CONVENTIONAL METHODS AND

ONTENTS

TECHNIQUES USED IN BACTERAIL

IDENTIFICATION

SUBMITTED TO:
Dr. Ghazala Naseem

SUBMITTED BY:
Wajiha Iram: 02
Nadia Jabeen: 09
Nosheen Sharif: 28

INSTITUTE OF MYCOLOGY AND PLANT

1
Topic

1- Introduction of bacteria
2- Identification of bacteria

a. Conventional method
i. Morphological characteraization
• Size, color, margin, elevation, form, surface, opacity

• Bacterial shapes

ii. Staining techniques

• Types of stains
• Types of staining
• Simple staining
• Gram staining
• Spore staining
• Capsule staining
• Flagella staining
iii. Biochemical characterizaztion
• Carbohydrate utilization
• Citrate utilization
• Gelatin utilization
• Starch hydrolysis
• Indole test
• MRVP test
• Triple iron & hydrogen sulphide production
• Urea test
• Catalase test
• Oxidase test
• Biochemical kit

iv. Additional non- biochemical tests

• Motility test

References

2
Introduction
Bacteria are a large group of unicellular, prokaryote, microorganisms. Typically a
few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to
rods and spirals. Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic
hot springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic
matter and the live bodies of plants and animals. There are typically 40 million bacterial
cells in a gram of soil and a million bacterial cells in a millilitre of fresh water; in all,
there are approximately five million (5×1030) bacteria on Earth, forming much of the
world's biomass. Bacteria are vital in recycling nutrients, with many steps in nutrient
cycles depending on these organisms, such as the fixation of nitrogen from the
atmosphere and putrefaction. However, most bacteria have not been characterized, and
only about half of the phyla of bacteria have species that can be grown in the laboratory.

Identification of bacteria
Identification of bacteria is done by
• Conventional method
• Advance techniques for microbial identification

Conventional methods
There are three conventional methods for bacterial identification:
• Morphological characterization
• Staining techniques
• Biochemical characterization

1- Bacterial morphological characterization

Morphological characterization of bacteria is generally carried out on the basis of

colony. Following are the key steps for the morphological identification of bacterial
colony.

Size
For the calculation of size of bacterial colony the measurement is taken from four
different sides, the average of four measurements will be the size of colony.

Color
The variation in the color of bacterial colony is not actually seen as in case of
fungus. The colors of bacterial colonies are mostly white, off white, yellow, and
sometimes orange. The bacterial colonies also have pink color in the case of lactobacillus
on blood agar.

3
Form
The shape of bacterial colony is irregular (ameoboidal), circular, ellipsoidal,
spindle (lens), punctiform, filamentous and rhizoidal.

Elevation
Elevation of bacterial colony may be flat, raised, convex, concave,
umbonate(dome shape), pulvinate.

Margin
The margin of bacterial colony is of six types, entire (round), undulate (wavy),
filamentous (rhizoidal), lobate (having lobes), reose (serrate, dentate), curled.

Fig 1. Form, Elevation and Margin of bacterial colony

Surface
The surface of bacterial colony may be either smooth or rough. The smooth
colony in case of bacteria is mostly shiny. The rough colony may be coarse or granular.
The coarse means finely crushed colony as compared to granular colony.

Opacity
The bacterial colony may be transparent and translucent.

4
Culture Reaction Description Culture Reaction Description

This microbe This microbe

forms medium forms slightly
sized colonies gummy/wet
Klebsiella
Escherichia coli with a regular looking colonies
pneumoniae
margin and that are circular,
convex convex with an
elevation entire margin.

Colonies of this
microbe are red Colonies are
Serratia in appearance, Pseudomonas circular, convex
marcescens circular and fluorescens with an entire
have an entire margin
margin

Colonies are
Colonies are
circular, convex
punctiform,
with an entire
Chromobacteriu Micrococcus convex with an
margin and
m violaceum luteus entire margin and
appear purple
appear yellow on
on most
most medium
medium.

Colonies are Colonies are

Enterococcus punctiform, Bacillus large, irregular
faecalis convex with an cereus and flat with an
entire margin. undulate margin

Colony morphology of different bacteria

5
Bacterial shapes
There are three basic shapes that bacterial cells adopt. They are round, rod shaped
or spiral. Round bacteria are referred to as cocci and rod shaped bacteria are known as
bacilli. The term 'bacillus' meaning a rod-shaped bacterium should not be confused with
the genus of bacteria known as 'Bacillus'. The shape of bacterial cells is of fundamental
importance in the classification and identification of bacteria. Although bacteria are of
three basic shapes, they display an astonishing variety of forms when viewed
microscopically.

6
Staining techniques
Staining Bacteria

Types of strains
Basic strains, due to their positive charge will bind electrostatically to negatively
charged molecules such as many polysaccharides, proteins and nucleic acids. Some
commonly used basic stains are crystal violet, safranin and methylene blue. Basic strains
may be used alone (a simple stain) or in combination (differential stain) depending on the
experiment involved. Acids stains bind to positively charged molecules, which are much
less common, meaning acidic strains are used only for special purposes.

Types of staining
• Simple staining
• Gram staining

1- Simple staining:

When a single staining-reagent is used and all cells and their structures stain in the
same manner, the procedure is caIled simple staining procedure. This procedure is of two
types: positive and negative.
• In positive staining, the stain (e.g., methylene blue) is basic (cationic) having
positive charge and attaches to the surface of object that is negatively charged.
• In negative staining, the stain (e.g., India ink, nigrosin) is acidic (anionic) having
negative charge and is repelled by the object that is negatively charged, and thus
fills the spaces between the objects resulting in indirect staining of the object.

Fig. 3 Simple staining of microbial cell

7
Procedure

Step 1. Bacteria from colonies

Clean a glass slide and place a small mark slightly off center using a grease
pencil. By using loop, transfer one small drop of water to the center of the slide,
being careful to be close to but not overlapping the grease pencil mark. Do not
transfer too much water because these drops will have to air dry. Sterilize loop and
touch a single colony and transfer the bacteria to the water droplet on the slide and
mix well. On mixing if see an opaque slurry of bacteria on the slide you have too
many bacteria for effective staining.

Bacteria from broth

Clean a glass slide. Mix the broth containing the bacteria well because the
bacteria may sediment to the bottom of the container. Use a sterile loop and
transfer one or two droplets of bacteria to the center of a cleaned glass slide.

Step 2. Drying

Allow the bacterial smear to air dry. Don’t heat the sample or below it to
hasten drying time because that could force bacteria into the air leading to
contamination and possible infection.

Step 3. Heat fixation

Holding the slide by one edge, pass it slowly through a bunsen burner flame.
Don’t move so slowly that the edge of the slide heat up to uncomfortable levels. This heat
fixation step denatures bacterial proteins causing the cells to stick to the slide while also
killing the bacteria making them safe for the following steps.

Step 4. Staining

Place the stain in a staining rack and cover the smear with the stain of
choice. Allow the stain to work for 30 seconds (some stains may have different
staining times but this time will work well for simple stains). Remove the stain by
rinsing with water from the squeeze bottle and gently blot the stain dry using
bibulous paper. The slide is now ready to look at under the microscope. Because
the bacteria were heat fixed, it will not be necessary to use a cover slip.

8
9
 Fig. 4 Preparation of smear and simple staining

Gram staining / Differential Staining

Bacteria may be conveniently divided into two further groups, depending upon
their ability to retain a crystal violet-iodine dye complex when cells are treated with
acetone or alcohol. This reaction is referred to as the Gram reaction: named after
Christian Gram, who developed the staining protocol in 1884. This reaction, however,
reveals fundamental differences in the structure of bacteria. Electron microscopy shows
that Gram-negative and Gram-positive bacteria have fundamentally different structures,
related to the composition of the cell wall, amongst other things.

Cells with many layers of peptidoglycan can retain a crystal violet-iodine

complex when treated with acetone. These are called Gram-positive bacteria and appear
blue-black or purple when stained using Gram's method. Gram-negative bacteria have
only one or two layers of peptidoglycan and cannot retain the crystal violet-iodine
complex. These need counterstaining with another dye to be seen using Gram's method.

The cell wall of Gram-positive bacteria lies beyond the cell membrane and is
largely made up of pepidoglycan. There may be up to 40 layers of this polymer,
conferring enormous mechanical strength on the cell wall. Other polymers including
teichoic and teichuronic acids also lie in the cell walls of Gram-positive bacteria. These
act as surface antigens.

Fig. 5 Gram-positive bacteria

10
 In contrast to Gram-positive cells, the cell envelope of Gram-negative bacteria is
complex. Above the cell membrane is a periplasm. This area is full of proteins including
enzymes. One or two layers of peptidoglycan lie beyond the periplasm. Gram-negative
bacteria are thus mechanically much weaker than Gram-positive cells. Beyond the
peptidoglycan of the Gram-negative cell wall lies an outer membrane. This has protein
channels - porins - through which some molecules may pass easily. The outer side of the
Gram-negative outer membrane contains lipopolysaccharide.

This provides the antigenic structure of the surface of Gram-negative bacteria and also
acts as endotoxin. It is this that is responsible for eliciting the symptoms of Gram-
negative shock if it gains access to the bloodstream. Porins and Outer Membrane Proteins
(OMPs) act as transporters through the outer membrane.

Fig. 6 Gram-nrgative bacteria

A few medically important bacteria do not stain easily using conventional stains,
and need to be heated to near boiling point in the chosen dye (carbol fuchsin for light
microscopy: rhodamine-auramine for fluorescence microscopy) for at least five minutes.
This is to allow the dye to penetrate the waxy cell walls. Having taken the stain, these
bacteria resist decolourisation with both acids and alcohol, and are known as acid-
alcohol fast bacteria. This is a property of mycobacteria. These include Mycobacterium
tuberculosis, the cause of tuberculosis; a chronic infection. Most common is pulmonary
tuberculosis, affecting the lung. The kidneys may be infected in renal TB, and there is a
rare form of osteomyelitis (bone infection) and meningitis caused by TB. In miliary
tuberculosis, the infection is disseminated through the body.

Another medically important mycobacterium is Mycobacterium leprae, the cause

of leprosy; a chronic infection of the skin and nerves. Nerve damage leads to a loss of
sensation, and ultimately to paralysis. This can lead to tissue damage that can lead to the
loss of fingers and toes.

11
Procedure

Step 1. Bacteria from colonies

Sterilize loop and touch a single colony and transfer the bacteria to the
water droplet on the slide and mix well.

Bacteria from broth

Clean a glass slide. Mix the broth containing the bacteria well because the
bacteria may sediment to the bottom of the container. Use a sterile loop and
transfer one or two droplets of bacteria to the center of a cleaned glass slide.

Step 2. Drying
Allow the bacterial smear to air dry. Don’t heat the sample or below it to
hasten drying time because that could force bacteria into the air leading to
contamination and possible infection.

Step 3. Heat fixation

Holding the slide by one edge, pass it slowly through a bunsen burner flame.
Don’t move so slowly that the edge of the slide heat up to uncomfortable levels. This heat
fixation step denatures bacterial proteins causing the cells to stick to the slide while also
killing the bacteria making them safe for the following steps.

Step 4. Primary stain

Cover the smear with crystal violet and incubate for 30 seconds. Rinse the
dye off with distilled water from squeeze bottle.

Step 5. Mordant
Cover the smear with gram iodine. After 20 seconds, rinse the slide with
distilled water.

Step 6. Decolorization
Rinse the stain with 95% ethanol. This step must be done very carefully.
Hold the slide at 45o angle over the staining rack and rinse with ethanol one drop
at a time. Watch the ethanol as it runs off the slide looking for blue color. Stop
dropping ethanol as soon as no more color is releases and rinse the slide
immediately with water. A few drops of ethanol too many and the gram positive
bacteria will also lose their crystal violet.

Step 7. Counterstain
Cover the bacteria with safranin for 30 seconds. Rinse with distilled water
and blot the slide dry with bibulous paper. Observed under microscope.

12
Spore staining

Bacterial spores (or endospores) are the toughest forms of life known. They are so
resistant to destruction that some scientists have proposed that life arrived on earth when
bacterial spores drifting through space fell to earth. Only G+ cells form spores,
specifically members of the genera Bacillus and Clostridium. Spores are formed by
bacteria to survive during period of deprivation, such as the loss of a food or water
supply. When a spore-forming-bacterium (SFB) senses that tough times are coming a
series of complex events are triggered that lead to the formation of a spore. Because a
thick, tough covering protects the spore, it is difficult to stain. Usually following
procedure is followed for spore staining.

Procedure
1. Using an inoculation loop, prepare a smear of microorganism; allow the smear to
air dry.
2. Heat fix the smear by passing it through the bunsen burner flame a few times.

3. Flood the smear with malachite green.

4. Place the slide on a tall staining rack. Pass the burner flame under the slide until
the stain steams; continue for 5 minutes, replenishing the stain as needed. Do not
allow the stain to boil or completely evaporate.

5. Remove the samples from the heat source and allow the slides to cool.

6. Rinse the slides with water.

7. Flood the sample with safranin for 30-60 seconds.

8. Rinse the slides with water.

9. Blot dry with bibulous paper. Examine under the microscope.

13
 Fig. 7 Bacillus subtilis, spore stain.

Capsule staining
Some bacteria have characteristic surface structures: capsules or flagella and
internal components: endospores, which may have taxonomic value for their
identification. When it is necessary to demonstrate whether or not a particular organism
possesses a capsule, is flagellated, or forms endospores, special staining techniques must
be used.

Many bacteria possess a capsule,

but it is often not visible. A few pathogenic
species, however, such as Streptococcus
pneumonia, Klebsiella pneumonia, and
Clostridium perfringens, have well-
developed capsules that contribute to
virulence by protecting the organisms from
host defense mechanisms, particularly
phagocytosis. Capsular materials often are
antigenic, providing the bacterial cell with
specific immunologic properties by which
they may be identified. Capsules can be
visualized microscopically by using a
simple, nonspecific negative staining
technique.

The negative staining procedure

is less distorting to cells. Stains like
Nigrosin are negatively charged (-) and are
repelled by the negative change (-) of the
bacteria. This prevents them from entering
the cell and staining the cytoplasm. This
technique produces a stained background
which surrounds the unstained microbe. Figure 8. Capsule staining of bacteria

14
 After staining the cells with Nigrosin, the slides are dried, and the slide
preparation is stained with safranin, a dye that does penetrate the cells and stains them.
When viewed under the microscope after this treatment, the bacteria are pink, while their
capusles appear as clear, unstained zones surround them, their outlines demarcated
against the black backgound proved by Nigrosin.

Flagella Staining
Bacterial flagella are composed of protein subunits called flagellin and are distinct
in structure from flagella found on eukaryotic cells. Flagella are anchored to the bacterial
cytoplasmic membrane and cell wall by basal bodies, and are assembled via flagellin
subunits traveling through the basal bodies, then through the center of the flagellum
itself, and are added to the distal end of the appendage. The intrinsic structure of the
flagellum is helical, making propulsion possible.

Flagella are of variable lengths that can extend several times the length of the
bacterium itself, with widths generally between 10 and 50 nm. Due to their narrow width
the best direct method of observing bacterial flagella is by the electron microscope,
because the normal limit of resolution of light microscopy is ~200 nm. For those who do
not have access to electron microscopy, bacterial flagella can be observed via the light
microscope in combination with stains. All flagella stains use mordants, like tannic acid
and potassium alum, to coat and thus thicken the flagellum in order to be within the limits
of size observable by light microscopy.

The Leifson flagella stain method uses tannic acid. For the Leifson flagella stain,
tannic acid and the dye form a colloidal precipitate that when absorbed by the flagellum
causes it to increase in diameter and become colorized, thus amenable to viewing by light
microscopy. The tannic acid-dye complex is more soluble in alcohol than water, and also
more soluble with decreased pH. The alcohol concentration in the Leifson solution is
sufficient to maintain solubility of the components. When the prepared sample is stained,
the alcohol evaporates faster than the water, and the concentration of the tannic acid and
dye increases to cause precipitation, leading to staining of the flagella (Silflow &
Lefebvre, 2001).

Salt concentration also affects the staining, presumably by altering charge of the
tannic acid-dye complex and the flagellum itself. The flagella stain is finicky because
many variables affect the outcome:

• Age of the bacterial culture

• Thickness of the culture on the microscope slide

15
 • Age of the staining solutions,
• pH
• Temperature
• Alcohol concentration
• Dye concentration
• Heat

Procedure

1. Take a prepared slide and using a wax pencil draw a rectangle around the dried
sample. Place slide on staining rack.

2. Flood Leifson dye solution on the slide within the confines of the wax lines. Incubate
at room temperature for 7 to 15 minutes. The best time for a particular preparation will
require trial and error.

3. As soon as a golden film develops on the dye surface and a precipitate appears
throughout the sample, as determined by illumination under the slide, remove the
stain by floating off the film with gently flowing tap water. Air dry.

4. View using oil immersion, at 1,000x magnification, by bright-field microscopy.

Bacterial bodies and flagella will stain red.

Fig. 9 Staining of Vibrio choleraeby using Liefson's flagellar stain method.

16
 Biochemical Tests for Identifying Unknowns

Carbohydrate Utilization
Bacteria produce acidic products when they ferment certain carbohydrates. The
carbohydrate utilization tests are designed to detect the change in pH which would occur
if fermentation of the given carbohydrate occurred. Acids lower the pH of the medium
which will cause the pH indicator (phenol red) to turn yellow. If the bacteria do not
ferment the carbohydrate then the media remains red. If gas is produced as a by product
of fermentation, then the Durham tube will have a bubble in it.

Following carbohydrate tests were performed:

• Glucose (Dextrose) test

• Lactose test
• Sucrose test

A B C

17
 Fig 10. Fermentation results from left to right; A: shows less acid formation than far
right tube, but gas is still made. B: no carbohydrate utilization to produce acid or gas.
C: shows acid was produced as evidenced by the yellow color, and gas was made

Citrate Utilization
Tests for the ability of bacteria to convert citrate (an intermediate of the Kreb’s
cycle) into oxaloacetate (another intermediate of the Kreb’s cycle). In this media, citrate
is the only carbon source available to the bacteria. If it can not use citrate then it will not
grow. If it can use citrate, then the bacteria will grow and the media will turn a bright
blue as a result of an increase in the pH of the media. To inoculate this slant, use the
transfer loop.

18
Gelatin Utilization
This media is used to test if bacteria can digest the protein gelatin. To digest
gelatin, the bacteria must make an enzyme called gelatinase. To inoculate this media, use
a transfer needle to stab the gelatin. After incubating the inoculated media for at least 48
hrs, transfer the tube into a refrigerator. The tube should be completely chilled prior to
observation. If the media is solid after refrigeration then the test is negative (the bacteria
did not digest gelatin). If the media is liquefied even after refrigeration, then the test
result is positive…the bacteria is able to digest gelatin.

A B

Fig.12. A: shows 'Serratia marcescens' which is positive for gelatinase production, as

evidenced by the liquidation of the media. B: 'Salmonella typhimurium' shows negative
results for gelatinase production, as evidenced by the solidity of the media.

19
Starch hydrolysis
This test is used to detect the enzyme amylase, which breaks down starch. After
incubation the plate is treated with Gram’s iodine. If starch has been hydrolyzed (broken
down) then there is a reddish color or a clear zone around the bacterial growth; if it has
not been hydrolyzed then there is a black/blue area indicating the presence of starch.
Simply use inoculating loop to spread bacteria onto plate surface. After the bacteria have
grown, add a few drops of Gram’s iodine to the plate and look for the color immediately
after adding the iodine.

Fig 13. Starch hydrolysis test in E.coli

Indole production
This test is done to determine if bacteria can breakdown the amino acid
tryptophan into indole. SIM media or TSB (tryptic soy broth) is inoculated using a
transfer needle. After incubating the bacteria for at least 48 hours, Kovac’s reagent is

20
added to the media to detect if indole has been made by the bacteria. The development of
a red/pink layer on top of the media is a positive result (the bacteria can breakdown
tryptophan to form indole). Failure to see a red layer is a negative result (indole was not
formed from tryptophan).

A B

FIG 14. A: showing red ring formation is positive for indole production. B: shows a negative
result.

MRVP (methyl red-Vogues Proskauer)

This test is used to determine two things. The MR portion (methyl red) is used to
determine if glucose can be converted to acidic products like lactate, acetate, and formate.
The VP portion is used to determine if glucose can be converted to acetoinThese tests are
performed by inoculating a single tube of MRVP media with a transfer loop and then
allowing the culture to grow for 3-5 days. After the culture is grown, about half of the
culture is transferred to a clean tube. One tube of culture will be used to conduct the MR
test, the second tube serves as the VP test.

A. MR (methyl red) test

21
 Methyl red is added to the MR tube. A red color indicates a positive result
(glucose can be converted into acidic end products such as lactate, acetate, and formate.
A yellow color indicates a negative result, glucose is converted into neutral end products.

B. VP (Vogues Proskauer) test

First alpha-napthol (also called Barritt’s

reagent A) and then potassium hydroxide (also
called Barritt’s reagent B) are added to the VP
tube. The culture should be allowed to sit for
about 15 minutes for color development to occur.
If acetoin was produced then the culture turns a
red color (positive result); if acetoin was not produced then the culture appears yellowish
to copper in color (a negative result).

Triple sugar Iron (TSI) - & Hydrogen sulfide production (H2S)

Looks at fermentation of glucose, lactose, and sucrose and checks if hydrogen
sulfide is produced in the process. Basically a pH indicator will change the color of the
media in response to fermentation, where that color change occurs in the tube will
indicate what sugar or sugars were fermented. The presence of a black color indicates
that H2S was produced. In this media, H2S reacts with the ferrous sulfate in the media to
make ferrous sulfide, which is black. To inoculate, use a needle to stab agar and then uses
a loop to streak the top slated region.

In addition to TSI media, SIM media can be used to determine if H2S is produced.
A black color in the SIM medium following inoculation and incubation indicates that H2S
is made by the bacteria.

SLANT COLOR Interpretation

RED does not ferment either lactose or sucrose
YELLOW ferments lactose and/or sucrose

BUTT COLOR/CONDITION

22
RED no fermentation of glucose
YELLOW some fermentation of glucose has occurred, acid has
been produced
GAS FORMED Seen as cracks in the agar, bubbles, or the entire slant
may be pushed out of the tube.
BLACK H2S has been produced

A B C D E F G H

FIG. 17. Triple sugar Iron (TSI) - & Hydrogen sulfide production (H2S). A: Uninoculated
control; B: Red slant and red butt, no black color i.e., no fermentation of glucose, sucrose or
lactose. No Hydrogen sulfide produced; C: Red slant and black butt i.e., no lactose or
sucrose fermentation, H2S has been produced; D: Red slant with yellow butt i.e., no
lactose or sucrose fermentation, lactose is fermented, no H2S has been produced ; E:
Yellow slant, yellow butt and black coloration i.e., Lactose, sucrose and glucose
fermented, and H2S has been produced; F & G: Yellow slant, yellow butt and lifting

23
and/or cracking of media, no black coloration i.e., Lactose, sucrose and glucose
fermented, H2S has not been produced but gas has been produced; H: Yellow slant,
yellow butt and no lifting and/or cracking of media, no black coloration i.e.,
Lactose, sucrose and glucose fermented, H2S has not been produced nor has gas
been produced.

Urea test
This test is used to detect the enzyme urease, which breaks down urea into
ammonia. Ammonia is a base and thus will raise the pH of the media if it is present. This
change in pH is indicated by a pH indicator called phenol red which is present in the
media. A color change from yellow to bright pinkish-red is positive; lack of color change
is a negative result. Inoculate the liquid media with a transfer loop.

A B C

Fig.19 urease test; A: shows positive reaction; B: shows negative reaction; C: shows un-
inoculated control.

Catalase Test
This test is can be used to detect the enzyme catalase. This enzyme is responsible
for protecting bacteria from hydrogen peroxide (H2O2) accumulation, which can occur
during aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to the
organism. Catalase breaks H2O2 down into water and O2. To perform the catalase test
simply smear a small amount of the test organism onto the lid of a Petri plate/culture
dish. Then add a drop of hydrogen peroxide to the smear. If bubbles become visible
(these would be the O2 bubbling up) then the test is positive and you can conclude that
the organism makes catalase. A lack of bubbles indicates the absence of catalase. *Note,
most aerobic organism make catalase.

24
Fig 21. Bubbling upon the addition of hydrogen peroxide is indicative of the presence of
catalase for this organism.

Oxidase test
To perform this test simply swab
some of your test culture into one of the
boxes on an oxidase dry slide. If a color
change to purple or blue is evident at 30
seconds-1 minute then the result is
positive. It is important that the test is read
by one minute to ensure accurate results
(avoid false negatives and false positives).
This laboratory test is based on detecting
the production of the enzyme cytochrome
oxidase by Gram negative bacteria. It is a hallmark test for the Neiserria. It is also used to
discriminate between aerobic Gram-negative organisms like Pseudomonas aeruginosa
and other Enterobacteriaciae.

Biochemical kit
Because for the identification of a bacteria at specie level 25-50 biochemical tests
are applied so it is laborious and compensated by the use of biochemical kits.
Biochemical kits comprises of;
• wells supplied with chemicals
• saline water for bacterial suspension
• chemicals for reaction

25
Procedure
The chemicals present in the contain sugars and antibiotics solutions. While the
gel, production of nitrate and nitrites, and production of CO2 . This is done with
saline water contains bacterial suspension made with help of spectrophotometer.
Then this suspension was poured in the wells and checks the hydrolysis the help
of different solutions. The most common used solutions are gel, nitrate solution
and VP solution.

All the data from these reactions are checked with help of a catalog which
contains different positive and negative reaction .by this catalog we get a cod
which is universal, this cod will tell us the species of bacteria. All these methods
are conventional methods. Now we use the modern techniques for the
identification of bacteria.

API-20 strip test

O
A L O C H U T I G G M I S R S M A A
N V
culture D D D I 2 R D N E L A N O H A E M R identification
P P
no. H C C T S E A D L U N O R A C L Y A
G
Escherichia
8101 + – + + – – – – + – – + + – + + + + – +
coli
Enterobacter
5B + – – – + – – – – + – + + + + + – – + +
agglomerans
Edwardsiella
8P44 – – + + – + – – + – – + + – – – + – – +
hoshinae

The types of biochemical tests we have explored here can be miniaturized. The
API-20 strip is one example of this type of test. Twenty tests are performed on this strip
by a simple procedure, saving time and money.

26
 • The first test is for the presence of the enzyme β-galatosidase, an enzyme
involved in lactose catabolism.
• The next three reactions (in order, arginine, lysine and ornithine) test for amino
acid decarboxylation. Decarboxylation is shown by an alkaline reaction (red color
of the particular pH indicator used).
• Hydrogen sulfide production (H2S) and gelatin hydrolysis (GEL) result in a black
color throughout the tube.
• A positive reaction for tryptophan deaminase (TDA) gives a deep brown color
with the addition of ferric chloride. Positive results for this test correlate with
positive phenylalanine and lysine deaminase reactions which are characteristic of
Proteus, Morganella and Providencia. The last nine tests are for carbohydrate
fermentation. The carbohydrates tests are (glucose, mannitol, inositol, sorbitol,
rhamnose, sucrose, melibiose, amygdalin and arabinose. Fermentation is shown
by an acid reaction (yellow color of indicator).

Additional Non-Biochemical Tests

Motility test
The motility test is not a biochemical test since we are not looking at metabolic
properties of the bacteria. Rather, this test can be used to check for the ability of bacteria
to migrate away from a line of inoculation thanks to physical features like flagella. To
perform this test, the bacterial sample is inoculated into SIM or motility media using a
needle. Simply stab the media in as straight a line as possible and withdraw the needle
very carefully to avoid destroying the straight line. After incubating the sample for 24-48
hours observations can be made. Check to see if the bacteria have migrated away from
the original line of inoculation. If migration away from the line of inoculation is evident
then you can conclude that the test organism is motile (positive test). Lack of migration
away from the line of inoculation indicates a lack of motility (negative test result).

A B

Fig. 23 A: Shows result for a non-motile bacterium; B: shows result for a motile organism.

27
 Identifying of microorganisms through these types of biochemical tests has been a
routine practice for many decades and the tests necessary to identify certain species of
groups of related species have become standardized. To make these test more convenient,
manufacturers have developed miniaturized versions that decrease the material cost and
make inoculation of the tests much more convenient. However, the tests still need to be
incubated to the prescribed period of time before being read, usually 24-48 hours.

References:
Donald B. 2007. Colony Morphology Resource Type. Microbiology and Molecular
Biology, 11: 12-34.
Fredrickson JK, Zachara JM, Balkwill DL. 2004. "Geomicrobiology of high-level nuclear
waste-contaminated vadose sediments at the Hanford site, Washington state".

Gram HC.1884. "Über die isolierte Färbung der Schizomyceten in Schnitt- und
Trockenpräparaten". Fortschritte der Medizin, 2: 185–9.

Hurlbert RE. 1999. Conclusion of Simple Staining & streaking: negative stain &
characterization of colonies. Microbiology, 1: 12-43.

John Heritage. 2006. Medical Microbiology - A Brief Introduction. Microbiology, 2: 24-

44.
Johnson R and Christine L. 2005. Preparation of smears and simple staining. Laboratory
Experiments in Microbiology, 2: 11-23.

Lourdes PN. 2005. Biochemical Tests for Identifying Unknowns. Microbiology &
Immunology, 1: 12-65.

Mcdowell A and Patrick S. 2005.Evaluation of Nonculture Methods for the Detection of

Prosthetic Hip Biofilms. Clin Orthop Rel Research, 437: 74 –82.

28
 Applied and Environmental Microbiology, 70: 4230–41.
Margaret E, Imbrook HE, Lan WL, Gail C. 1999. staining bacterial flagella easily.
Journal of Clinical Microbiology, 2612-2615.

Rappe MS, Giovannoni SJ. 2003. "The uncultured microbial majority". Annual Review of
Microbiology 57: 369–94.

Whitman WB, Coleman DC, Wiebe WJ.1998. "Prokaryotes: the unseen majority".
Proceedings of the National Academy of Sciences of the United States of America,
95: 6578–83.

William MJH. 2004.Spore Stain Tutorial Resource Type. Microbe library, 1: 23-12.

29