-324, Oxford: Oxford University
Press.
Yip, R. 1994. Iron deficiency: contemporary scientific
issues and international programmatic
approaches. J. Nutr. 124 (S8) : S1479-S14.
—❋❋❋—
Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 75-78
© Global Science Publications

EFFECT OF SUN EXPOSURE ON BLOOD PRESSURE IN

HUMAN SUBJECTS

MUKESH KUMAR*, SATISH PRAKASH** AND RUPESH TIWARI***

* Department of Physiology, Subharti Medical College, Meerut, U.P., India

** Department of Biophysics, Meerut College, Meerut, U.P., India
*** Department of PSM, Subharti Medical College, Meerut, U.P., India

(Received 15 June 2011; Accepted 17 August 2011)

Key words : Sun exposure, Human subjects, Blood pressure

Abstract - The paper presents results of experimental studies dealing with the effect of sun exposure
on blood pressure in human subjects outdoors. The field experiments were carried out in the month of
April, May and June 2006 in Meerut District. Detailed history about age, height weight work, exercise,
alcohol consumption, smoking and duration of exposure to sunlight in 221 volunteer subjects (age-20-
40 yrs.) were taken. Subjects were classified into eight groups according to the duration of exposure to
sunlight. Systolic (BPS) and Diastolic (BPD) Blood pressure were recorded in all groups using mercury
manometer. Decrease in blood pressure was statistically significant (p<0.05) and positively co related
(r ho = 0.78) with the number of hrs exposed to sunlight. The most obvious conclusion of this study is
that sunlight exposure greater than 3 hrs/ day is a major factor in the prevention of Hypertension
particularly for dark skinned people. The optimum exposure for prevention of Hypertension is 7-hrs/
per day. It seems that mechanisms that play role in blood pressure control during sun exposure are
intensive sweating, metabolism of cholesterol and decreased level of stress.

INTRODUCTION of cancer, is fortunately blocked by the ozone layer

The aim of the paper is to observe the effect of
duration of sun exposure on blood pressure.
Exposure to sun light is very important factor in
human life. Many people wonder about the safety
of UV light. In the 20th century, we were taught
that UV light is dangerous and to be afraid of it, yet
our ancestors lived outside and sun did not make
them sick (Linaya Hahn, 1995). Physically, it is a
type of electromagnetic wave with three
wavelengths: UV-A (320-400nm), UV-B (280-320
nm) & UV-C (less than 280 nm) ( Kime 1980 &
Liberman 1991) . UV-B stimulates the production
of Vitamin D3 in our skin and is essential for the
absorption of calcium in the bones (Adams et al.
1982 & Neer et al. 1971). Those with greater amount
of pigment- melanin in the skin (as in Indians)
require six times the amount of UV-B light to
produce the same amount of vitamin D as
compared to lighter skinned people (Jablonski et al.
2000). Most of the UV-C, which can increase the risk
of the earth’s atmosphere.
Hypertension is a silent killer and can lead to
heart attack, stroke, kidney failure and even
blindness etc. According to WHO and ISH,
cardiovascular disease will rank as first among the
causes of the global burden of disease by 2020
(Indian express, 27 July 1993). In the West, the high
probability is still between 40-60 years, while in
India it is dipped to early 30s.
According to 10 year data from 1990-1999, the
percentage of coronary heart disease patients in
Indian urban area is 7.6 to 12.6% while in rural area
it is 3.1 to 7.4 % (Jurger Kiefev, 1990).
In India 25% of urban Indians suffer from
hypertension. Stress, sedentary life style, junk food
etc. and besides this, the increasing tendency to
escape from sunrays in urban people has certainly
increased the possibility of heart diseases (TOI. 26
March, 2001).
These data show that despite other factors, there
may be a close relation between solar radiation

*Corresponding author - Dr. Mukesh Kumar, Professor, Department of Physiology, Subharti Medical College,
NH-58, Delhi-Haridwar By pass road, Meerut, E-mail : mkumar5031@rediffmail.com
76 KUMAR ET AL.

and heart diseases because in rural area the people according to the duration of exposure to sun light.
are in more contact with the sunrays in
Sun exposure
comparison to urban.
duration (hrs) 1 2 3 4 5 6 7 8&
more
METHODS
Groups I I I I I I I V V V I VII VIII
The field experiments were carried out in the No. of subjects 39 25 28 45 16 23 09 36
month of April, May and June 2006 in Meerut
district. There were 221 male volunteer subjects for Systolic (BPS) and Diastolic (BPD) Blood pressure
analysis. The population samples in the study were were recorded in all groups using mercury
taken from all social classes of rural and urban manometer. Mean Systolic, Diastolic with Standard
areas. Subjects in the age group of 20- 40 years with Deviation and Average blood pressures were
BMI of 18.5-24.9 Kg/m2 were eligible for our study calculated in each group. Statistical Analysis:
and those having any history of hypertension, Differences in blood pressure in each group were
CHD, Diabetes and other cardiac related problems analyzed using student t-test and the co-relation
were excluded. between sun exposure and blood pressure was
After the informed consent, subjects were analyzed using r value.
questioned in detail about name, age, height,
weight, work, exercise, smoking, alcohol RESULTS
consumption and duration of exposure to sun light.
The subjects were classified in to eight groups Results are shown in following Table and Graph.

Table 1. Daily Sun Exposure & Blood Pressure

Sun Exposure Gorups No. of Subjects Systolic Blood Pressure Diastolic Blood Pressure
Duration (Hrs.)
Mean ± S.d. (Mm. Hg.) Mean ± S.d. (mm. Hg)

1. I 39 132 ±2.39 80±3.82

2. II 25 136±2.47 76±3.63
3. III 28 122±2.21 72±3.44
4. IV 45 128±2.32 70±3.35
5. V 16 124±2.25 72±3.44
6. VI 23 120±2.18 68±3.25
7. VII 09 112±2.03 67±3.20
8. VIII 36 126±2.29 74±3.53
 Effect of Sun Exposure on Bloodpressure in Human Subjects
DISCUSSION
Our findings reveal that increased duration of
exposure to sunlight leads to decrease in blood
pressure in different groups. Decrease in blood
pressure was statistically significant (p<0.05)
between the groups and it was correlated with the
duration of exposure to sun light.
Sunlight lowers blood pressure in normal
individuals and this effect is more pronounced in
individuals with high blood pressure (Mercola,
2004).
Quantity of Melanin varies from race to race,
individual genetic level, and in terms of the
personal adjustment. Pigmentation in the human
skin determines the tolerance of UV-B radiation.
Melanin increases with the exposure to sun light.
Transmission of radiation through deeply
pigmented skin is only 5% as compared to 20%-30%
in white skin. Indian and Pakistani children living
in United States show that their capacity to
produce vitamin D is same as whites, but like
blacks, they require longer exposure to UV light
because of increased melanin content (Jablonski et
al. Oct. 2002).
In human body, the prominent factors, which
cause high blood pressure are cholesterol, sodium
and stress.
The levels of cholesterol, sodium and stress can
be controlled by exposure to sunlight. The UV-B
radiation of sunlight (290-315 nm wave length)
decreases blood pressure, by lowering levels of
cholesterol by synthesizing Vitamin-D3 (Adams et
al. 1982), level of serum sodium by increased
sweating and level of stress by increasing level of
endorphins (Mercola, 2004).
Both vitamin D 3 and cholesterol (which is
needed to make the sex hormones) are derived from
the same substance in the body-a chemical called
SQUALENE, which is found in the skin. In the
presence of sun light, this squalene is converted to
vitamin D3 but in its absence, it is converted to
cholesterol (Forman et al. 2005). So, every molecule
of vitamin D3 created in skin’s blood stream leads to
decrease in one molecule of cholesterol.
Studies show that sunlight not only lowers
blood pressure and cholesterol levels but also
increase the cardiac output by as much as 39%.
Also during sun exposure the capillaries dilate,
permitting increased flow of blood to the skin and
thus the increase in capillary volume keeps blood
pressure normal.
77
The predominant electrolyte of sweat is sodium.
Sweating is the body’s most efficient mechanism of
eliminating sodium and believed to be beneficial for
the cases of mild hypertension.
Apart form cholesterol and sodium being the
prominent factors for high blood pressure; the
researchers theorize that UV exposure leads to the
release of chemicals in the brain called endorphins,
which are linked to both pain relief and euphoric
feelings. The researcher believes that decrease
vitamin-D production actually results in increase
parathyroid hormone production that actually
serves to increase the blood pressure. Another
study found that vitamin-D is a negative inhibitor
of the rennin-angiotensin system and this serves to
lower blood pressure (Mercola, 2004).
The reasons for higher incidence of
cardiovascular problems, particularly coronary
artery disease, in winters have remained
controversial but investigators have mostly
pointed to the environmental cold (Bull, 1973).
Evidences show that the hypertension, the major
cause of cardiovascular deaths in population,
increases during cold season when the availability
of solar radiation is very less (Anderson et al. 1970
& Baker-Blocker 1982). Environmental cold induces
vasoconstriction, which in-turn raises arterial
blood pressure and consequent increase in
myocardial oxygen demand.
Published data shows a linear rise in blood
pressure at increasing distance from equator; and
geographical and seasonal changes in blood
pressure are inversely associated with solar
radiation (Peggy, 2000). Mortality is usually lowest
in the region of 15-20 oC and rises both with
lowering and rising of temperature (Eugene Rogot,
1962). In northern India, which represents a wide
range of climates, the lowest mortality is seen in
the temperature range of 20-25oC.
CONCLUSION
The most obvious conclusion of this study is that
sunlight exposure greater than 3hrs / day is a major
factor in the prevention of Hypertension
particularly for dark skinned people. The optimum
exposure for prevention of Hypertension is 7hrs/
day.
REFERENCES
Adams, J.S., Clemens, T.L., Parrish, J.A. and Holick, M.F.
78 KUMAR ET AL.
March25, 1982. Vitamin-D Synthesis and
Metabolism after Ultraviolet irradiation of normal
and vitamin-D deficient subjects. New England
Journal of Medicine. 306 (12) : 722-25.
Anderson, T.W. and Le Richa, W.H. 1970. Cold weather
and myocardial infarction. Lancet. 1 : 291-296
Baker-Blocker, A. 1982. Winter weather and
cardiovascular mortality in Minneapolis- St. Paul.
Ame. J Public Health. 72 : 261-265
Bull, G.M. 1973. Meteorological correlation with
myocardial and cerebral infarction & respiratory
disease. Brit J Prev Soc Med. 27: 108-113
Mercola, 6 July 2004. Sun light can lower your blood
pressure. Eurek Alert.
Eugene Rogot and Stephan J. Padgett, 1962-1966.
Association of coronary and stroke mortality with
temperature and snowfall in selected areas of the
Unite States. Am J Epidemiol. 103 : 565-575.
Forman, J.P., Bischoff, H.A., Willatt, W.C. and Stampfer,
M.J. 2005. Vitamin-D intake and risk of incident
Hypertension: Results from three large
prospective cohort studies. Hypertension. 46 (4) :
676-682.
—❋❋❋—
Indian Express, 27 July 1993. Killing you silently. In
Lifeline: Hypertension.
Jablonski, Nina, G. and Chaplin. 2000. The evolution of
human skin coloration. Journal of Human Evolution.
106 : 39 -57.
Jablonski, Nina, G. and George Chaplin, 2002. Human
skin color. Scientific American. 287 (4) : 74
Jurger Kiefev, 1990. Biological Radiation Effects. Springer
Berlin Heidelberg: 292
Kime, Z.R. 1980. Sunlight. World Health Publications,
Penryn, CA.
Liberman, J. 1991. Light : Medicine of the Future. Santa Fe:
Bear.
Linaya Hahn, L.N.C. 1995. Solving the Puzzle. Spectrum
Press, Chicago: 89-91.
Neer, R.M., Davis, T.R.A., Walcott, A. and Koski, S.
Jan.22, 1971.Stimulation by artificial lighting of
calcium absorption in elderly human subjects.
Nature. 229.
Peggy, C.W. 2000. Seven Countries Study Research
Group. New England Journal of Medicine. 342 : 1-8.
TOI. 26 March 2001. Hypertension Hits Daily Efficiency.
AIIMS Study, AIIMS, New Delhi.
Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 79-82
© Global Science Publications

PCR BASED DETECTION OF COLLETOTRICHUM

GLOEOSPORIOIDES (PENZ.) PENZ. AND SACC. THE CAUSATIVE
AGENT OF LEAF SPOT DISEASE OF MURRAYA KOENIGII L.

MIDHILA PADMAN AND G. R. JANARDHANA*

Mycology and Plant Pathology Laboratory, Department of Studies in Botany,

University of Mysore, Manasagangotri, Mysore570 006, Karanataka, India

(Received 28 June 2011; Accepted 17 August 2011)

Key words : Colletotrichum gloeosporoides, Murraya koenigii, Leaf spot disease.

Abstract - The curry leaf tree is vulnerable to leaf spot disease characterized by black necrotic spots on
leaves. Not much work has been done on leaf spot of Murraya koenigii. Hence this research work has
emphasized in elucidating the disease causing agent and determining their role in producing the
observed symptoms. Isolates of Colletotrichum spp. from infected curry leaf were characterized by
molecular methods and compared with morphological identification. The two its universal primer pair
used for PCR 5’- TCC GTA GGT GAA CCT GCG G- 3’ and 5 - TCC TCC GCT TAT TGA TAT GC- 3’
determined the causative agent as Colletotrichum gloeosporoides.

INTRODUCTION seedlings (Rhizoctonia solani) and leaf spot

Murraya koenigii, L. (Rutaceae) commonly known as
curry leaf plant used extensively for its slightly
pungent, bitter and feebly acidic leaves as an
important leafy vegetable for flavouring foodstuffs
in Indian cookery system. The plant originated in
the Tarai region of Uttar Pradesh (India) and is
being cultivated in Burma, Sri Lanka, China,
Australia and Pacific Islands. It is known to possess
anti-inflammatory, antidysenteric, antioxidant and
diverse pharmacological properties (Joseph and
Peter, 1985). The extracts of leaves, roots and bark
are stomachic and carminative (Nadkarni, 1954).
The leaves contain essential oils, which are
fungitoxic and are used as a fixative for a heavy
type of soap perfume. The root extract is
antibacterial and antifeedant. It is also widely used
in Indian Ayurvedic and Unani prescriptions and
several indigenous system of medicine as an anti-
diabetic agent in many Asian countries (Khan et. al,
1995). This ethno pharmaceutically important
medicinal plant is vulnerable to a few diseases and
pests, such as sap rot (Fomes pectinatus), collar rot of
(Phyllostictina murrayae) and leaf spot disease
(Rangaswamy, 1975). The leaf spot disease caused
by Colletotrichum gloeopsporioides (Penz.) Penz. and
Sacc. In Penz. (teleomorph Glomerella cingulata
(Stoneman) Spauld and Schrenk) reduces its use in
food industry (Smith and Black ,1990).
Traditional methods used for the detection and
identification of Colletotrichum gloeoporioides relied
primarily on morphology, such as size and shape of
conidia, colony color, presence or absence of setae,
and the existence of telomorphs (Sutton, 1992).
Morphotaxonomic criteria are not accurate enough
to identify the pathogen and are time consuming.
Further, the traditional methods have not been
satisfactory for differentiating Colletotrichum
species. Recently, the use of molecular marker
techniques has improved the accuracy and speed of
identification (Mc.Donald and Mc.Dermott, 1993). In
the present investigation, polymerase chain
reaction (PCR), uses primers designed from the
internal transcribed spacer (ITS) regions of nuclear
ribosomal DNA (nrDNA) of Colletotrichum species
are used as rapid and reliable markers for the
detection of Colletotrichum gloeopsporioides .

*Corresponding author - Janardhana, G. R., Mycology and Plant Pathology Laboratory, Department of Studies in
Botany, University of Mysore, Manasagangotri, Mysore-570 006, Karnataka-India, Email: grjbelur@gmail.com.
80 PADMAN AND JANARDHANA
MATERIALS AND METHODS
Collection of Diseased Samples
Samples were collected from different places in
Mysore. The diseased leaf samples were collected
and brought to laboratory in sterile plastic covers.
A detailed study on the leaf spot disease was done.
The leaf spot disease of Murrayya koenigii develop as
small scattered dead areas on leaves, black in
colour, which vary in size from 1-2mm diameter,
which appear on younger leaves. The black spots
enlarge to become dark black necrotic spots. Later
many such large necrotic spots coalesce to form
large necrotic areas, and infected leaves develop
chlorosis. Young twigs can then be affected with
similar symptomatic expression of development of
necrotic spots, which coalesce to form large necrotic
areas and cover the entire length of the twig. The
symptoms of the infection show up during early
winter thereby affecting the curry leaf quality.
Fresh leaf samples showing characteristic necrotic
and chlorotic spots will be selected, surface
sterilized and cut into small pieces and plated on
PDA medium and incubated.
Isolation of fungal pathogen
The fungal pathogen Colletotrichum gloeoporioides was
isolated on Potato Dextrose Agar (PDA) medium
from leaf spot affected Murraya koenigii (Fig. 1).
Briefly, the portion of infected leaves were surface
sterilized wih1.5% sodium hypochlorite solution
and cut into discs of 5mm diameter and placed on
solidified PDA medium and incubated at ambient
temperature. The fungal colonies expressed after 6-
8 days, (Fig. 2) were sub cultured and identified up
to species level by fungal keys and manuals.
DNA isolation and PCR. For PCR detection, DNA
samples isolated from two leaf spot affected curry
leaf samples and three isolates of Colletotrichum
gloeosporioides used for PCR amplification (Zhang et
al., 1998). About 200mg of leaf samples or mycelial
mats of isolated fungal pathogen Colletotrichum
gloeosporioides were taken in micro centrifuge tubes
along with 500 µL of lysis buffer (preheated at 65oC)
and samples were ground with blunt end of
disposable pipette tips at 65 oC for 15 min. The
supernatant was treated with 500µL mixture of
phenol: chloroform (1:1) and vortexed for 1min and
centrifuged (3000rpm, 5 min at 4oC). DNA was re-
extracted with an equal volume of ice cold 99.5%
isopropanol, incubated at -20oC (60 min) and the
pellet was rinsed with 80% ethanol, air-dried, re-
suspended in 40µL of nucleic acid free water and
used directly for PCR.
PCR conditions and DNA amplification
Primers for PCR were synthesized and obtained
from Bangalore Genei, Bangalore. The two its
universal primer pair used were 5’- TCC GTA GGT
GAA CCT GCG G- 3’ and 5 - TCC TCC GCT TAT
TGA TAT GC- 3’. The PCR was performed using
Advanced Thermus 25 Thermocycler (Peqlab,
Germany), PCR mixture (50µL) was prepared with
2µl of DNA sample with 10X PCR buffer, 2.5mM
MgCl2, 200µM dNTPs, 20 pmol of each forward and
reverse primers and 1.5 U of Taq DNA polymerase.
The PCR conditions were 94oC (5min) for initial
denaturation, followed by 25 cycles of denaturation
at 94 o C (45 sec) and primer annealing at 46 o C
(1min), primer extension at 72oC (1 min) and final
extension at 72oC (1min) (Chen et al., 2006).
Agarose Gel Electrophoresis
The amplified PCR products were separated on
1.5% agarose containing 4µL of ethidium bromide,
visualized and documented in a Biorad UV Trans-
illuminator (Fig. 4).
RESULTS
Isolation of fungal pathogen
The fungal colony of Colletotrichum gleosporioides,
showed dense aerial white to grey mycelium
bearing mass of conidia and acervuli (Fig. 3). The
microscopic observations revealed the hyaline
mycelia having cross walls with large number of
acervuli (200-640µm x 225-615µm) with presence of
characteristic setae. The acervuli were scattered as
globose bodies singly or in cluster. Conidia were
hyaline, oblong with obtuse ends (13.5-15.5µm x
3.75µm).
DNA isolation and PCR
The expected 558-bp PCR products were detected
in two leaf samples of infected curry leaves and
three samples of Colletotrichum gloeosporioides isolates.
The results clearly indicated the association of the
Colletotrichum gloeosporioides with leaf spot disease of
Murraya.
DISCUSSION
Murraya koenigii is grown in tropical and
 PCR Based Detection of Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. the Causative Agent 81

Fig. 1:a Murraya koenigii leaves showing necrotic leaf spot symptoms. b) Fungal pathogen Colletotrchum
gloeosporioides growing on PDA medium isolated from infected leaves. c) The fruiting body acervulus with brown
setae (45X). d)1.5% Agarose gel showing the expected 558-bp amplified DNA products. M-100 bp marker and
Lanes-1, 2 PCR amplified products from leaves infected with Colletotrchum gloeosporioides and lane 3 - Control
(Fusarium oxysporum), lanes 4, 5 and 6 PCR amplified products of DNA from isolates of Colletotrchum gloeosporioides.

subtropical East Asia, to New Caledonia and
North-eastern Australia (Xie et al., 2006). There is
an increasing demand for curry leaves and its
products globally for its use in variety of
applications. The leaf spot disease is severely
affecting the quality and the profitability of curry
leaves for its use in food and pharmaceutical
applications. In the present investigation, it was
verified that the leaf spot pathogen in the affected
leaf samples and the fungal isolates both could be
detected using PCR.
The disease is characterized by the small,
circular or irregular necrotic spots of 1-2mm
diameter on young leaves, turned to black brown
as the disease progresses and become chlorotic
killing the photosynthetic young shoots. The
disease is manifested during early winter there by
affecting the leaf quality. It is a sporadic disease and
the pathogen is worldwide in its distribution
causing diseases on a wide range of hosts including
cereals, legumes, vegetables, perennial crops and
fruit trees and ornamentals. The fungus grows
favorably in high temperature and humid moist
weather. The conidia are released and spread only
when acervuli are wet and generally spread by
splashing and blowing rain or by coming in
contact with insects, animals and tools (Agrios,
1978).
In this report, a rapid and reliable detection
technique to identify the associated pathogen in
infected curry leaf was developed and it could be
employed as molecular tool for quick and accurate
detection in place identification methods based
solely on lengthy, laborious morphological and
microscopic studies. Hence the protocol described
in this work is of immense importance for the
82 PADMAN AND JANARDHANA

detection of the causative agent of the leaf spot
disease of Murraya koenigi.
REFERENCES
Agrios, G.N. 1978. Plant Pathology. New York: Academic
Press, pp. 120-301.
Chen, L.S., Chu, C., Liu, C.D., Chen, R.S. and Tsay, J.G.
2006. PCR-based identification and differentiation
of anthracnose pathogens, Colletotrichum
gloeosporoides and C. truncatum from vegetable
soybean in Taiwan. J. Phytopathology. 154 : 654-662.
Joseph, S. and Peter, K.V. 1985. Curry leaf (Murraya
koenigii), perennial nutritious, leafy vegetable.
Economic Botany. 39 : 68-73.
Khan, B.A., Abraham, A., and Leelamma, S. 1995.
Hypoglycemic action of Murraya koenigii (curry
leaf ) and Brassica juncea (mustard). Indian J.
Biochem. Bioph. 32 : 106-108.
Mc.Donald, B.A. and Mc.Dermott, J.M. 1993. The
population genetics of plant pathogenic fungi.
Bioscience. 43 : 311-319.
Nadkarni, K.M. 1954. Murraya koenigii. In: Indian
Materia Medica, pp. 195-196.
Rangaswamy, G. 1975. Diseases of Crop Plants. 2 nd ed.
Prentice-Hall of India, New Delhi, 319.
Smith, B.J. and Black, L.L. 1990. Morphological, cultural,
and pathogenic variation among Colletotrichum
species isolated from strawberry. Plant Dis. 74 : 69-
76.
Sutton, B.C. 1992. The genus Glomerella and its
anamorph Colletotrichum. In: Colletotrichum Biology,
Pathology and Control. J.A. Bailey and M. J. Jeger,
eds. C.A.B. International, Wallingford, England, pp.
1 - 26.
Xie, J.T., Chang, W.T., Mehendale and Sangeeta, R. 2006.
Curry leaf (Murraya koenigii) reduces blood
cholesterol and blood glucose levels in ob/ob
mice. The American Journal of Chinese Medicine. 34 :
279-284.

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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 83-86
© Global Science Publications

BIO EFFICACY OF AQUEOUS RHIZOME EXTRACT OF

KYLLINGA NEMORALIS (FORSTER) DANDY AND
TRICHODERMA VIRIDE PERS. FR AGAINST
SEED BORNE FUNGI OF OKRA

P.M. BEEBI RAZEENA* AND RASHEED AHMAD

Department of Applied Botany, Mangalore, University, Mangalagangothri 574 199, India

(Received 15 June 2011; Accepted 25 September 2011)

Key words : Kyllinga nemoralis, Trichoderma viride, Seed borne pathogens, Okra.

Abstract - The present investigation was undertaken to study the bio efficacy of aqueous rhizome
extract of Kyllinga nemoralis. Common weed of Kerala and Trichoderma viride, against seed borne
mycoflora of okra (Abelmoschus esculentus (L) moench) cultivated in Malabar, Kerala, India. Seeds of
both local and hybrid (salkeerthi) varieties collected during rainy and summer season of 2004 and 2005.
Invitro cullture studies of Kyllinga nemoralis rhizome extract showed 97.7% inhibition of Aspergillus flavas,
94.4% inhibition of Apergillus niger and Fusarium moniliforme and 88.8% inhibition of Macrophomina phaseolina,
were the four selected dominant pathogenic fungi with a corresponding increase in efficiency in treated
seeds under in vitro conditions. The treated seeds also showed an increase in germination (97%) where
as under conditions in vitro Trichoderma viride showed inhibition of Fusarium moniliforme and
Macrophomina phaseolina (100%) and Aspergillus flavus and Aspergillus niger (83.3%). Germination
efficacy of treated seeds invitro showed 83.7%. In both cases an enhanced vigour index was observed.
Use of the above mentioned bioagent showed a drastic reduction of tested pathogenic fungi.

INTRODUCTION multipronged approach. Therefore developing bio-

Abelmoschus esculentus (L) Moench commonly
referred to as lady’s finger belongs to the family
malvaceae is an annual herb primarily valued for
its edible pods. It is grown as a garden crop plant
throughout the tropical and sub-tropical part of
the world. The pods are rich in protein and
mucilage. A mucilaginous preparation from the
pod has found application as a plasma replacement
or blood volume expander in medicine
(Anonymous, 1973).
The plant faces serious menace of fungal
pathogen causing heavy losses in different
agricultural conditions. Moreover repeated use of
fungicides has led to the problems of environ-
mental pollution and resistance to the fungicides
by pathogen. Control of seed borne pathogenic and
saprophytic fungi is possible only through a
control measures as an effective broad based, cheap
and eco-friendly method which is easily accessible
to farmers, is desired, hence the present study.
MATERIALS AND METHODS
Isolation and identification of pathogens
Fungal pathogens (Aspergillus flavus Link; Fr,
Aspergillus niger vanTieghem, Fusarium moniliforme
schldon, Macrophomina phaseolina (Tassi) (Goid) were
isolated from diseased okra seeds on Potato
Dextrose Agar. The pure cultures of pathogens were
identified and subsequently multiplied and
maintained in petridishes.
Germination percentage and vigour index
The criteria applied to differentiate germinated

*Corresponding author - P.M. Beebi Razeena, “NISHANA” Near Sir Syed College, P.O. Karimbam,
Taliparamba 670142, Kannur Dist., Kerala, India; Email: razeenaansari@gmail.com
84 RAZEENA AND AHMAD

seeds from ungerminated seeds were length of
shoot and root. Those seeds showing equal length
of shoot & root were considered to be germinated.
The root and shoot length were measured on the
11th day to calculate the seed vigour. Percentage of
germination was calculated by observing the
shoot length/root length only the vigour index was
calculated by using the following formula.
Vigour index (V1) = [Average shoot length+ Average
root length] xGermination percentage (Abdul Baki
and Anderson 1973).
Isolation and identification of Trichoderma viride
Trichoderma viride was isolated from okra garden by
serial dilution method on nutrient agar to get 1x10
colony forming unit and purified by single spore
isolation. The fungal culture was purified and the
indentification was confirmed. Subsequently
purified cultures were multiplied and maintained
in petridishes containing Potato Dextrose Agar
media.
Screening of antagonist
The antagonism between the aqueous rhizome
extract of Kyllinga nemoralis and Trichoderma viride
against dominant pathogenic fungi (Aspergillus
flavus, Aniger, Fusarium moniliforme and Macrophomina
phaseolina) isolated from okra seeds during summer
and rainy seasons of 2004 and 2005 tested by
adding the extract of Kyllinga nemoralis to the P.D.A
medium. 20 mL of sterilized and melted Potato
Dextrose Agar media. was poured into 90 mm of
sterilized petridishes. After solidification the plates
were inoculated with test fungus using 5 mm
diameter disc of actively growing colony.
In another experiment to petridish containing
P.D.A. were inoculated with 5 mm diameter disc of
test fungus at one end of the petridish and 5 mm
diameter of disc of Trichoderma viride was made at the
opposite end. The plates were incubated at a 25+2oC
for seven days and the observation was recorded.
Each treatment was replicated thrice. At the end of
the incubation period radial growth of the
pathogen and the test fungus was measured both
towards the inter action side and side of the
pertridish. Percentage inhibition of fungi was
determined by applying the formula of Skidmore
(1976),
Where C represents the distance in mm of
fungal growth from the point of inoculation
towards the colony margin and C1, is the growth of
the fungus towards antagonistic.
RESULTS AND DISCUSSION
In vitro studies of aqueous rhizome extract of

Table 1. Percent inhibitory action of Kyllinga nemoralis and Trichoderma viride against dominant pathogenic
fungi isolated from Abelmoschus esculentus, seeds under in vitro condition
Fungi Kyllinga nemoralis Trichoderma viride % of mycelial
aqueous extract growth on
control
% of % of mycelial % of % of mycelial
inhibition growth inhibition growth
Aspergillus flavus 97.7 2.3 83.3 16.7 100
Aspergilllus niger 94.4 5.6 83.3 16.7 100
Fusarium moniliforme 94.4 5.6 100 100 100
Macrophomina phaseolina 88.8 11.2 100 100 100

Table 2. Percent germination and vigour index of Kyllinga nemoralis rhizome extract and Trichoderma viride
treated seeds of Abelmoschus esculentus

Kyllinga nemorlis Trichoderma viride Control

Germination percentage 97 83.7 77

Vigour index 01139 1234 1024
 Bio Efficacy of Aqueous Rhizome Extract of Kyllinga nemoralis (Forster) Dandy and Trichoderma viride 85

Fig. 1 Effect of rhizome extract of Kyllinga nemoralis and Trichderma viride on the growth of Aspergillus flavus,
Aspergillus niger, Fusariun muniliforme and Macrophomina phaseolina isolated from Abelmoschus esculentus
seeds under in vitro conditions

Kyllinga nemoralis showed significant antifungal
activity. 97.7% inhibition of Aspergillus flavus, 94.4%
inhibition of Aspergillus niger and Fusarium monilforme
and 88.8% inhibition of Macrophomina phaselina
whereas 100% mycelial growth on control plate
(Fig. 1, Table 1). Invivo germination (97%) and
vigour index (1139) and on control it was 336 Table
2. As per gas chromatography analysis essential
oil from Kyllinga nemoralis rhizome showed that
major components are cyperone 43-85% and
caryophyllene, 20.66% followed by humulene
11.126%, followed by seleinene 4.04% and eugenol
3.04%.
Invitro studies using Trichoderma viride showed
100%, inhibition of Fusarium moniliforme and
Macrophomina phaseolina and 83.3% inhibition of
Aspergillus flavus and Aspergillus niger where as 100%
macelial growth on control plate (Fig. 1 & Table 1).
Trichoderma viride was also found effective in
protecting the seeds and seedlings. Trichoderma viride
treated seeds exhibited high levels of pre-
emergence mortality compared to control,
86 RAZEENA AND AHMAD
germination efficiency was 83.7% and high vigour
index 1234 but in control germination percentage
was 32 and vigour index was 336 (Table 2) Gupta et
al. (1989) also reported that Aspergillus flavus, A. niger
and Fusariun muniliforme were internally seed borne
and produce varying degree of seed and seedling
mortalities.
Present experiments undoubtedly proved that
aqueous rhizome extract of Kyllinga nemoralis and
Trichderma viride were effective in controlling seed
borne and seed transmitted pathogenic and decay
causing saprophytic fungi of okra cultivated in
Malabar, Kerala. Compared to Trichoderma viride
aqueous rhizome extract of Kyllinga nemoralis was
most effective and efficient biocontrol agent. Hence
developing effective above mentioned strategies for
broad based integrated control of seed-borne
—❋❋❋—
pathogens and decay causing saprophytes by eco
friendly methods were the need of the present and
future generations.
REFERENCES
Abdul Baki, A.A and Anderson, 1973. Vigour
determination in soybean seeds by multiple
criteria. Crop Science. 13 : 630 - 633.
Anonymous, 1973. Wealth of India Raw-materials vol.
5. Council of scientific and industrial research.
New Delhi 80-85.
Gupta, K., Sindhu, I.R. and Naaz, S. 1989 Seed
mycoflora of Abelmoschus esculentus (L) Moench. 1-
survey and enumeration. Acta Botanica Indica. 17
(2) : 200-206.
Skidmore, A.M 1976. Interactions in relation to
biological control plant pathogens. Microbiology of
Aerial Plant Surfaces, (Eds Dickson, C.H. and Prece
T.E.)
Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 103-104
© Global Science Publications

PRELIMINARY PHYTOCHEMICAL ANALYSIS AND

ANTIMICROBIAL SCREENING OF CASSIA TORA LINN.

D. EAZHISAIVALLABI, R. AMBIKA AND P. VENKATALAKSHMI*

Department of Biochemistry, S.T.E.T Women’s College, Mannargudi 614 001, India

(Received 24 August 2011; Accepted 28 September 2011)

Key words : Antimicrobial activity, Phytochemical analysis, Cassia tora Linn,

Abstract - In the present study the methanolic and aqueous extracts of Cassia tora were subjected to
preliminary phytochemical analysis and antimicrobial activities against certain pathogenic
microorganisms. The phytochemical analysis revealed the presence of Alkaloids, Carbohydrates,
Glycosides, Saponins, Sterols, Fats and Oils, Phenols, Tannins, Flavanoids and Proteins. The
antimicrobial activity was more in methanolic extract than the aqueous extract.

INTRODUCTION soxhlet extraction separately and successively

Cassia tora Linn, is an annual herb (30-90 cm), is
widely found in the tropical regions of India as
wasteland rainy season weed. The leaves are
pinnate and its flowers are pale yellow in colour
with five petals. The common vernacular names of
Cassia tora are Tagarai (Tamil), Charota (Hindi) and
Chakramarda (Sanskrit).
According to Ayurveda, the leaves and seeds are
acrid, laxative, antiperiodic, ophthalmic, liver-
tonic, cardio-tonic and expectorant. The leaves and
seeds are useful in leprosy, ringworm, flatulence,
colic dyspepsia, constipation, cough, bronchitis,
cardiac disorders.
The medicinal values of C.tora is known from
time immemorial (Chopra et al.,1982).The objective
of the present study is to investigate the
effectiveness of the leaf extracts of C.tora (aqueous
and methanolic extracts) against few selected
pathogens using appropriate standard methods.
MATERIALS AND METHODS
Plant material and Extracts Preparation
The fresh leaves of C.tora were collected from near
by areas of Thanjavur, Tamilnadu, was brought to
the laboratory and shade dried to crisp. They were
then subjected to pulverization to get coarse
powder. The coarse powder was subjected to
with methanol and water. These extracts were
concentrated to dryness in flash evaporator under
reduced pressure and controlled temperature (40-
50oC). Both extracts were put in separate air tight
containers and stored in a refrigerator.
Qualitative phytochemical analysis
Qualitative phytochemical analyses were done
using the procedures of Kokate (1995). Alkaloids,
Carbohydrates, Tannins and Phenols, Flavanoids,
Gums and Mucilages, Phytosterol, Proteins and
Aminoacids, Fixed oils, Fats, Volatile oil and
saponins were qualitatively analyzed.
Microbial cultures
The microorganisms used in the present study
were procured from National Chemical Laboratory
(NCL), Pune.
Screening for Antimicrobial activity
The disc diffusion method was followed for
antimicrobial susceptibility tests. The plates were
prepared by pour plate technique using Muller
Hinton agar for bacterial strains and potato
dextrose agar for fungal strains with the proper
concentration of the inoculums. The filter paper
discs impregnated with aqueous and methanolic
extracts of C. tora were placed at suitable distance
on the plate. The plates were incubated at 37oC for

*Corresponding author - Email: venkatalakshmisathish@gmail.com

104 EAZHISAIVALLABI ET AL.

Table 1. Preliminary phytochemical analysis of aqueous extract showed greater antifungal activity towards
and methnolic extracts of C.tora
Trichoderma reesei the zone of inhibition was found to
S.No. Test Result be moderate with S. cerevisiae.
Phytochemicals can be used as a natural
1. Alkaloids +
2. Carbohydrates +
blueprint for the development of a drug (Didry et
3. Glycosides + al., 1998). The preliminary phytochemical studies
4. Saponins + revealed the presence of alkaloids, carbohydrates,
5. Sterols + glycosides, saponins, sterols, fats and oils, resins,
6. Fats & Oil + phenols, flavanoids and proteins (Table 1).
7. Resins + Regarding the antimicrobial activity among the
8. Phenols + two extracts, methanolic extract was more potent
9. Tannins + than the aqueous extract. Further studies are
10. Flavanoids + required to establish the exact nature and
11. Proteins +
mechanism of the active principles present in the
12. Diterpenes -
extract.
+ = presence and - = absence of phytochemicals

Table 2. The antimicrobial activity of aqueous and methnolic extracts of C.tora

S. No Name of the organism Zone of inhibition (mm)

Aqueous Methanolic Cephalosporin Amikacin Fluconazole

extract extract (50mg) (50mg) (20mg)

Bacteria
1. Staphylo coccus aureus 13 15 11 13 -
2. Escherichia coli 14 16 18 17 -
3. Rhodo coccus 13 16 17 13 -
4. Bacillus subtilis 12 18 20 16 -
Fungi
1. Aspergillus niger 11 14 - - 19
2. Trichoderma reesei 12 11 - - 20
3. Saccharomyces cerevisiae 17 16 - - 19
- = no activity
ACKNOWLEDGEMENT
24 h and were examined after 24 h.The zone of
inhibition was measured in mm and recorded. Authors are thankful to Dr. V. Dhivaharan,
Correspondent, S.T.E.T Women’s College,
RESULTS AND DISCUSSION Mannargudi for the help rendered by him during
the course of the project.
The bioactive components including thiocyanate,
nitrate, chloride and sulphate, beside other water REFERENCES
soluble components which are naturally occurring
in most plant materials, are known to be Didry, N., Dubreuil, L., Trotin, F., Pinkas, M. 1998.
bactericidal and fungicidal in nature thus Antimicrobial activity of the aerial parts of
conferring the anti microbial property to plants Drosera pellata smith on oral bacteria. J.
Ethnopharmacol. 60 : 91-96.
(Lutterodt et al., 1999).
Chopra, R.N., Nayar, S.L.,Chopra, I.C. 1986. Glossary of
The methanol extract showed higher anti-
Indian Medicinal Plants. CSIR, New Delhi. 29.
microbial activity than aqueous extract and it was Lutterodt, G.D., Ismail, A., Besheer, R.H., Baharudin,
evident from the zone of inhibition. The H.M. 1999. Antimicrobial effects of Psidium guajava
antibacterial activity of the methanolic extract of extracts as one mechanism of its antidiarrhoeal
Cassia tora is maximum for B.subtilis (18 mm zone of action. Malaysian J. Med. Sci. 6 (2) : 17-20.
inhibition). Among the fungal species, the plant Kokate, C.K. 1996. Practical Pharmacognosy, Vallabh
Prakashan, Pune.4th ed. 107-109s.
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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 109-122
© Global Science Publications

MICROBIOLOGICAL STUDIES AND MACROINVERTEBRATE

FAUNA OF THE LOTIC SYSTEMS IN AND AROUND
COURTALLAM, TIRUNELVELI DISTRICT (TAMIL NADU)

R. DRUSILLA, A. KUMARESAN* AND M. NARAYANAN**

* School of Veterinary Medicine, Faculty of Medical Science, The University of West Indies,
St.Augustine, Trinidad and Tobago, West Indies.
** Department of Examinations, Sadakathullah Appa College,
Rahmath Nagar, Tirunelveli 627011, Tamil Nadu, South India, India

(Received 18 August 2011; Accepted 30 September 2011)

Key words : Total coliforms, Faecal coliforms, Escherichia coli, Macroinvertebrates, Lotic system.

Abstract - Microbial populations and macroinvertebrate fauna of river Chittar were investigated for
two years. Microbes analysed were:Total coliforms, Faecal coliforms and Escherichia coli. Results
revealed the following trend: Total coliforms >Faecal coliforms > Escherichia coli. Macroinvertebrate
fauna included crustaceans, insect nymphs, aquatic insects and molluscs. The percentage of
crustaceans varied between 16.28 and 18.50, insect nymphs from 24.60 to 26.98, molluscs between 26.01
and 29.37 and aquatic insects from 28.24 to 30.01. Shannon – Wiener index of diversity of
macroinvertebrates ranged between 1.361 and 1.374 while Simpson’s index of Dominance varied form
0.256 to 1.40.

INTRODUCTION worked on benthic macroinvertebrates and

Bacteriological parameters are of great importance
in the hygienic evaluation of water (Bonde, 1977).
With regard to water microbiology, coliform
bacteria have been used as bacteriological tool to
measure the occurrence and intensity of faecal
contamination in drinking water nearly for the
past 70 years (Amutha and George, 1988). So, the
analysis of Coliforms which include Faecal
coliforms and Escherichia coli drew the attention of
research workers to ascertain the microbiological
quality of water. Further, the presence of enteric
bacteria in the aquatic environment has received
more importance because pathogenic members of
the group threaten the very human life.
The importance of bottom-living macroinver-
tebrates and their significant role in the trophic
cycle of water body were recognized quite early
(Hora,1936). Macroinvertebrates are the most
valuable indicators of environmental quality in
aquatic systems because of their developmental
stages and comparatively stable mode of life.
Investigators like Guafin and Tarzwell (1956)
brought out the significance of these organisms as
indicators of pollution so also Mason (1981) and
Vattakeril and Diwan (1991).
Microbiological studies have been carried out in
the river Cooum of Madras by Selvanayagam et al.
(1988);Vaigai river of Tamil Nadu (Amutha and
George, 1988); Tambaraparani river, Tamil Nadu
(Haniffa et al., 1993; Martin, 1994 and Martin et al.,
2000); Narmada river at Jabalpur, Madhya Pradesh
(Shrivastava et al.,2000); Narmada river at
Hoshangabad, Madhya Pradesh (Singh et al., 2001);
Godavari river, Maharashtra (Bhosle & Rao,2001);
Mandakini river, Chitrakoot, Madhya Pradesh
(Rani et al., 2004); Pamba river, Kerala (Harikumar
and Madhavan, 2006); Venkatapura river, Uttara
Kannada, Karnataka (Karthick and Ramachandra,
2007); Periyar river at Alwaye (Nanthakumar et al.,
2007); Karuvannur river, Kerala (Sreekumar et al.,
2009) and Tungabhadra and Hundri rivers of
Andhra Pradesh (Rajeswari and Saraswathi, 2009).
Studies on macroinvertebrates are meagre.
Nirmalakumari and Nair (1984) have carried out
investigation on macroinvertebrates in the Chackai
110 DRUSILLA ET AL.
canal, Trivandrum, India; Cooum river, Madras
(Selvanayagam, et al.1988; Jebanesan and
Selvanayagam, 1994); Pune rivers, India
(Yazdundoost and Katdare, 2000); Arjuna river,
Sivakasi, Tamil Nadu (Rajan and Murugan, 2001);
Manjar river, Osmanabad, Maharashtra (Hiware
and Jhadav,2001); Ithikkara river of Kerala (Sheeba
and Ramanujan, 2005); Pamba river of Kerala
(Harikumar and Madhavan, 2006); Darna river,
Maharashtra (Bhamre and Deoray, 2008) and
Santubong river,Sarawak, Malaysia (Ashraful et al.,
2009).
The present study deals with the enumeration
and distribution of some indicators of enteric
pathogens, faecal pollution and total coliforms in
MPN index and also the dynamic nature of
macroinvertebrate fauna in six stations at a stretch
of 25 km of the lotic systems in and around
Courtallam, Tirunelveli district, Tamil Nadu.
MATERIALS AND METHODS
The presence and abundance of microbes and
macroinvertebrates are estimated in six stations
beginning from Five falls stream water to Chittar
river at Kadabokathy village, covering a length of
25km. Five falls stream originates in Pothigai hills
of Western Ghats at an altitude of 1,060m. River
Chittar, an east - flowing river starts from Pothigai
hills of southern part of Western Ghats at an
altitude of 2,074 m and meanders through a
distance of 120 km in Tirunelveli district and joins
Tambaraparani river at Seevalaperi village which
in turn drains into the Bay of Bengal. Chittar river
receives Gundaru river coming from Shencottah at
Tenkasi - entry point, while it joins the Old falls
stream at Kadabokathy village.
Station 1 - 25m away from Five falls stream
Station 2 -River Chittar close to Main falls of
Courtallam .
Station 3 - Confluencing point of Five falls stream
and river Chittar from Main falls.
Station 4 - Confluencing point of river Chittar with
Gundaru river (from Shencottah) at Tenkasi-entry
point
Station 5 -Chittar river at Tenkasi–exit point
Station 6 - Confluencing point of river Chittar with
Old falls stream.
Water samples were collected once a month in
300 mL sterile glass-stoppered bottles and kept in
ice-box for bacteriological examination from all the
six stations of these lotic systems. Total coliform
bacteria were determined by MPN (Most Probable
Number) method.
Total Coliforms - MPN Method
The collected water samples were inoculated in
each Mac Conkey’s broth tubes, i.e.10 mL water
sample was inoculated into each of the five tubes
containing 10mL double strength Mac Conkey’s
broth. 1 mL water sample was inoculated into each
of the five tubes containing 5mL single strength
broth and 0.1 mL water sample inoculated into
each of the five tubes containing 5mL single
strength Mac Conkey’s broth. All the test tubes
were incubated at 37 oC for 24 to 48 hrs. After
incubation, all the test tubes were observed for acid
and gas production. The production of acid and gas
indicates the presence of coliforms and thus the test
is positive.
Faecal coliform Analysis (IM Vic Test)
Tryptone broth, glucose-phosphate broth and
citrate agar were inoculated with loopful of
inoculations from positive MPN tubes for faecal
coliforms from the above experiment. After
inoculation at 37oC for 24 hrs, results were taken.
Escherichia coli Analysis
From positive faecal coliform tubes, loopful of
inoculants were inoculated on EMB (Eosine
Methylene Blue) agar plates. After incubation,
results were noted. Purple dark-centered colonies
with a greenish metallic sheen were typical of
Escherichia coli.
Macroinvertebrates
The benthic and water column macroinvertebrate
collection were made in all the six stations. They
were seen only in 3rd, 4th,5th and 6th stations of this
lotic system. Collection of samples was done once a
month. All the benthic fauna were collected in a
square meter area with Surber sampler due to the
shallow nature of the river water. On the same day,
animals were sieved through 0.5m.m mesh
(Martin, 1994) and separated manually using
forceps and preserved in 5% and 10% formalin
according to the size. In the laboratory, the sorted
benthic animals were identified. An average of
three samples of zoobenthic organisms were taken
monthly from each station and mean density value
of the three replicates was given as individuals per
square meter (ind./m2). Macroinvertebrates were
identified following Ward and Whipple (1959);
 Microbiological Studies and Macroinvertebrate Fauna of the Lotic Systems in
Monographs (APHA,1976); Tonapi (1980) and
Michael (1986). The procedure given by Drusilla et
al. (2009) for calculating the Shannon - Wiener
index of diversity and Simpson’s index of
Dominance for zooplantton density was followed
for estimating these indices for macroinvebrates of
these lotic systems.
RESULTS AND DISCUSSION
Total Coliforms
Coliform bacteria (group) include all organisms
that are gram-negative, non-spore forming rod
which ferment lactose and occur in large number in
the colon of warm -blooded animals (Tate and
Trussel, 1977). In the present study, the MPN index
of Total coliforms, Faecal coliforms and Escherichia
coli count/100 mL for the four downstream stations
were estimated. Conspicuous absence of the above
cited coliforms was noticed in monsoon seasons
(south -west and north - east monsoons) but they
were recorded only in post and pre-monsoon
months. Total coliform count ranged from 0 to 240
MPN/ 100 mL and maximum value was noted in
stations 5 and 6 during the study. Coliform
bacteria were absent in the first two stations and in
the downstream stations during monsoons. Lower
limit of 75/100 mL was recorded in station 3 in the
first year of study.
The lower limit of 110/100mL MPN of
Tambaraparani river of Tamil Nadu, recorded by
Haniffa et al.,1993) and the lower limit of 30 MPN/
100 mL of Tambaraparani river, Tamil Nadu
(Martin,1994) fall within the values of present
study. As in the present work, in river
Tambaraparani of Tamil Nadu, Martin (1994)
recorded the maximum total coliform count during
pre-monsoon in some stations, while in some
stations there did occur monsoon (south-west and
north-east) maxima too. But high level of 11,000
MPN/100mL of total coliform in Tambaraparani
river (Haniffa et al.,1993) is not comparable to the
very low values of total coliforms of this lotic
system. The MPN of coliform of Suruliar river,
Tamil Nadu ranged from 720.0 to 12,153/100mL as
recorded by Sivasubramani and Mahadeven
(1995). The above quoted data are much higher than
the range of total coliform count of river Chittar.
Total coliform levels were always higher than
faecal coliform counts since coliform bacteria can
originate from non-faecal source such as plants and
111
soils (Geldrich, 1974). Further, Kataria et al. (1997)
opined that high values of MPN index of total
coliform in Halati river of Madhya Pradesh were
owing to the entry of more contaminated sewage
during pre-monsoon season. It is true with regard
to the present lotic system also. Similar to the
results of total coliforms of present investigation, in
a river at Dakshina Kannada, Karnataka,
Madhyastha et al. (1999) registered minimum value
of< 2 MPN/100mL and an upper limit of >1,600
MPN/100mL of total coliforms which is far beyond
comparison with the values of present work. Total
coliform count in Pamba river, Kerala was above
500 MPN/100 mL in one of the stations due to
Sabarimala pilgrimage during post and pre-
monsoons (Koshy and Nair, 1999), while in the
same river at Kozhencherry of Kerala, the total
coliform count varied between 0 and 1,600 MPN/
100mL (Koshy and Nair, 2000). The elevated values
of total coliforms noted in the above said rivers
were due to high sewage pollution and anthropo-
genic activities. The lower range of coliforms of 11
to 115 MPN/100mL of Morna river at ‘Akola’,
Maharashtra reported by Musaddiq (2000) falls
within the range of total coliforms obtained in the
present study.
Shrivastava et al., (2000) recorded total coliform
count as 170 to 1,600 MPN/100mL in Narmada
river at Jabalpur, Madhya Pradesh, India, while
Singh et al.(2001) registered a wide range of total
coliforms ranging from 24 to 9,200 MPN/100mL in
Narmada river at Hoshangabad, Madhya Pradesh,
India. The above mentioned lower limits of total
coliforms in Narmada river are comparable with
the values of present work whereas the upper
limits do deviate much. High faecal contamination
due to human activities would have led to the high
coliform count in Narmada river as suggested by
the authors. In Godavari river of Maharashtra,
Bhosle and Rao (2001) estimated MPN of total
coliforms as 175 to 900/100 mL due to sewage
entry. On the other hand, during ‘Holi Mela’ in
Godavari river at Nanded, Gyananath et al. (2000)
recorded MPN index as 1,300 to 1,800/100 ml due to
heavy anthropogenic input apart from sewage
inflow. The above quoted high values are not
comparable with the very low values of present
running water system which was less polluted at
the time of investigation. In the upstream and
midstream of Panchganga river in Kolhapur city of
Maharashtra, the average MPN index of total
coliforms ranged as 1,234/100mL and 753/100mL
112 DRUSILLA ET AL.
respectively, while the maximum count went upto
1,600 MPN/100mL (Lomate and Samant, 2005). The
above cited data are very high and not comparable
with the low values of present work.
Contrary to the results of total coliforms of
present lotic system, the findings of microbial
quality of river Pamba at Kerala revealed the MPN
of coliforms/100 mL as 460 to 46,000 (Harikumar
and Madhavan, 2006) and the elevated data in
Pamba river were due to bathing of crowds of
pilgrims of Sabarimala. Udhayakumar et al. (2006)
estimated the MPN of coliforms as 460/100mL in
Cauvery river of Namakkal and Erode districts of
Tamil Nadu. Venkatapura river basin of Uttara
Kannada district of Karnataka showed the
presence of coliform contamination which proved
the human interference in that aquatic system
(Karthick and Ramachandra, 2007) and such a
condition did prevail in river chittar also. The
maximum number of total coliform found in
Periyar river water at Alwaye, Kerala was 2,400
MPN/100mL and minimum number as 22 MPN/
100mL (Nanthakumar et al., 2007) The above said
maximum value of total coliforms is very high
compared to the low values of present work but the
minimum number of total coliforms falls within
the range estimated in this river Chittar. In
Tungabhadra and Hundri rivers of Andhra
Pradesh, Rajeswari and Saraswathi (2009) recored
the MPN of coliform count varying between 190
and 4,600/100mL and 1,80,000 to 3,50,000/100mL
respectively with maximum result at summer
season as it was noted in this lotic system. High
data of coliform bacteria in Tungabadhara river
was as result of washing of clothes and
indiscriminate use of detergents while in Hundri
river in Andhra Pradesh during summer might be
owing to contamination of water with municipal
sewage (Rajeswari and Saraswathi, 2009). In
Shipra river near Ujjain of Madhya Pradesh, Parag
Dalal et al. (2009) reported the MPN index of
coliforms to be above the permissible limits
throughout the study period.
In north Indian Yamuna river at Delhi, India, the
range of total coliforms was from 850 to 2,40,000
MPN/100 mL and 40 to 3,500 MPN/100 mL in
Yamuna river at Panchnada, Uttar Pradesh, India,
as registered by Sharma et al. (2000a) and Narain
and Chauhan (2000) respectively. In river Nunia of
West Bengal, Chatterjee and Raziuddin (2001)
reported total coliform count as 170 to 1731 MPN/
100 mL. Only the lower limit of total coliforms of
the above said four north Indian rivers are
comparable with the results of present work. The
MPN index of total coliform bacteria in the Hathli
stream of Hamirpur in Lower Himalayas varied
between 1,100 and 12,00,000/100 ml (Sharma et al.,
2003) while in river Beas in Shivalik hills of
Himachal Pradesh (Hamirpur area of Outer
Himalayas) the MPN index of coliforms ranged from
280 to 1,600+/100 mL (Sharma, 2004). The data
obtained in Hathli stream of Lower Himalayas do
confirm the grossly polluted nature of the stream.
Begum et al. (2004) estimated an average count of
total coliforms as 679.17±75.90/100 ml in the river
Brahmaputra of Guwahati. But the winter data of
300.0± 51.63/100 mL of total coliforms is slightly
higher than the pre-monsoon maxima of 240/100
mL of this river Chittar. Unlike the pre-monsoon
maxima of total coliform count/100mL of this lotic
system, in Brahmaputra river of Guwahati, Begum
et al. (2004) recorded maximum total coliform count
only in monsoon season (1033 ± 125.60). Very high
results of total coliform count in Brahmaputra
river might be due to dust, soil sewage, factory
waste and other decomposing organic matters
(Begum et al., 2004).
In the Nirjuli stream and Dikrong river of
Arunachal Pradesh, India, Sahu et al. (2006)
recorded the MPN index of coliforms ranging from
21 to 27/100 mL and 43 to 52/100 mL respectively.
The above quoted low values fall within the range
obtained in this river Chittar. Total coliform
bacteria of Badi Naula stream and Raj Naula
stream of Kumaun Himalaya varied from 12 to 220
MPN/100 mL and 17 to 350 MPN/100mL
respectively and the maximum count was
observed during rainy season (Paliwal and Sati,
2007). The above cited high values of total coliforms
of the running water systems might be owing to
the input of faecal organic waste from the
catchments area with rain water and increased
percolation rate of faecal matter from the septic
tank (Paliwal and Sati, 2007) Total coliform bacteria
data ranged from 0.7x102 to 28.0x102 MPN/100mL
in Kosi river water of Central Himalaya (Sati and
Paliwal, 2008). The above said data are very high
compared to the results obtained in this lotic
system. Oluwande et al.(1983) registered total
coliform count / 100mL in the water of five
Nigerian rivers varying between 1.82x 102 and
3.70x105 and Adewoye and Lateef (2004) recorded
coliform bacilli as 1800 MPN /100mL in Oyun
river (both in the upstream as well as downstream)
 Microbiological Studies and Macroinvertebrate Fauna of the Lotic Systems in
of north central Nigeria. The above quoted data of
bacterial count are very high and are not
comparable with the results of river Chittar.
Faecal coliforms
During periodical monitoring, faecal coliforms
ranged between 39 and 150 MPN/100mL from
stations 3 rd to 6th in the post-monsoon and pre-
monsoon months in river Chittar. Maximum value
of 150 MPN/100 mL was noticed in stations 5 and 6
both the years. Complete absence of faecal coliforms
was noticed in the first two stations in all the
seasons while during monsoons in the downtream
stations. Minimum number of 39 MPN/100 mL was
noted in station 3 during pre-monsoon in the first
and second year of study.
Faecal coliform count recorded in this river
Chittar exceeded the water quality standard of one
coliform /100 mL of sample indictated in Drinking
water Act by George & Shroeder (1985). Maximum
faecal coliform count of 150 MPN/100 mL of this
lotic system is very low compared to the upper
limit of 93,000 MPN/100mL given for
Tambaraparani river of Tamil Nadu by Martin
(1994). High level might be as a result of pollution
by sewage discharge and human activities. But the
minimum value of 4 MPN/100 mL recorded by
Martin (1994) falls within the range obtained in
this lotic system. Faecal coliform bacteria of
Suruliyar river, Tamil Nadu varied between 9 and
4,920 MPN/100 mL due to human impact and
sewage discharge into the river (Sivasubramani
and Mahadevan, 1995). Though the minimum
value of faecal coliform count of the above cited
river falls within the range of values of present
work, the upper limit deviate much from the
results of river Chittar so not comparable.
Unlike the low values of faecal coliform bacteria
of this lotic system, in Narmada river, Jabalpur of
Madhya Pradesh, Shrivastava et al.(2000) registered
faecal coliform count ranging from 110 to 1,600
MPN/100mL. Similarly, in north Indian Yamuna
river at Delhi and Panchnada district of Uttar
Pradesh, India, the faecal coliform count varied
between 150 and 13,800 MPN/100 mL and from 0 to
1,40,000 MPN/100 mL as recorded by Sharma et al.
(2000a); Narain and Chauhan (2000) respectively.
Minimum value of faecal coliforms in Yamuna
river at Delhi coincides exactly with the maximum
value of this lotic system. High faecal coliform
count in Narmada river, Jabalpur and Yamuna
river at Delhi and Panchnada district of Uttar
113
Pradesh, India might be owing to anthropogenic
activities and sewage entry. In river Nunia (a
tributary of Damodar river), West Bengal, faecal
coliform count ranged between 5 and 127 MPN/100
mL (Chatterjee and Raziuddin, 2001) and the
above cited data fall within the results of faecal
coliforms of present work. Chatterjee & Raziuddin
(2001) opined that dumping of untreated sewage
into the river might have caused an increase in the
microbial populations in water. In Mandakini river
in Chitrakoot of Madhya Pradesh, Rani et al. (2004)
recorded the MPN index of faecal coliforms ranging
from 826.0 to 2,400/100 mL due to the discharge of
human wastes. Maximum value of MPN index of
2,400/100 mL was registered during fair (mela)
days (Rani et al., 2004). Contrary to the findings of
present work, in the polluted Oyun river of north-
central Nigeria, Adewoye and Lateef (2004)
registered high bacterial count during rainy season
and low count during dry season.
Escherichia coli
Faeces of man and animal contain millions of
bacteria/g of faeces and majority of which is
Escherichia coli, a natural inhabitant of the
intestines. Absence of Escherichia coli in water is a
positive indication of the absence of pathogenic
bacteria (Sundari et al,. 2004). Escherichia coli is the
common member of coliforms. Survival of
Escherichia coli in water samples indicate the chance
of pathogenicity and causes diarrhoea in children
(Shah and Patel, 1989). Although Escherichia coli is
known to be non-pathogenic bacteria, some strains
of Escherichia coli may cause diarrhoea in adults also
(Kumar and Saha, 1989) .
Escherichia coli was absent during monsoons
(south-west and north-east) in all the stations but
they were noticed only during post and pre-
monsoons in stations 3 rd , 4 th , 5 th and 6 th. These
bacteria were absent in stations 1 & 2 throughout
the study period. Maximum count of Escherichia coli
of 75 MPN/100mL was recoded in stations 5 and 6
in both the years of study. A low value of 20 MPN/
100 mL was noticed in station 3 during post-
monsoon and pre-monsoon of first and second
year of study respectively in this river Chittar.
Escherichia coli count of this lotic system ranged
from 0 to 75 MPN/100mL. In the water sample
collected near Main falls of Courtallam (Station 2)
there was no Escherichia coli but in the Main falls as
Courtallam, Kumaresan and Bagavathiraj (1996)
had reported Escherichia coli count as 5 + 1/100 mL.
114 DRUSILLA ET AL.

Results of present work are too low when Family : Baetidae

compared to the high counts of 1.07 x 104/100 mL in Mayfly nymph (Baetis sp.)
river Ganga at Patna and 4 x 101 to 42x104 in Mula Order : Hemiptera
Mutha river system, Maharashtra, as recorded by Family : Nepidae
1. Ranatra elongata
Kumar and Sinha (2000) and Tape et al. (2001)
2. Laccotrephes maculates
respectively. Both the data indicated above are not Family : Belostomatidae
comparable with the low values of this river and Diplonychus indicus
elevated data in the above quoted rivers might be Family : Notonectidae
owing to untreated sewage pollution. Sreekumar et Anisops bouveri
al. (2009) recorded Escherichia coli in the surface Family : Gerridae
water samples of main river and major tributaries Gerris sp.
of Karuvannar river (Thrissur district of Kerala) Family : Corixidae
ranging form 2.0 to 120.0/ 100mL but there was Corixa sp.
Order : Coleoptera
complete absence of Escherichia coli during monsoon
Family : Dytiscidae
in 3 stations out of 7 stations studied. This range is Dytiscus sp.
lower than the range of Escherichia coli obtained in Phylum : Mollusca
this lotic system. In the polluted Oyun river of Class : Pelecypoda
north-central Nigeria, Adewoye and Lateef (2004) Order : Integripalliata
recorded the presence of Escherichia coli bacteria also. Family : Unionidae
1. Lamellidens marginalis
Macroinvertebrates 2. Lamellidens consobrinus
The presence and abundance of organisms in any Class : Gastropoda
Order : Pectinibranchiata
ecosystem depend on a wide range of environ-
Family : Ampullariidae
mental conditions. Of the six stations studied, in Pila globosa
the first two stations macroinvertebrate fauna Family : Lymnaeidae
were absent and more number of species were Limnaea sp.
found in 5th and 6th stations compared to 3rd and
4th stations. The various groups of macroinverte- In the first two stations macroinvertebrates
brates were represented in the order of: aquatic were absent due to the turbulent nature of the
insects > molluscs > insect nymphs > crustaceans. water. Members of macroinvertebrates were seen
A survey on the macroinvertebrate fauna of this only in stations 3rd, 4th, 5th, and 6th. A survey on the
lotic system is listed below. macroinvertebrate fauna of this lotic system
showed the following trend: aquatic insects>
List of Macroinvertebrates collected
molluscs > insect nymphs > crustaceans
Phylum : Arthropoda Crustaceans
Class : Crustacea The number of crustaceans (Prawns and Crabs)
Order : Decapoda varied between 2.0/m and 8.0/m2 in the four
Family : Palaeomonidae downteram stations. Prawns (Macrobrachium idea)
Species : Macrobrachium idea (Prawn)
and crabs (Paratelphusa hydrodromus and P.Jacquenti)
Family : Potamonidae
1. Paratelphusa hydrodromus (Crab)
were seen adhered to macrophytes or grasses or
2. Paratelphusa jacquenti (Crab) under the pebbles and stones present on the banks
Phylum : Arthropoda of the lotic system. Yearly total ranged from 42.0 to
Class : Insecta 66.0/m2 in stations 3rd,4th,5th and 6th for the two year
Order : Odonata study period. First two stations were lacking in
Family : Libellulidae crustaceans throughout the study. Minimum value
Dragonfly nymph of 2.0/m2 was found in station 3 at the time of north
(Diplocodes trivialis) - east monsoon while maximum density of 8./m2
Family : Coenagrionidae
was obtained in 4th, 5th, and 6th stations during
Damselfly nymph (Ischnura sp.)
the period of investigation.
Order : Plecoptera
Family : Nemouridae Pre-monsoon and post-monsoon values of
Stonefly nymph (Neoperla sp.) crustaceans are more than monsoon results in all
Order : Ephemeroptera the four downstream stations studied. The
 Microbiological Studies and Macroinvertebrate Fauna of the Lotic Systems in
percentage of crabs and prawns varied between 16.
28 and 18.50. In the stations 3 rd , 4 th ,5 th and 6 th,
prawns were not reported during monsoon
seasons. In the present study, crustaceans were
found to be the least among macroinvertebrate
fauna. Crabs were noticed throughout the study
period in the last four stations but in varying
numbers. On two way analysis of Variance, the
results did show statistically significant variation
(p<0.01) between stations and months.
During monsoons, prawns were absent in all
the four downstream stations. This may be due to
high velocity of water current in this lotic system &
similar observation was made in Tambaraparani
river by Martin (1994). In the present study, the
density of crustaceans ranged from 2.0 to 8.0/m 2
while in Tambaraparani river of Tamil Nadu, it
ranged from 1.0 to 44.0/m2 (Martin et al., 2000). As
in the present findings, in river Tambaraparani, the
pre-monsoon maxima of crustaceans was recorded
by Martin et al. (2000). The lowest density of
crustaceans in Tambaraparani river was due to
high rainfall (13.40cm) and such a situation
prevailed in the present study also (rainfall : 26.50
cm). In river Tambaraparani, 4 genera of
crustaceans were reported by Martin et al. (2000)
whereas only 2 genera of crustaceans were
recorded in the present investigation. Crustaceans
were totally absent in station 2 of Tamparaparani
river owing to the discharge of textile mill effluents.
In Pune rivers of India, Yazdandoost and Katdare
(2000) registered 2 genera of crustaceans namely,
Paratelphusa and Macrobrachium as in the river
Chittar.
The torrential waters of Garhwal region of
Himalaya did harbour crabs in the rapid-
stenothermal region of station 1 (Nautiyal, 1986).
On the contrary, the results of present work reveal
the complete absence of crabs in the first two
stations due to rapid flow of water, less
availability of nutrients and lack of microflora and
fauna. But crabs were present in the last four
downstream stations which were placid-
eurythermal in nature just like the torrential
waters of Garhwal region of Himalaya (Nautiyal,
1986). Sharma et al. (2000b) reported the occurrence
of shrimps in the high altitude station of Berach
river system, Udaipur, which was without human
interference. In the present study only one species
namely, Macrobrachium idea was seen while in the
upper Brahmaputra river of Assam, Biswas and
Boruah (2000) recorded 6 species of prawn
115
belonging to the genus, Macrobrachium. The
diversity of species in upper Brahmaputra river
might be owing to high nutrient content in the form
of microflora and fauna. In Mahi river, Gujarat,
Nanda et al. (2005) reported small sized
Macrobrachium sp. during pre-monsoon as it was
seen in river Chittar.
Insect nymphs
The number of insect nymphs ranged from 2.0/m2
to 14.0/m2 for the four downstream stations. In the
first two upstream stations, insect nymphs were
totally absent. Yearly density of insect nymphs
varied between 62.0 and 104.0/m 2 in the last four
downstream stations in the two year study period.
Minimum density of 2.0/m 2 in the monthly
collection was found in station 3 during north -
east monsoon in the first and second year of study.
Maximum density of 14.0/m2 was recorded in
stations 5 and 6 during pre-monsoon period of first
and second year of study. Monsoon values were
lower than pre-and post-monsoon values. The
percentage of insect nymphs ranged from 24.6 to
26.98. Though the insect nymphs are considered as
“Macrozooplankton”as per the reports given by
Martin (1994); Singh (1997) and Martin et al. (2000),
they are dealt with under the section of
“Macroinvertebrates”
The nymphs of dragonfly, Diplocodes trivialis
and damselfly nymph (Ischnura sp.) were perennial
members and were minimum during monsoon
periods. Mayfly nymphs (Baetis sp.) and stonefly
nymphs (Neoperla sp.) were not present during
monsoons in majority of the downstream stations
in the present investigation. Two way Analysis of
Variance showed statistically significant variation
(P<0.01) between stations and months.
Seasonality is one of the factors affecting the
community structure. A high current velocity and
water level lead to washing of fauna from the
sediments. This is true with regard to the present
study because no fauna was observed in the first
two stations. The upper sections of this river
system differ from lower sections in terms of food
materials available to the benthos. Similar to the
findings of present work, Ray et al. (1966) and Singh
and Shrivastava (1989) have recorded a peak
density of macroinvertebrate population in early
summer (pre-monsoon) before the floods and the
lowest density during monsoon in Ganga and
Yamuna rivers at Allahabad (Uttar Pradesh) and
Ganga river between Buxar and Ballia region
116 DRUSILLA ET AL.
respectively. In Chackai canal of Trivandrum,
Nirmalakumari and Nair (1984) observed that
most of the dragonfly naids were present attached
to the weeds. Sample collection stations of present
work were mostly confluencing points of two
streams/rivers & the naids collected in the present
study were seen attached to the few macrophytes
(Eichhornia crassipes) on the banks of the river.
Among the insect nymphs, the naids of
dragonfly (Diplocodes trivialis ) was found to inhabit
the moist edges of the river banks. This
phenomenon was noticed throughout the study
period in all the four downstream stations. A
notable observation was that the naids were well
high in number during pre-monsoon and low in
monsoons. In river Tambaraparani in Tirunelveli
district of Tamil Nadu, Martin (1994) also recorded
maximum density of naids during pre -monsoon
which might be due to rich nutrient content and
the minimum density of naids in monsoons owing
to dilution of nutrients in the river water.
Maximum values of odonate naids were noticed in
stations 5 and 6 in the present study which might
be as a result of more nutrients compared to the
two upstream stations (stations 3 rd and 4 th). In
Ithikkara river of Kerala, Sheeba and Ramanujan
(2005) recorded the presence of nymphs of mayfly,
stonefly, damselfly and dragonfly as it was
reported in this running water system. The
percentage of insect nymphs ranged from 0.29 to
14.78 in Ithikkara river of Kerala while in this lotic
system it varied between 24.60 and 26.98 Actually
a higher percentage prevailed in this fluvial system
which might be owing to the entry of agricultural
runoff and sewage with high density of microflora
compared to Ithikkara river, Kerala. As it was
stated by Harrison and Hynes (1988) for Ethiopean
mountain streams and rivers and in freshwater
lakes of Mysore, Karnataka (India) analysed by
Padmanabha and Belagali (2007), the nymphs of
Ephemeroptera are inhabitants of clear water and
such a situation did exist in river Chittar also.
In the torrential waters of Garhwal region of
Himalaya, Nautiyal (1986) reported that dragonfly
naids were found to inhabit the moist edges of the
river banks while mayfly nymphs and stonefly
nymphs creeping and crawling on the banks of the
river. Similar situation did prevail at times during
post- and pre- monsoon seasons in this lotic system
also. In the north Indian river, Ganga at Patna
(Bihar), Singh (1997) registered the disappearance
of dragonfly nymphs from sites below the
discharge point of sewage. In the upper Berach
river basin of Udaipur, north India, dragonfly and
damselfly nymphs were observed in all the
stations excepting station 3, which was considered
almost a “biological desert” with heavy metal
pollution (Sharma et al., 2000b).
Learner et al. (1971) reported that the nymphs of
aquatic insects like stoneflies, mayflies, caddisflies
and water beetles were restricted to the upstream
stations of the river Cynon, a polluted tributary of
the river Taff (South Wales). Since all the stations of
present study were not that much polluted as the
upstream stations of the river Cynon, the insect
nymphs were recorded in the four downstream
stations only and not in the sewage-free two
upstream stations. Further, Eichhornia crassipes
would have given protection against water current
and naturally these insect nymphs were seen in
the downstream stations of river Chittar. Wiley et
al. (1997) recorded Baetis sp. in Michigan trout
stream which was strongly influenced by climatic
and hydrological conditions. As it was seen in this
lotic system, in river Basantar, at Samba of Jammu
and Kashmir, Dutta et al. (2001) reported seasonal
occurrence of nymphs of Baetis sp. (Ephemeroptera)
and nymphs of Neoperla sp. (Plecoptera). In the
streams of Cascade Mountains and Willamette
Valley of Western Organ (U.S.A), the nymphs of
Baetis sp. was identified by J.Li et al. (2001).
Aquatic Insects
Results of aquatic insects varied between 4.0/m2
and 14.0/m2 for the four downstream stations in the
two year study period.Yearly total density of
aquatic insects ranged from 74.0 to 116.0 m2 in the
stations 3rd, 4th, 5th, and 6th. Minimum number of
4.0/m2 was noticed in station 3 during north-east
monsoon whereas maximum density of 14.0 /m2
was observed in stations 5 and 6 during pre-
monsoon months in the first and second year of
study. Anisops bouveri (notonectid) was the most
prevalent group among aquatic insects and corixa
sp. occupied the second position in river Chittar.
Laccotrephes maculates and Ranatra elongata (nepids)
were more during pre-monsoon season compared
to the values obtained in monsoons. Among the
macroinvertebrates, maximum percentage was
noticed in aquatic insects and it ranged from 28.24
to 30.01. In stations 1 and 2, aquatic insects were
totally absent. Aquatic insects revealed statisti-
cally significant difference (P<0.01) between
stations and months on Two way Analysis of
 Microbiological Studies and Macroinvertebrate Fauna of the Lotic Systems in
Variance.
Aquatic insects are referred to as water quality
indicators (Ward, 1984). Certain groups namely,
Belostomids, Notonectids and Nepids may be used
as bio-agents in the control of mosquito larvae
(Hoffman, 1927; Menke, 1979 and Narayanan et
al.,1998). Aquatic hemipterans such as Gerridae,
Nepidae and Corixidae and Pleidae nourish fishes
(Jebanesan and Selvanayagam, 1994). The reports of
Fernando (1959) on Ranatra elongata and Popham
(1964) on corixids revealed that aquatic insects
migrate from one habitat to another during spells
of warm summer weather. The above said
phenomenon can be attributed to as extent to this
lotic system also because the hemipterans present
in the downstream stations might have migrated
or got washed by water current from the pond just
prior to the 4 th station and be present more in
density compared to 3 rd station. Beyond the 4 th
station, (i.e) 2 km ahead, full-fledged growth of
Eichhornia crassipes was seen on the banks for a
distance of 2 km specially during post-and pre-
monsoon seasons. So, water current would have
washed these aquatic insects to the downstream
stations. This might be the cause for more number
of aquatic insects in the 5th and 6th stations of this
lotic system. Further, the nutrient content due to
agricultural runoff and sewage entry was high in
the last two downstream stations apart from a
few of these hydrophytes on their banks and these
might have led to the increase in the density of
aquatic insects in this river.
In the river Cooum of Madras, Tamil Nadu,
hemipterans were absent in the polluted zone
(Selvanayagam et al.,1988). But in river
Tambaraparani of Tamil Nadu, hemipterans were
totally absent in station 2 due to textile mill
effluents draining into it (Martin et al., 2000). But in
the present investigation, there was no such gross
pollution so hemipterans did occur in all the four
downstream stations. In this Chittar river, only 6
genera of hemipterans were recorded but Martin
et al. (2000) reported 8 genera of hemipterans which
might be owing to rich nutrients because of sewage
entry in that station. Further, north-east monsoon
minima of the four downstream stations were as a
result of heavy rains and high velocity of water
current and such low values cannot be compared
to the north -east monsoon maxima of hemipterans
found in Tambaraparani river of Tamil Nadu
(Martin et al., 2000). The maximum value of 14.0/m2
observed in the present study is no way
117
comparable to the minimum value of 91.0/m2 of the
above cited Tambaraparani river ( Martin et al.,
2000). Nirmalakumari and Nair (1984) noticed that
nymphs of hemipteran insects attached to aquatic
weeds in the Chackai canal, Trivandrum. Lausbury
(1957) also reported the association of hemipterans
with the vegetations of the water ways. Prior to
station 5 also there were macrophytes (Eichhornia
crassipes) in abundance on the river banks to which
the aquatic insects might have clinged and got
washed to downstream stations 5 and 6 by water
current. These macrophytes provide suitable
breeding place for the aquatic insects (Martin et al.,
2000)
Among north Indian rivers, in Ganga river at
Bhagalpur region, Jhingran (1991) recorded the
entire insect population consisted only of
hemipterans. In this lotic system, hemipterans did
dominate but water beetles (Dytiscus sp.) were very
scanty. These water beetles were absent in all the
four downstream stations during monsoon months
which might have been due to rapid currents of
water. Jhingran (1991) suggested that densities of
aquatic insects may be related to seasonal
fluctuations in water level with high number in
winter and low value in summer. Contrary to the
above view, variations did occur in the density of
aquatic insects in the present study with pre-
monsoon maxima, which might be as a result of
rich nutrient content in that season by agricultural
runoff and sewage entry.
In the rapid stretch of Alaknanda station of
torrential waters of Garhwal region of Himalaya,
Nautiyal (1986) reported the presence of aquatic
beetles (Dytiscids) at a depth of 1 to 2 feet. Besides
aquatic beetles, the Nayar station of torrential
waters of Garhwal region of Himalaya harboured
Nepids, Belostomatids (Diplonychus sp.). In the
above said Nayar station, on the sandy or muddy
patches on the river bed, corixids were present. In
the river Chittar also the above mentioned aquatic
insects were present during post-and pre-monsoon
but a few species were not seen during monsoons
as a result of high velocity of water current. In
north Indian Berach river system, Udaipur Sharma
et al.(2000b) identified Hydrophilus sp. in the metal-
polluted station which was considered almost a
“biological desert”. In the river Basantar, Samba of
Jammu & Kashmir seasonal presence of Hemiptera
(Corixa sp.) and Coleoptera were reported by Dutta
et al. (2001) as in this river Chittar of Tamil Nadu.
Macrobenthic diversity and density were high
118 DRUSILLA ET AL.
during September to November with optimum
water flow in river Basantar, Samba of Jammu and
Kashmir The rich fauna is due to the presence of
large number of pebbles, gravels and stones
(brought from the upper catchment during
monsoon) and rich algal growth (Dutta et al., 2001).
But in river Chittar, the situation is diametrically
opposite because there was low density of aquatic
insects during monsoons and high density during
pre-monsoon when there was low water content
but rich nutrients owing to sewage entry and
agricultural runoff.
Molluscs
Results of monthly variations of molluscs
(Pelecypods and Gastropods) ranged from 4.0/m2 to
14.0/m2 for the stations 3rd to 6th. First two stations
were devoid of molluscs throughout the study
period. Yearly numerical density ranged from 68.0
to 104.0/m 2 for all the four downstream stations
during the period of study. Minimum value of 4.0/
m2 was recorded in station 3 during north-east
monsoon both in the first and second year of study.
Maximum density of 14/m 2 was obtained in
stations 5 and 6 in the pre-monsoon period for both
the years. Compared to monsoon values (south-
west and north-east), the values of molluscs
registered during pre-and post-monsoon were
higher in the four downstream stations and the
percentage varied between 26.01 and 29.37. The
pelecypods (Lamellidens marginalis and Lamellidens
consobrinus) were the perennial species, while the
gastropods, Pila globosa and Limnaea sp. were found
to be scanty during monsoons because of high
velocity of water current as a result of heavy spell
of monsoon rains. Two way Analysis of Variance
showed distinct difference between stations and
months (P<0.01).
Present study revealed the absence of mollscan
forms in the first two stations due to the turbulent
nature of water. However, there was low density of
molluscs compared to aquatic insects in the four
downstream stations. Oxygen depletion has shown
to be a major constraint of freshwater pelecypods
(Ingram,1957). The presence of pelecypods indicates
good dissolved oxygen content and favourable
physico-chemical conditions in the river Chittar.
On the other hand, gastropods were found to be
more tolerant of organic pollution (Singh, 1997). In
this lotic system, the apple snail, Pila globosa was
found to be meagre in number during monsoons so
also Limnaea sp. due to increased speed of water.
Unlike the present investigation, in
Tambaraparani river of Tamil Nadu, Martin (1994)
recorded molluscs in all the stations. The numerical
density ranged from 5.0 to 34.0/m2 in all the eleven
stations studied. But the molluscs observed in the
present study varied between 4.0 and 14.0/m2 in
the last four downstream stations which do
confirm the less nutrient level of present lotic
system compared to river Tambaraparani. As in to
the present observation, river Tambaraparani also
showed maximum density of molluscs in pre-
monsoon months which might be due to abundant
nutrients. Low density of molluscs in monsoon
months might be owing to high velocity of water
current in both the rivers. Only the minimum value
of molluscs (5.0/m2) of Tambaraparani river of Tamil
Nadu is comparable to the minimum value of
present study, whereas maximum number of 34.0/
m2 of molluscan forms in Tambaraparnai river is
not comparable with the very low maximum
value of 14.0/m2 of molluscs of the present study.
High density of molluscs in Tambaraparani river
might be as a result of organic enrichment by
domestic sewage. Only 4 species (2 species of
pelecypods and 2 species of gastropods) were
observed in the river Chittar which might be due
to its “Oligotrophic” nature. Results of molluscs
revealed higher density in stations 5 & 6 compared
to 3 rd and 4 th stations owing to low velocity of
water current (Drusilla et al., 2004) and increased
rate of agricultural runoff (nutrients) from the crop
fields of the riparian areas entering this lotic
system. But during monsoon period, the
population of mollucs diminished in number owing
to flushing of this river by the spates. Yazdandoost
and Katdare (2000) recorded two genera of
pelecypods (Lamellidens and Parreysia) in Pune rivers,
India. In Arjuna river, Sivakasi of Tamilnadu,
Rajan and Murugan (2001) reported the presence of
Lamellidens marginalis. varying between 1.0 and 11.0.
The density of above said bivalve is similar to the
density of bivalve collected in river Chittar of Tamil
Nadu. Bhamre and Deoray (2008) registered the
occurrence of the bivalve, Parreysia favidens in the
Darna river near Nashik road, Maharashtra, India.
Among north Indian rivers, in Ganga river,
Pahwa (1979) collected 4 species of pelecypods and
10 species of gastropods which might be as a result
of rich nutrient content because of pollution load
while Bilgrami and Datta Munshi (1979) obtained
only 2 species of pelecypods and 8 species of
gastropods in river Ganga. In the torrential waters
 Microbiological Studies and Macroinvertebrate Fauna of the Lotic Systems in
of Garhwal region of Himalaya, Nautiyal (1986)
reported gastropods being present in rapid -
stenothermal region. Jhingran(1991) recorded 3
species of pelecypods and 6 species of gastropods in
river Ganga near Patna which are higher than the
values of present work. According to Singh (1997)
gastropods are facultative fauna and important
indicators of organic pollution prevailing in Ganga
river at Patna (Bihar). In the upper Brahmaputra
river of Assam, Biswas & Boruah (2000) registered
6 species of molluscs. As in the present studies, Pila
globosa was the dominating species among
gastropods in the above said river. On the contrary,
in the river Chittar, gastropods were found in all
the four downstream stations but were scanty
during monsoons which might be owing to high
speed of water flow as a result of heavy rains and
low availability of food resources in monsoon
seasons. In Ganga river at Hathidah, 7 genera of
gastropods and 2 genera of pelecypods & odonates
while in Damodar river at Durgapur 5 genera of
gastropods and one genus in pelecypods and
odonates were collected by Biswas and Konar
(2007). The above quoted data are higher than the
genera of molluscs observed in river Chittar of
Tamil Nadu. As in the present findings, in the
western bank of the river Hooghly, Banerjee and
Banerjee (2007) reported seasonally molluscan
population more during pre-and post-monsoon
than monsoon season. In Santubong river,
Sarawak, Malaysia, Ashraful et al. (2009) recorded
the presence of green mussel, Perna viridis.
Shannon-Wiener diversity index of macroin-
vertebrates ranged from 1.361 to 1.374 in the
downstream stations of present investigation. But
in river Tambaraparani, Tamil Nadu, Martin et al.,
(2000) obtained a wide diversity of organisms with
Shannon-Wiener diversity index varying between
1.0 and 3.02. Shannon-Wiener diversity index of
macroinvertebrates in the river Damodar at
Durgapur ranged from 0.514 to 1.011 in three
stations (1 st , 3 rd , & 4 th) with 7 to 10 species of
macroinvertebrates while in river Darmodar at
Narankuri the diversity index varied between
0.993 and 2.102 with 20 species in one station
(Biswas & Konar, 2007). The above said values are
comparable with the present work to an extent. In
the streams of Cascade Mountains and Willamette
Valley of western Oregon (U.S.A), the Shannon
diversity index ranged from 1.40 to 3.31 for
Ephemeroptera, Plecoptera and Trichoptera (Li et
al., 2001). High diversity index and population
119
density of nymphs of aquatic insects in the above
cited rivers might be due to more amount of
nutrients.
Simpson’s Dominance index varied between
0.256 and 0.261 in the present findings while in
Tambaraparani river of Tamil Nadu it was from
0.11 to 0.4 as reported by Martin et al. (2000) and
the range is quite low in river Chittar of Tamil
Nadu.
CONCLUSION
Results of present study on the coliform bacteria of
the lotic systems in and around Courtallam
revealed the absence of these microbes in the first
two upstream stations. However, coliform bacteria
including faecal coliforms and Escherichia coli were
present in the four downstream stations in an
increasing order from 3rd station to 6th station.
Monsoon dilution of water did minimise the MPN
index of coliforms, while during post - monsoon
and pre-monsoon the MPN/100ml of coliform
bacteria has been enhanced due to the entry of
sewage in the four downstream stations.
Rapid flow of water current would have led to
the absence of macroinvertebrates in the first two
upstrems stations. The density of macroinverte-
brates during south-west & north-east monsoons
was low compared to post-monsoon and pre–
monsoon values. Enrichment of water with
agricultural runoff and sewage could have
increased the number of macroinvertebrate fauna
from station 3 to station 6. The trend seen in the
density of macroinvertebrates was : aquatic insects
> molluscs > insect nymphs > crustaceans.
ACKNOWLEDGEMENT
The First author is extremely grateful to the
Professors of Department of Zoology and Dr. Jacob,
Reader in Zoology, Department of Zoology of
St.Xavier’s College, Palayamkottai, Tirunelveli Dt,
Tamil Nadu for lending the helping hands in
identifying the macroinvertebrates of this lotic
system. The author is much thankful to
Dr.Thiruvan and Dr.(Mrs) Shanthi Thiruvan for
making arrangements in their laboratory of
Shanthinikethan Nursing Home, Shencottai,
Tirunelveli Dt. to analyse the categories of microbes
present in the water samples of this river Chittar.
The help offered in the estimation of microbial
criteria of the water samples of river Chittar by the
120 DRUSILLA ET AL.
staff members of ICAR Department of Sri
Parasakthi College for Women, Courtallam,
Tirunelveli Dist, Tamil Nadu is thankfully
acknowledged.
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© Global Science Publications

IN-VITRO ASSESMENT OF CHOLIC ACID PERMEABILIZING

ABILITY IN COMBINATION WITH ANTIBIOTICS TO
COMBAT MICROBIAL RESISTANCE EXHIBITED BY
BIOFILM PRODUCING BACTERIA
A.I. SHEIKH, SHWETA MISHRA AND M. MUSADDIQ

P.G. Department of Microbiology, Shri Shivaji College, Akola 444 001, (M.S.) India.

(Received 20 July 2011; Accepted 15 September 2011)

Key words : Biofilm, Drug resistance, Pathogenic organism, Cholic acid.

Abstract - Biofilm are complex aggregation of bacteria marked by the excretion of a protective and
adhesive matrix. They develop almost anywhere in water and solid or solid and gases meet as well as
living surfaces, which means they are virtually everywhere, implying their ubiquitous nature. When
they form biofilm, bacteria seem to gain super power. In human terms, biofilm spells all kind of big
trouble, for instance they fog our contacts, help to rot our teeth and cause a host of diseases from
cystic fibrosis and ulcers to colitis and middle ear infection. Microorganism growing in biofilm includes
Pseudomonas, Staphylococcus, Streptococcus, Candida, Klebsiella, and Salmonella etc. The nature of biofilm
structure and physiological attributes of biofilm organism have found to confer an inherent resistance
to antimicrobial agents. Considering the wide variety of microorganism that are able to form biofilms
and the mentioned aspects including its environmental, medical, commercial significance the present
work was undertaken. Pathogenic organisms were isolated, identified and antibiogram studies were
made in presence of usually recommended antibiotics. Screening for biofilm producers was made.
With a view to search out for intervention strategies combination of cholic acid was analyzed with
antibiotics. The present investigation supports that a link exist between pathogenic organism, biofilm
production and drug resistance. It also lead us to conclude the ability of cholic acid in combination with
antibiotics to inhibit biofilm producers.

INTRODUCTION attachment, structural heterogeneity, genetic

Biofilms have been described in many systems
since Antony Van Leeuwenhoek examined the
animalcules in the plaque on his own teeth in the
seventeenth century, but the general theory of
biofilm predominance was not promulgated until
1978.
Microorganisms in natural environments, faced
with physical, chemical and biological threats,
often found themselves up against a wall, both
literally and figuratively adherence to the surfaces
can lead to formation of biofilm. A biofilm is a
structured community of microorganisms
encapsulated within a self-developed polymeric
matrix and adherent to a living or inert surface.
Biofilms are also often characterized by surface
diversity, complex community interactions and an
extracellular matrix of polymeric substances
(Allison et al., 2000).
The clinical evidences point to reveal that
biofilm is a source of several infectious diseases.
Biofilms can contain many types of microorganism,
e.g., bacteria, archaea, protozoa, fungi and algae;
each group performing specialized metabolic
functions. Detachment of cells or cell aggregates,
production of endotoxin, increased resistance to the
host immune system and provision of a niche for
the generation of resistant organisms are all biofilm
processes which could initiate the disease process.
Biofilms have been found to be involved in a wide
variety of microbial infections in the body, by one
estimate 80% of all infections. Infectious processes

*Corresponding author - E-mail : dr.m.musaddiq@rediffmail.com; shaikhasimiqbal1@gmail.com

124 SHEIKH ET AL.
in which biofilms have been implicated include
common problems such as urinary tract infections,
catheter infections, middle-ear infections, Burn
wound infection, Cystic fibrosis, and less common
but more lethal infections of permanent indwelling
devices such as Prostheter, catheters, contact lenses
and heart valve etc. Several microbes like
Staphylococcus, Streptococcus, Candida, Klebsiella, and
Salmonella etc. are found to grow as a host
associated biofilm that cause persistent human
infection (Anaissie et al., 1995).
The nature of biofilm structure and the
physiological attributes of biofilm organisms confer
an inherent resistance to antimicrobial agents,
whether these antimicrobial agents are antibiotics,
disinfectants or germicides. Mechanisms
responsible for resistance may be one or more of the
following Delayed penetration of the antimicrobial
agent through the biofilm matrix, altered growth
rate of biofilm organisms, other physiological
changes due to the biofilm mode of growth.
Intervention strategies are of great economic
relevance because traditional antibiotic therapy is
usually not sufficient to eradicate the biofilm based
infections. One major reason for persistence seems
to be the capability of the bacteria to grow within
biofilms that protects them from adverse
environmental factors. Eradication of slime can be
achieved by means of minimization of the initial
contamination of medical devices or inhibition of
quorum sensing or by degradation of matrix.
Combination therapy of antibiotics was used to
combat the resistance disaster. But considering the
ill effect of high dosage of antibiotics and their cost,
the concept did not proved satisfactory (Van
Laethem et al.,1983).
Several investigators analyze the utility of
clavulanicacid in combination with antibiotics
which proved effective but clavulanic acid is
effective only in case of Penicillin resistance,
attention was switched onto analyzed the
potentiating ability of cholic acid in combination
with antibiotics. In nature, cholic acid is produced
in the liver from cholesterol. The liver converts
cholesterol into the conjugated salts of glycocholic
and taurocholic acid which are excreted into the
bile (Van Landuyt, Denolf and Lambert, 1981).
However little information is available on
potentiating effect of cholic acid on antibacterial
activity of antibiotics against multi-drug resistant
biofilm producers. Therefore in present
investigation the efficacy of antibiotic in
combination with cholic acid on their antibacterial
activity is analyzed.
MATERIALS AND METHODS
Clinical samples (viz. pus, skin swabs, serum etc.)
were collected in sterilized tryptone soya broth and
exposed to further analysis. Isolation and
identification of causative pathogens was made via
standard conventional method.
Antibiogram in presence of usually
recommended antibiotic was studied by Kirby -
Bauer Disc diffusion method (1966). Isolates were
categorized as susceptible, moderately susceptible,
and resistant based upon interpretive criteria
developed by the Clinical & Laboratory Standards
Institute (CLSI) and results were recorded as per
zone of inhibition. Antibiotics used were : Penicillin
G (10mcg), Nalidixic acid (30mcg), Kanamycin
(10mcg), Colistin (10mcg), Gentamycin (30mcg),
Chloramphenicol (50mcg), Colistin (10mcg ),
Tetracycline (30mcg) etc.
Screening and detection of biofilm producers
was made by Congo red Agar (CRA) method
(Vandenesch et al., 1995). Isolates that produce
black colonies with dry, crystalline consistency
were regarded as slime positive where as those
showing pink colonies were slime negative.
Comparative analysis of the antibiogram exhibited
by biofilm producers and non biofilm producers
was made.
Growth response exhibited by multiple drug
resistant isolates was studied in presence of cholic
acid fortified in sensitivity test medium at sub-
bacteriostatic concentration of 0.05 % and results
were recorded.
RESULTS AND DISCUSSION
Table 1. Nature of clinical samples utilized for isolation
purpose
Name of Organism Clinical Sample Utilise
Staphylococcus aureus Burn wound biopsy, Blood,
Pus, Skin infection.
Pseudomonas aeruginosa Pus, Urine, Burn wound
biopsy
Klebsiella pneumoniae Pus, Urine, Burn wound
biopsy
Proteus vulgaris Pus, Urine, Burn wound
biopsy
Enterobacter aerogens Burn wound biopsy
Salmonella typhi Serum
 In-vitro Assesment of Cholic Acid Permeabilizing Ability in Combination with Antibiotics 125

In the present attempt in all the 60 clinical Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella
samples [viz. pus, urine, skin swabs etc.] were pneumoniae, Salmonella typhi, Enterobacter aerogens and
collected and further subjected to isolation and Proteus vulgaris etc. based on the various
identification studies of relevant organisms i.e., morphological and biochemical characteristics
(Table 2).
Table 2. The results (%) of slime production. Slime factor production of obtained isolates was
Organism Biofilm Biofilm investigated by both the methods used for
Producer Non- qualitative analysis viz. (CRA and ST method). It
producer has been considered that testing for biofilm
formation would be a useful marker for the
Pseudomonas aeruginosa 50 50
pathogenicity of obtained isolates. Amongst the
Staphylococcus aureus 47 53
Salmonella typhi 20 80 obtained isolates Pseudomonas aeruginosa (50%),
Klebsiella pneumoniae 20 80 followed by Staphylococcus aureus were found to be
Proteus vulgaris 25 75 eminent biofilm producers by CRA method, slime
Enterobacter aerogens 10 90 production was also observed in Klebsiella

Table 3. Antibacterial sensitivity pattern (%) exhibited by biofilm producers.

Name of antibiotics Name of organism

S. aureus P. vulgaris P. aeruginosa Salmonella typhi K. pneumoniae E. aerogens

Penicillin G 23 49 18 18 68 62
Kanamycin 50 42 8 25 76 81
Nalidixic acid 30 56 50 45 57 94
Colistin 100 100 100 100 100 100
Gentamycin 41 42 22 59 57 47
Chloramphenicol 25 100 34 95 81 100
Tetracycline 97 12 58 75 51 91

Table 4. Antibacterial sensitivity pattern (%)exhibited by non-biofilm producers.

Name of antibiotics Name of organism

S. aureus P. vulgaris P. aeruginosa Salmonella typhi K. pneumoniae E. aerogens

Penicillin G 34 52 21 54 74 68
Kanamycin 57 46 14 48 81 86
Nalidixic acid 33 58 54 60 64 100
Colistin 1000 100 100 100 100 100
Gentamycin 47 47 27 70 59 49
Chloramphenicol 37 100 37 84 83 100
Tetracycline 100 27 100 100 71 100

Table 5. Antibacterial sensitivity pattern (%) exhibited by biofilm producers in presence of cholic acid.

Name of antibiotics Name of organism

S. aureus P. vulgaris P. aeruginosa Salmonella typhi K. pneumoniae E. aerogens

Penicillin G 28 50 28 25 78 64
Kanamycin 61 100 86 50 77 88
Nalidixic acid 42 62 86 50 61 100
Colistin 100 100 100 100 100 100
Gentamycin 47 50 43 62 60 52
Chloramphenicol 25 100 43 78 81 100
Tetracycline 100 100 71 79 58 100
126
pneumoniae and Salmonella typhi (20%). Least number
isolates of Enterobacter aerogens (10 %) were found
to produce biofilm by CRA method.
Bacterial identification, screening of biofilm
producers followed by susceptibility test are
important for selection of appropriate
antimicrobial agent for treatment. Considering this
susceptibility/resistant pattern of slime producers
as well as non slime producers was recorded and
presented in Table 4 & 5 respectively. Antibiogram
of obtained isolates was analyzed by Kirby Bauer
Disc Diffusion technique. Results revealed that
slime producing S. aureus isolates were completely
sensitive to Colistin (100%) followed by
Tetracycline. The isolates were found least sensitive
to Penicillin G (23%). Kanamycin was found
satisfactory in inhibiting slim producing S. aureus
isolates followed by (41%) and Nalidixic acid (30%)
(Souli et al., 1998).
Pseudomonas aeruginosa (slime producing) isolates
were found almost completely resistant to
Kanamycin (8%) and Penicillin G (18%). Nalidixic
acid was found satisfactory (50%) in inhibiting the
organism followed by Chloramphenicol (34%),
Tetracycline considerably inhibited (58%) the
growth.
Chloramphenicol remarkably inhibited growth
of P. vulgaris (100%), E. aerogens (100%), and K.
pneumoniae (81%). Gentamycin also inhibited P.
vulgaris (42%), E. aerogens (47%), and K. pneumoniae
(57%) satisfactory. S. typhi slime positive isolates
were found completely sensitive to Colistin (100%),
followed by Chloramphenicol (81%), while isolates
showed resistance to Penicillin (18%), and
Kanamycin (25%). E. aerogens was found as the most
sensitive among all the isolates analyzed, while
Penicillin G was found as least effective antibiotic.
With respects to non-biofilm producers
Tetracycline and Colistin were found to be the drug
of choice as they were able to control complete
growth of all the isolates.
Chloramphenicol (37%), Nalidixic acid
(33%), Penicillin G (34%) were found least effective
with respect to S. aureus isolate, while satisfactory
performance was exhibited by Kanamycin (57%)
followed by Gentamycin (47%). Similarly the same
above three antibiotics expressed a varied but
satisfactory sensitivity pattern against P. vulgaris.
Tetracycline (27%) was found least effective against
this organism. None of the antibiotics accepts
Nalidixic acid (54% & 60%) was found remarkably
effective against P.aeroginosa and S. typhi respectively.
SHEIKH ET AL.
All the analyzed antibiotics inhibited growth of E.
aerogens, K. pneumoniae and S.typhi considerably
(Anwar et al., 1992).
As per review our results are in correlation with
several investigators working on line. The
observation lead to conclude that recommendation
of Penicillin G antibiotic in therapeutic procedure
with special relevance to P. vulgaris, S. aureus,
Pseudomonas aeruginosa and S. typhi infections can
lead to enhancement in drug-resistance. Colistin
can be considered as the drug of choice especially in
case of infection associated with slime producers.
It is noticed that slime production and multi-
drug resistance are associated with each other.
Keeping this in mind the potentiating effect
induced by cholic acid on the antibacterial activity
of antibiotics was analyzed and recorded against
slime producer isolates. Pseudomonas aeruginosa
(slime producing) isolates were found completely
sensitive to Colistin (100%) followed by Nalidixic
acid (86%) and Kanamycin (86%) while Tetracycline
considerably inhibited (71%) the growth,
Gentamycin and Chloramphenicol were found
satisfactory (43%) in inhibiting the organism
followed by penicillin G with least susceptibility
(28%). Chloramphenicol, Colistin and Tetracycline
remarkably inhibited growth of P. vulgaris (100%),
followed by Nalidixic acid (62%). Penicillin G and
Gentamycin also inhibited P. vulgaris (50%)
satisfactory. Colistin, Chloramphenicol and
Tetracycline were found remarkably inhibited the
growth of E.aerogens (100%), followed by Kanamycin
(88%) and Gentamycin (64%).
S.typhi slime positive isolates were found
completely sensitive to Colistin (100%), followed by
Chloramphenicol (78%) and Tetracycline(79%)
while isolates showed less susceptibility to
Penicillin (25%) and Kanamycin (50%) satisfactory.
E. aerogens was found as the most sensitive among
all the isolates analyzed, while Penicillin G was
found as least effective antibiotic.
Slime producing S.aureus isolates were
completely sensitive to Colistin (100%) followed by
Tetracycline. The isolates were found least sensitive
to Penicillin G (23%). Kanamycin was found
satisfactory in inhibiting slim producing S.aureus
isolates followed by (41%) and Nalidixic acid (30%).
Thus in the present study it has been observed
that cholic acid at its’ sub-bacteriostatic
concentration (0.05%) is effective in potentiating
antibacterial activity of antibiotics. The underlying
mechanism of this in-vitro potentiation still remains
 In-vitro Assesment of Cholic Acid Permeabilizing Ability in Combination with Antibiotics
undefined but it may be due to enhanced
potentiation of antibiotic into the bacterial cell.
Present investigation shows that potentiation of
antibacterial activity is highly structure specific
which is in accordance with to the earlier
observations made by Sylnic and Puzakova (1980).
They stated the bile acid increases the bactericidal
activity of certain antibiotics. However their work
was independent of presence/absence of slime
production. Hence the results of the present piece of
work entitled "In-vitro assessment of cholic acid
permeabilizing ability in combination with
antibiotics to combat microbial resistance
exhibited by biofilm producing bacteria". Embark
the possibility of utility of cholic acid at sub-bacte-
riostatic concentration (0.05%) in combination
therapy with antibiotics to eradicate infection
associated with slime production.
REFERENCES
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operation in Biofilms. Cambridge: Cambridge
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L.M., Barret, F.F., Melton, D.M. and Beachey, E.H.
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Devenport, D.S., Massanari, R.M., Pfaller, M.A., Bale, M.
J., Streed, S.A. and Hierholzer, W.J. 1986.
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clinically significant infections with coagulase
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Freeman, D.J., Falkiner, F.R., Kaene, C.T. 1989. New
method for detecting slime producing by
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Lagast, H., Husson, M. and Klastersky, J. 1985.
Comparetive in-vitro activities of azlocillin,
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Sytnik, I.A. and Puzakova, E.V. 1980. Combined effect of
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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 14, No. (1) : 2012 : 137-142
© Global Science Publications

EFFECT OF CHROMIUM ON VARIOUS BIO-CHEMICAL

PARAMETERS OF PITHOPHORA SP.

N.H. BRAHMBHATT* AND RINKU V. PATEL

V. P. & R.P.T.P. Science College, S.P. Unversity, Vallabh Vidyanagar, Gujarat, India.

(Received 6 April 2011; Accepted 15 November 2011)

Key words : Pithophora sp. Chromium, Bio-chemical parameters, Wastewater

Abstract - The industrial wastewater and algae were collected from Ankleshwar GIDC area. The
collected sample of Pithophora sp. were treated with Chromium ion at different concentrations and at
different duration of incubation periods. Investigations of the various bio-chemical parameters and its
effect and accumulation of Chromium by Pithophora sp was studied. In conclusion, Pithophora sp. was
able to grow in Chromium contaminated sites and to accumulate significant amounts of Chromium with
a high transfer factor.

INTRODUCTION fungi, and many develop complex biofilm

Pollution of water sources is increasing rapidly
due to waste discharge from industries. Among
different kinds of pollution, occurring in nature,
metals constitute as one of the major environmental
pollutants. Bioremoval of heavy metals is one of the
most promising technologies involved in the
removal of toxic metals from the industrial waste
streams and natural waters. The more promising
bioremediation strategies, which are being
developed for metal removal, are; the identification
of organisms, which have enhanced capability to
sequester metals and the expression of, trace metal
binding factors in transgenic organisms. Sewage
treatment, activated sludge plants, biofilters,
biofilm reactors, fixed and suspended film systems,
lagoon treatments, stream meanders, nitrification
and denitrification treatments, biological
phosphate removal processes, wetlands and reed-
bed technologies, composting, and in situ and ex
situ bioremediation processes all rely on the
activities of microbes to break down organic
substances. The main types of microbes associated
with metals in terrestrial and aquatic habitats are
sulfate-reducing bacteria (SRB), sulfur-oxidizing
bacteria, iron-oxidizing/reducing bacteria,
manganese -oxidizing bacteria, bacteria secreting
organic acids and slime, and various algae and
communities on surfaces of materials (Beech &
Sunner et al., 2004; Gu et al., 2009). Where microbial
reduction of a metal to a lower redox state occurs,
mobility and toxicity may be decreased for several
elements (Lovley, 2001; Lloyd & Lovley, 2001;
Finneran et al., 2002 a, b; Lloyd et al., 2003; Holden &
Adams, 2003; Wall & Krumholz, 2006), e.g. U (VI) to
U (IV) and Cr (VI) to Cr (III) (Phillips et al., 1995;
Smith & Gadd, 2000).
Toxic action of Zinc on growth and enzyme
activities of rice seedlings was shown by Nag et al.,
(1984). Kalplan et al., (1987) have shown that algal
extracellular polysaccharides also chelate or bind
metal ions. Response of the ascorbate peroxidase of
Selenastrum capricornutum to copper and lead in storm
waters is showm by Wong et al., (2001). Proline
alleviates heavy metal stress in Scenedesmus armatus
(Enany-el and Issa, 2001). Biosorption of Cr (III) by
microalgae and macro algae (Michalak et al., 2007).
Removal of Cu (III) and Pb (III) by Pithophora
oedogonia biomass (Singh and Kumar, 2008).
Biosorbents for heavy metal removed and their
future (Wong et al., 2009). Kumar and Chakraborty
(2009) have reported effect of light stress on
Peroxidase, Succinate dehydrogenease and total
Chlorophyll content in Andrographis paniculata. Such
properties of mineral surfaces as micro topography,
surface composition, surface charge and

*Corresponding author -Email: naina_bbhatt@yahoo.co.in

138 BRAHMBHATT AND PATEL
hydrophobicity play an important role in
thigmotropism, microbial attachment and
detachment, and are therefore critical for
colonization and biofilm formation, and the
ecology of microbial populations associated with
mineral substrates (Vaughan et al., 2002; Gleeson et
al., 2005, 2006, 2010; Bowen et al., 2007; Brown et al.,
2008).
MATERIALS AND METHODS
The algae collected from Ankleshwar GIDC
(Gujarat Industrial Development Corporation
industrial area) were sides, washed the algae under
running tap water and removed the other
epiphytic algae attached it. The algal culture was
carried out as per AWPA-AWWA-AWPC, (1989).
After media preparation the culture were placed
in incubator at 25+ 2 oC at 1400 lux in a 14/10 hr
Light Dark Cycle. In this experiment, short-term
toxicity test was used. The stock solution of 1000
ppm for Cr metal was prepared in double distilled
water by dissolving the salt of metal. The salt was
used to obtain desired metal in solution CrCl 2
(Chromium Chloride). The following concen-
trations of metal were used 2, 5, 10, 20 and 30 ppm
of this metal. Algal sample harvested after 3, 6 and
9 days of incubation were washed repeatedly, dried
and digested in HNO3/HClO4 mixture (4:1, v/v) for
heavy metal analysis. Estimation of Chromium
was done on ICP [Inductively Couple Plasma
spectrometer, Perkin Elmer Corporation (ICP
optima 3300RL)].
Peroxidase was determined following
Sadasivam and Manikam, (1996) and Sugar was
determined by GOD-POD test. Catalase and Proline
concentrations were determined following
published procedures (Thimmaiah, 1999). Protein
content was determined in accordance with the
method of Lowry et al. (1951). Chlorophylls (“a”,
“b” and total Chlorophyll) concentrations were
determined following published procedures
(Arnon, 1949).
RESULTS AND DISCUSSION
The observations recorded for the bio-chemical
response and heavy metals accumulated by
Pithophora sp to various industrial wastewaters.
The bio-chemical test were carried out with
Pithophora sp for different concentration and
different duration of incubation period. Pithophora
sp. accumulated higher value of Cr 30 ppm
concentration, at 6 days (Table 1 & Fig. 1) & lower
value 5-ppm concentration, at 3 days. There are
several reports of algae accumulating heavy metals
from the aquatic environment. However, the rate of
metal uptake varies depending upon the kind of
metal and algal species involved. Silva et al. (2003)
found that Sargassum accumulated 6.23 mmol/L Cr
and 5.45 mmol/L Cu ions. Spirulina was found to
accumulate 83% of Cr (VI) from electroplating
wastewater (Hamer, 1986). Ramaswamy and
Somashekar (1982) reported the distribution of 23
genera of algae, from effluents of an electroplating
unit indicating that these algae might be absorbing
heavy metals from the effluents. They also found
that Pithophora was the dominant algae in the
electroplating wastes and accumulated 15 mg/L, 12
mg/L, 15 mg/L, 6 mg/L, 0.55 mg/L, 12 mg/L and 30
mg/L of Zn, Cu, Pb, Ni, Co, Cd and Cr respectively.
In peroxidase activity, accumulation of Cr is
increased in different concentrations at 3 days, but
it is decreased after 6 days (Table 2 & Fig. 2).
Catalase-Peroxidase has a protective role against
environmental H2O2 generated by algae or bacteria
in the ecosystem (Tichy and Vermaas, 1999). In
pollution related studies, the estimation of the
enzyme activities can be considered as one of the
important measurements since pollutant metals
disturb the oxidative balance in algae and hence
proved to be an important palliative measure in
the induction of antioxidants. Following 3 days of
exposure, the catalase activity decreased gradually
from two to 30-ppm concentration of the metal ions
(Table 3). It decreased gradually after 6 days and 9
days of exposure.
Accumulation of Cr ion increased proteins
content from 2 ppm to 10 ppm, but after 10 ppm, it
was decreased each day (Table 4 & Fig. 4) . As per
Gardea et al., (1998) Synechococcus sps was reported
to revel good metal ion binding properties of Cd,
Cu, Cr and Pb and they identified class II
metallothionein genes for these proteins. They also
demonstrated that the over expression of these
proteins increased metal tolerance and improved
the ability to sequester toxic metal ions. Enany and
Issa (2001) investigated the accumulation of proline
and correlated it with protein content. Gawrinath
and Rao (1989) reported the inhibition of pigment
synthesis in some of the plankton diatoms exposed
to Cr in the concentration range of 2.73 to 2.86 mg/
L. In our results, Pithophora metal concentration
increased after accumulation of Cr and proline
 Effect of Chromium on Various Bio-Chemical Parameters of Pithophora sp. 139

Table1. Estimation of Cr (mg/l) ion after accumulation at Table 5. Estimation of proline (mg/gm) at Cr ion after
different concentrations (ppm) in Pithophora sp. accumulation at different concentrations (ppm) in
Pithophora sp.
No. Concentration 3 Days 6 Days 9 Days
No. Concentration 3 Days 6 Days 9 Days
1. Control 0.1132 0.1132 0.1132
2. 2 ppm 0.2764 0.1820 1.2074 1. Control 0.880 0.880 0.879
3. 5 ppm 0.1747 0.4834 0.4370 2. 2 ppm 0.869 0.785 0.781
4. 10 ppm 0.5422 0.3067 0.1843 3. 5 ppm 0.862 0.766 0.759
5. 20 ppm 1.5632 0.8946 1.2727 4. 10 ppm 0.860 0.749 0.732
6. 30 ppm 1.0334 2.4816 2.3619 5. 20 ppm 0.853 0.730 0.721
6. 30 ppm 0.851 0.722 0.700

Table 2. Estimation of peroxidase (mg/gm) at Cr ion

after accumulation at different concentrations (ppm) in Table 6. Estimation of sugar (mg/gm) at Cr ion after
Pithophora sp. accumulation at different concentrations (ppm) in
Pithophora sp.
No. Concentration 3 Days 6 Days 9 Days
No. Concentration 3 Days 6 Days 9 Days
1. Control 0.890 0.809 0.882
2. 2 ppm 0.895 0.791 0.790 1. Control 20.7 20.7 20.7
3. 5 ppm 0.898 0.773 0.770 2. 2 ppm 20.2 20.9 21.7
4. 10 ppm 0.899 0.751 0.746 3. 5 ppm 24.5 23.6 22.3
5. 20 ppm 0.905 0.749 0.737 4. 10 ppm 25.7 24.2 23.6
6. 30 ppm 0.909 0.743 0.731 5. 20 ppm 20.4 22.2 24.7
6. 30 ppm 19.7 20.9 21.1

Table 3. Estimation of catalase (mg/gm) at Cr ion after

accumulation at different concentrations (ppm) in exposure (Table 6). As shown in Fig. 6, at these
Pithophora sp. exposure periods, maximum value for sugar was
recorded at 10 ppm after 3 days of exposure.
No. Concentration 3 Days 6 Days 9 Days Chlorophyll content is most important pigment of
1. Control 25.3 25.0 24.9 photosynthesis. After accumulation Cr, ion
2. 2 ppm 25.0 24.0 23.6 chlorophyll content is decreased. In Pithophora,
3. 5 ppm 24.2 24.1 23.1 chlorophyll-‘a’ is higher than chlorophyll-‘b’.
4. 10 ppm 23.0 23.6 22.7 Maximum reduction for chlorophyll was recorded
5. 20 ppm 22.7 22.9 22.0 at 2 ppm after 3 days of exposure (Table 7).
6. 30 ppm 21.2 22.6 21.4 Following exposure of the 6 and 9 days, the amount
of total chlorophyll decreased gradually as the
Table 4. Estimation of protein (mg/gm) at Cr ion after
concentration increased from lower to higher. At
accumulation at different concentrations (ppm) in this exposure, the amount of chlorophyll was much
Pithophora sp. less in the treated filaments as compared to that of
control. As represented in Fig. 7, the increased
No. Concentration 3 Days 6 Days 9 Days exposure period from three to 9 days, reduced the
1. Control 10.69 10.59 10.11 amount of total chlorophyll.
2. 2 ppm 10.52 10.09 10.01
3. 5 ppm 11.11 11.01 10.89 ACKNOWLEDGEMENTS
4. 10 ppm 11.01 10.79 10.56
5. 20 ppm 10.89 10.13 10.00 The authors are grateful to UGC, New Delhi for
6. 30 ppm 10.01 9.58 9.22 providing financial assistance to carry out the
investigations under the special assistance
content decreased each day (Table 5 & Fig. 5). programmed of S.P.University, Gujarat.
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140 BRAHMBHATT AND PATEL

Table 7. Estimation of chlorophylls (mg/gm) at Cr ion after accumulation at different concentrations (ppm) in
Pithophora sp.

No. Concentration 3 Days 6 Days 9 Days

Chl-a Chl-b Total chl Chl-a Chl-b Total chl Chl-a Chl-b Total chl

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© Global Science Publications

BIODEGRADATION OF PETROLEUM HYDROCARBON BY TWO

ASPERGILLUS SPP. AND TWO PENICILLIUM SPP. ISOLATED
FROM THE CONTAMINATED SOIL AND WATER OF
SHIP BREAKING YARD

KARTIK DHAR, SHOMA DUTTA AND M.N. ANWAR *

Department of Microbiology, University of Chittagong, Chittagong 4331, Bangladesh.

(Received 27 March 2011; Accepted 25 September 2011)

Key words : Petroleum hydrocarbon, Biodegradation, Fungi.

Abstract - Petroleum hydrocarbon pollution caused by ship breaking activities around Bhatiari coast of
Chittagong is one of the major environmental problem in Bangladesh. Currently available chemical and
mechanical methods have limited effectiveness and expensive. Bioremediation of petroleum
contamination by using fungi is cost-effective, environment friendly and easy to operate. Two
Aspergillus spp. (Aspergillus candidus and Aspergillus fumigatus); and two Penicillium spp. (Penicillium fuscum
and Penicillium waksmani) were isolated from the oil contaminated sites of Bhatiari and characterized. All
the four species have the ability of complete decolourization of Redox reagent (2,6-dinitrophenol
indophenol). Marked cultural and morphological differences were observed when the isolates were
grown on different hydrocarbon source containing media. The isolates had been found to show
biodegradation of kerosene, diesel and octane with in defined period of time. Greasy spot test was used
for primary indication of degradation. For more specific estimation of degradation rate
spectrophotometric method was carried out. The isolate Penicillium waksmani showed highest percent
(60.5% and 59.85%) degradation of kerosene and diesel. Aspergillus fumigatus was found to degrade
highest percent (61.29%) of octane. These results suggest that the fungal population could be used for
bioremediation of petroleum hydrocarbon pollution.

INTRODUCTION the Amoco Cadiz, which went aground in the

Ship breaking has earned a good reputation for
being a profitable industry in Bangladesh but there
are a number of environmental and human health
hazards. Most of the oil pollution caused by
discharging the wastes of the scrapped ships
directly into its adjacent areas which are
ultimately draining into the Bay of Bengal. Oil
residues and the other refuses are being spilled,
mixed with the sea water and left floating along the
entire seashore which may cause serious
environmental damage (Barua, 2006). We are
aware of disastrous oil spills including the
following accidents involving oceanic
supertankers: Louisiana Oil Spill 2010; the
Prestige-Spain in November 2002; the Torrey
Canyon, which ran aground in 1967; the Metula,
which wrecked in 1973 in the Strait of Magellan;
English Channel in 1978; the Exxon Valdez, which
ran onto a reef in Prince William Sound in southern
Alaska in 1989; and the Braer, which spilled 93,000
tons (84,000 metric tons) of crude oil off the
Shetland Islands of Scotland in 1993 (Source:
science.jrank.org).
Petroleum hydrocarbon degrading bacteria and
fungi are widely distributed in marine, freshwater
and soil habitats. Similarly, hydrocarbon
degrading cyanobacteria have been reported
(Lliros et al., 2003 and Chaillana et al., 2004),
although contrasting reports indicated that
growth of mats built by cyanobacteria in the Saudi
coast led to preservation of oil residues (Barth,
2003). Typical bacterial groups already known for
their capacity to degrade hydrocarbons include
Pseudomonas, Marinobacter, Alcanivorax, Microbulbifer,
Sphingomonas, Micrococcus, Cellulomonas, Dietzia and

*Corresponding author -Email: anwarmn51@yahoo.com

144 DHAR
Gordonia groups (Brito et al., 2006). Molds belonging
to the genera Aspergillus, Penicillium, Fusarium,
Amorphoteca, Neosartorya, Paecilomyces, Talaromyces,
Graphium and the yeasts Candida, Yarrowia and Pichia
have been implicated in hydrocarbon degradation
(Lliros et al., 2003).
Recently, there is an increased interest in
promoting environmental methods in the process
of cleaning oil-polluted sites. Bioremediation offers
a very feasible and an effective technology for
treatment of oil pollution. The majority of the
molecules in the crude oil and refined products are
biodegradable by hydrocarbon degrading bacteria
and fungi which are widely distributed in marine,
freshwater & soil habitats. Fungal bioremediation
have been successful for clean up of petroleum
hydrocarbon which has versatility. The present
study focus on fungal bioremediation and was
undertaken to isolate, identify and characterize
petroleum hydrocarbon degrading fungi from
contaminated soil and water of ship breaking yard
and also to evaluate their biodegradation
efficiencies.
MATERIALS AND METHODS
Sampling site and collection of samples
Soil and water of ship breaking areas are
continuously polluted with petroleum
hydrocarbon. Samples were collected from
petroleum hydrocarbon polluted soil and water of
these ship scraping areas of Bhatiari and Kumira
coast under Sitakunda Upazilla of Chittagong
district. 20 different sites were selected to get fair
idea about hydrocarbon utilizing microorganisms.
Soil and water samples of the contaminated sites
were collected by sterile equipments, aseptically
bottled and properly labeled. Proper precautions
were taken to avoid contamination. The time, date,
location of the sample, pH, temperature of the
sampling sites was recorded. Samples were
preserved carefully in refrigerator at 4 °C before
and after microbial analysis.
Isolation of fungi
Three different kinds of media were used for the
isolation and purification of fungi from the
collected samples. Potato dextrose agar (PDA) and
Sabouraud Dextrose agar (SDA) and Czapek Dox
agar (Thom and Raper, 1945) medium were utilized
for isolation of fungi. Tetracycline was added to
prevent bacterial growth and permitted selective
ET AL.
isolation of yeasts and moulds (Walker and
Colwell, 1976; Paul and Clark, 1988; Harrigan and
McCance, 1990). Sterile physiological saline i.e.
0.85% (w/v) sodium chloride was used as diluent
for inoculum preparation. On the basis of colony
morphology different colonies were selected and
the isolated fungi were purified through repeated
plating method. Well separated pure cultures of
fungi were then obtained. Purity was further
confirmed by microscopic observation.
Identification of oil degrader
Oil agar medium was prepared according to the
mineral salts medium (MSM) composition of Mills
et al., (1978) as modified by Okpokwasili and
Okorie; (1988). The medium was prepared by the
addition of 1% (v/v) sterilized crude oil to sterile
MSM, which has been cooled to 45oC under aseptic
condition. Tetracycline was added to prevent
bacterial growth.
Seven different types of media were prepared for
characterization of the selected isolates. These
media are PDA, Czapek Dox agar, Winsted agar,
Bushnell Hass agar and three types of modified
Czapek Dox agar from which sucrose was replaced
by mobil, diesel and octane respectively. The above
mentioned media were prepared and autoclaved at
121°C for 20 minutes. In case of petroleum
hydrocarbon containing media, filtered sterilized
petroleum hydrocarbons were mixed with media
after autoclaving the media. Cultural and
morphological characteristics of the isolates were
examined after 7-10 days of incubation.
Redox reagent decolourization test
A modified method of Desai et al. (1993) was utilized
for the bio degradation test. Isolates were grown
in Czapek agar. Two agar plugs 1-2 cm each of a
pure growth of each isolate were inoculated into
Bushnell Hass broth incorporated with sterile
crude oil 1% v/v, redox indicator (2,6-dinitrophenol
indophenol) 2% and Tween 80 0.1%. The inoculated
broth was incubated at room temperature (26 ±
2°C) with constant shaking at 180 rev/ minute. The
aliquots in the flasks were monitored daily for
colour change from deep blue to colourless.
Greasy spot test (Williams, 1994)
Bushnell-Hass mineral salt supplemented with 2%
filtered sterilized hydrocarbon was used. Brown
paper bag or normal paper was utilized in this
experiment. The degradation of hydrocarbon was
 Biodegradation of Petroleum Hydrocarbon by Two Aspergillus spp. and Two Penicillium spp.
estimated by comparing the circumference of
greasy spots of treatment with control tubes after 5,
7, 10 days of incubation period.
The rates of degradation was calculated by using
the following equation:
Percentage of degradation (%)=
Diameter of control spot - Diameter of test spot]
X 100
Diameter of control spot
Estimation of petroleum hydrocarbon degradation
The isolates were cultured in three sets of 150 mL
conical flask containing 25 mL of mineral salt
medium (Mills et al., 1978). The first and second sets
of conical flasks were supplemented with 0.2% of
kerosene and diesel respectively while the third set
was supplemented with 0.16% of octane after
sterilization (Juwarkar and Khirsagar, 1991). The
cultures were maintained at 150 rpm for 7 days at
3 oC. After 7 days of incubation cell free culture
broth was extracted with three volumes of toluene
and extract was made up to 10 mL and the OD
(optical density) was measured at 420 nm. The
percentage of degradation was calculated from
standard value. The standard value was obtained
from the absorbance at 420 nm of toluene having
specific amount of hydrocarbon.
The percentage of degradation was calculated by
the following equation:
Percentage of degradation (%) =
Standard absorbance -treatment absorbance
x 100
standard absorbance
RESULTS AND DISCUSSION
General descriptions of the collected samples are
given in Table 1. The pH of the collected sample had
been found in the range of 3.9 to 8.7 while the
temperature was found in the range of 26°C to 32°C.
From twenty petroleum contaminated soil and
water samples, 18 fungal colonies were isolated.
Among them, four fungal isolates were selected
presumptively as petroleum hydrocarbon utilizers
on the basis of their growth on oil agar medium
supplemented with petroleum hydrocarbon as the
sole source of carbon and energy. Selected isolates
were designated as S5bC2, S8C2, S11C3 and S11C5.
The isolates were provisionally identified and
found to be closely related to the species Penicillium
145
fuscum (S5bC2), Penicillium waksmani (S8C2),
Aspergillus candidus (S11C3) and Aspergillus fumigatus
(S11C5) while compared with the standard
description given in the “Manual of Soil Fungi”
(Gilman, 1957).
Redox reagent decolourization test
All the isolates confirmed as true petroleum
hydrocarbon utilizers by their ability of complete
decolourization of Redox reagent (2,6-dinitrophenol
indophenol). The isolate Penicillium waksmani (S8C2)
decolourized the reagent with 5 days, indicating
that it is the best petroleum degrader. S5bC2
(Penicillium fuscum) took 7days for decolourization.
Other two isolates took 10 days for
decolourization.
Cultural and morphological characterization of the
isolates on different hydrocarbon containing solid
media
The isolates were cultured on different carbon
source (including petroleum hydrocarbon
containing media) where marked cultural and
morphological differences were observed. On PDA
and Czapek Dox agar all isolates showed abundant
growth indicating that they preferably use
carbohydrate or sugar as their carbon source. Well
developed colonies, compact and highly branched
mycelia, high level of sporulation with in shortest
period of incubation, well organized fruiting body,
normal size conidiophores, dense spores masses,
normal size and shaped individual conidia were
observed on both media.
On Winsted’s media the isolates S11C5
(Aspergillus fumigatus) showed good growth
indicating efficient utilization of carboxymethyl
cellulose (CMC). Penicillium fuscum (S5bC2) and
Aspergillus candidus (S11C3) showed moderate
growth.
In Bushnell Hass medium where kerosene was
the sole source of carbon the isolates S11C5
(Aspergillus fumigatus) showed relatively good
growth. The isolate S5bC2 (Penicillium fuscum)
showed moderate growth.
The isolate S5bC2 (Penicillium fuscum), S11C3
(Aspergillus candidus) and S11C5 (Aspergillus fumigatus)
exhibited good growth on Czapek agar having
diesel as carbon source. The isolate S5bC2
(Penicillium fuscum) and S11C5 (Aspergillus fumigatus)
showed relatively good growth on Czapek Dox
agar containing octane (as sole C and energy source)
where as the other two showed scanty growth. All
146 DHAR ET AL.

Table 1. Date, time, type, description, pH and temperature of the collected samples

Sample Date and Type of Description pH Tempe-

No. time of sample rature (°C)
collection

1. 12-01-09 Soil Soil taken from about 50 feet away from the 5.24 27
10.05 am center of the ship breaking site at Bhatiari.

2. 2-01-09 Soil Soil taken from a canal at the ship breaking yard 5.53 26
10.20am which was dry at the time of sample collection.
3. 07-02-09 Soil Soil taken from oil spilled ground i.e. Just 4.2 30
09.30 am under a ship which was being broken at the
time of collection.
4. 07-02-09 Water Sample taken from a stagnant water 3.9 32
10.00 am reservoir from the ship breaking yard.
5. 20-03-09 Soil Soil taken from a engine room of 6.2 29
1.00 pm a ship breaking yard.
6. 20-03-09 Soil Engine oil spilled ground. 7.2 29
1.20 pm
7. 20-03-09 Soil Soil taken from the shore side portion of 7.1 30
1.35 pm the ship breaking yard.
8. 20-03-09 Soil Upper portion of the soil taken from the ground 7.3 30
1.50 pm where unused oil was collected in drums.
9. 20-03-09 Water & Taken from the shore side of the yard. 6.4 30
2.10 pm Soil
10. 22-04-09 Soil Soil taken from 1-2 inch beneath the surface 8.7 32
1.30 pm ground where unused oil was collected in drums.
11. 22-04-09 Soil Taken from the 'Kwailla' canal from 7.4 31
1.40 pm Madam Bibir Hat at Bhatiari.
12. 22-04-09 Soil & Taken from the bank of 'Kwailla' canal from 6.5 30
1.55 pm Water Madam Bibir Hat at Bhatiari.
13. 22-04-09 Soil Taken from a black oily and viscous ground. 7.6 31
2.12 pm
14. 22-04-09 Soil Greasy, black oil spilled ground 6.8 31
2.25 pm
15. 22-04-09 Water Canal in front of GlaxoSmithKline factory which 6.5 29
2.35 pm has linked with the ship breaking yard.
16. 22-04-09 Soil Surface soil taken from a moist, damp site which 7.1 31
2.50 pm gives characteristics kerosene and diesel like smell.
17. 22-04-09 Soil Soil taken from 2-3 inch beneath the surface of a 7.3 29
3.05 pm moist, damp site, which gives characteristics
kerosene and diesel like smell.
18. 22-04-09 Soil Surface portion taken from a sandy damp site. 6.8 32
4.10 pm
19. 22-04-09 Soil Deep portion taken from a sandy damp site. 6.5 30
4.30 pm
20. 22-04-09 Soil Soil taken from a site where engines 6.4 31
4.42 pm have been gathered

isolates except S8C2 (Penicillium waksmani) showed
moderate to relatively good growth on Czapek agar
containing mobil as the sole source of carbon and
energy. S8C2 (Penicillium waksmani) failed to grow on
mobil. Moreover, S8C2 (Penicillium waksmani)
showed scanty growth in all other media where
petroleum hydrocarbon were used as C and
energy source.
In cases of scanty growth on petroleum
hydrocarbon (kerosene, diesel, octane or mobil), the
isolates showed slow growth rate, sharp decrease
of mycellial branching, prolonged sporulation
time, decreased rate of growth and shortened
conidiophore size. Spore mass density also lowered
and conidia faded from its normal colour. The
above facts indicate that, the isolates can utilize
 Biodegradation of Petroleum Hydrocarbon by Two Aspergillus spp. and Two Penicillium spp. 147

Table 2. Showing the diameter of the oil spots after 5, 7 and 10 days of incubation in Mineral salt medium
supplemented with kerosene, diesel and octane.

Isolate Diameter of the greasy spot (cm)

5 days 7 days 10 days

Kerosene Diesel Octane Kerosene Diesel Octane Kerosene Diesel Octane

S5bC2 1.2 1.3 1.7 1.0 1.2 1.1 0.9 0.9 0.7
(55.55%) (40.90%) (32%) (61.53%) (45.45%) (56%) (65.38%) (57.14%) (70.83%)
S8C2 1.3 1.5 1.7 1.0 1.0 1.1 0.9 0.7 0.8
(51.85%) (31.81%) (32%) (61.53%) (54.54%) (56%) (65.38%) (66.67%) (66.67%)
S11C3 1.3 1.6 1.2 0.8 1.0 1.0 0.6 0.9 0.9
(51.85%) (27.27%) (52%) (69.23%) (54.54%) (60%) (76.92%) (57.14%) (62.5%)
S11C5 1.4 1.2 1.8 1.3 1.0 1.2 1.0 0.9 0.6
(48.14%) (45.45%) (28%) (50%) (54.54%) (52%) (61.53%) (57.14%) (75%)
Control 2.7 2.2 2.5 2.6 2.2 2.5 2.6 2.1 2.4
(100%) (100%) (100%) (100%) (100%) (100%) (100%) (100%) (100%)

Table 3. Percentages of Kerosene, diesel and octane
degradation after 7 days of incubation (absorbance at
420 nm)
Isolate Percentage of Degradation (%)
Kerosene Diesel Octane
S5bC2 58.13 50.70 53.23
S8C2 60.50 59.83 51.61
S11C3 47.67 56.34 53.22
S11C5 41.86 45.07 61.29
petroleum hydrocarbon but comparatively slowly
as compared to carbohydrate substrates.
Determination of petroleum hydrocarbon
degradation by greasy spot test
The greasy spot test was performed to evaluate the
relative ability of the fungal isolates to breakdown
supplied petroleum hydrocarbon. There was a
sharp decrease of the diameter of the greasy spot
over time. The longer the incubation period, the
smaller the diameter of the greasy spot. According
to the results of this test, S11C3 (Aspergillus candidus)
was found to be the best kerosene degrader. The
isolate S8C2 (Penicillium waksmani) was found to be
the highest degrader of diesel and the isolate S5bC2
(Penicillium fuscum) showed highest octane degra-
dation. Highest (76.92%) kerosene degradation was
recorded for S11C3 (Aspergillus candidus). Diesel was
also well degraded by the isolates. The isolate S8C2
(Penicillium waksmani) was found to be the highest
(66.66%) degrader of diesel within 10 days of
incubation. A highest (70%) octane degradation
was recorded for S5bC2 (Penicillium fuscum) (Table 2).
Specific estimation of petroleum hydrocarbon
degradation by spectrophotometric method
For more specific estimation of petroleum
hydrocarbon degradation spectrophotometric
method was used. By this method, S8C2 (Penicillium
waksmani) was found to be the best degrader of
kerosene and diesel. The highest degradation of
octane was recorded for S11C5 (Aspergillus
fumigatus). All the isolates showed moderate (above
40%) degradation of kerosene, diesel and octane.
(Table 3) The peak kerosene degradation (60.5%)
was recorded for S8C2 (Penicillium waksmani).
Highest (59.85%) degradation of diesel was showed
by S8C2 (Penicillium waksmani). Octane was also
found to degraded fairly well by the isolates. S11C5
(Aspergillus fumigatus) was the highest (61.29%)
degrader with in 7 days. (Table 3).
The ability of Aspergillus spp. and Penicillium spp.
in degrading petroleum hydrocarbons is well
known (Bartha & Atlas, 1977). The results of the
above two degradation estimation tests suggest
that the selected isolates can utilize petroleum
hydrocarbon as the sole source of carbon and
energy, in the presence of inorganic nutrients
which supply nitrogen and phosphorous.
Although no organic nutrient was added, the
organisms showed significant amount of
petroleum hydrocarbon degradation. As it is
proved in the laboratory scale that inorganic salts
is sufficient to support growth of the oil degrader,
there is a high probability of showing such
phenomenon in natural habitat. We can conclude
that environmental modification like inorganic
nutrient amendment in the pollution sites
148 DHAR
(Bhatiari, Kumira etc.) may be useful for
bioremediation and protection of these habitats
from petroleum pollution.
The isolates when cultured on solid media
having petroleum hydrocarbon as sole source of
carbon and energy showed moderate to scanty
growth even after prolonged incubation period. But
the same isolates when cultured on liquid media,
showed good growth and fair rate of oil
degradation. The limitation of using solid media
was reported by Walker and Colwell (1976). From
the mentioned phenomenon of differences in
growth pattern in solid and liquid media, suggest
the use of liquid media instead of solid media for
isolating the oil degrader.
From the present study it is revealed that fungi
of the sampling sites have a potentiality of
degrading petroleum hydrocarbon pollutants. So,
they can be used for the bioremediation of the
contaminated sites. Extensive degradation of
petroleum pollutants is generally accomplished by
mixed microbial population, rather than single
microbial species. After Investigation of optimal
conditions and further investigation on real
contaminated soils, where possible, under field
conditions; the fungal population could be used for
bioremediation.
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