Gram positive and Gram Negative

Clinical microbiology laboratory plays several important roles

in the management of antibacterial infections. Isolation,

identification of pathogenic microorganisms in cultures and

subsequent antibacterial susceptibility testing always assist in

selecting appropriate antimicrobial agent and prevention of

unnecessary complications. The most important primary test to

perform directly on the some special samples such as

cerebrospinal fluid and positive cultures in gram staining which

serves as the most rapid and simplest test to characterize

microorganisms.

The most fundamental technique for classifying bacteria is

the gram stain, developed in 1884 by Danish scientist Christian

Gram. It is called a differential stain because it

differentiates among bacteria and can be used to distinguish

among them, based on differences in their cell wall.

In this procedure, bacteria are first stained with crystal

violet, then treated with a mordant—a solution that fixes the

stain inside the cell (e.g., iodine-KI mixture). The bacteria

are then washed with a decolorizing agent, such as alcohol, and

counterstained with safranin, a light red.

The walls of gram positive bacteria (for example, Staphylococcus

aureus) have more peptidoglycans (the large molecular network of

repeating disaccharides attached to chains of four or five amino

acids) than do gram-negative bacteria. Thus, gram-positive

bacteria retain the original violet dye and cannot be

counterstained.

Gram negative bacteria (e.g., Escherichia coli) have thinner

walls, containing an outer layer of lipopolysaccharide, which is

disrupted by the alcohol wash. This permits the original dye to

escape, allowing the cell to take up the second dye, or

counterstain. Thus, gram-positive bacteria stain violet, and

gram-negative bacteria stain pink.

The gram stain works best on young, growing populations of

bacteria, and can be inconsistent in older populations

maintained in the laboratory (Jrank.org).

It is also one of the most important methods used in the

clinical microbiology laboratory. It is used for initial

assessment of specimens prior to culture and is an important

step in the workup and identification of pathogens isolated from

clinical specimens. Gram stains guide workup pathways and

identification of organisms; thus, they are extremely important

to the accurate processing and workup of specimens.

According to Buckley et. al, (2005), it is therefore highly

likely that the information provided by the Gram staining will

help to assess the adequacy of preliminary diagnosis and

antimicrobial therapy selected after collecting culture

specimens and before final identification of the microorganism.

In recent reports, the impact of Gram staining results on

patient mortality has been documented. On the other hand, there

remains the possibility that Gram staining results do not match

with the final identification of microorganisms. This would

carry a risk leading to inadequate antimicrobial therapy and

potentially affecting patients' clinical course and mortality.

The aim of this mini review is to analyze and discuss the

clinical significance and limitations of reporting Gram staining

results for sample meant for bacteriological investigations.

According to the study of Ramesh CH et al.,

(2010) the bacterial test organisms used in thier study were

two gram-positive species Staphylococcus aureus (ATCC 25923),

Bacillus subtilis (ATCC 6633); gram-negative species Escherichia

coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) grown in

Mueller Hinton agar at 37°C for 24 hours (Merck). Fungal species

was unicellular Candida albicans (ATCC 60192) grown in potato

dextrose agar (Hi Media) at ambient temperature for 72 hours.

They used agar well diffusion method in determining the

aniti microbial activity of methanolic extract of mushrooms,[11]

with slight modification to suit the conditions of this

experiment.They dissolved methanolic extract in

dimethylsulfoxide (DMSO) to a final concentration of 10 mg/mL,

and filter sterilized through 0.45-μm membrane filter. Small

wells (6 mm in diameter) were made in the agar plates by sterile

cork borer. One hundred microliters of the extract of each

isolate of mushrooms was loaded into the different wells.

Concentration of target microbial cells suspensions was adjusted

to 104 cfu/mL. In order to determine the antimicrobial efficacy

of the fractions, aliquot of test culture (100 μL) was evenly

spread over the surface of the solidified agar. Bacteria were

cultured on Mueller Hinton agar; and fungi on potato dextrose

agar. Ampicillin (10 μg/mL) for bacteria, nystatin (100 U) for

fungi were used as positive control and DMSO was used as

negative control for test microorganisms. All the preloaded

plates with respective extract and test organism were incubated

at 37° C, for 24 hours for bacteria and at 27° C. 48 hours for

fungi. After incubation period, zone of inhibition was measured

in millimeters. All the tests were carried out in triplicate and

their means recorded.

 Antibacterial Assay

In the last three decades, the search for natural bioactive

compounds that can serve as antioxidant and antimicrobial agents

had increased tremendously. The reasons for these are increasing

understanding of the harmful nature of reactive oxygen species

(ROS) produced during oxidation processes, harmful nature of

synthetic antioxidant such as butylated hydroxyanisole (BHA) and

butylated hydroxytoluene (BHT) and the increasing resistance

posed by microorganisms to synthetic antibiotics. Extracts from

mushroom have received attention based on their safety and

records of health promotion. Mushrooms produce a wide range of

secondary metabolites with high therapeutic value. Health

promoting properties such as antioxidant, antimicrobial,

anticancer, cholesterol lowering and immunostimulatory effects,

have been reported for some species of mushrooms. Both fruiting

body and the mycelium contain compounds with wide-ranging

antioxidant and antimicrobial activities (Oyetayo, May 2008).

The antioxidative and free radical scavenging properties of

the phenolic content of mushroom methanol extracts have been

reported, suggesting possible protective roles of these

compounds, due to their ability to capture metals, inhibit

lipoxygenase and scavenge free radicals. Recently, Ferreira et

al. reported the antioxidative properties of two mushrooms,

Lactarius deliciosus (L.) Gray and Tricholoma portentosum (Fr.)

Que2 l obtained from northeast Portugal. Moreover, the activity

of the exudates from mushroom mycelia against protozoa such as

the parasite that causes malaria, Plasmodium falciparum and

other microorganisms had been reported. Chinese Shiitake

mushroom (Lentinus edodes) has also been reported to possess

both anti-tumour and antimicrobial properties . In an earlier

study, Suay et al. reported that extracts of more than 75%

polypores mushroom species surveyed showed antimicrobial

activity and 45% of 204 mushroom species inhibited wide variety

of microorganisms. Hence, mushrooms may be a source of new

antimicrobial capable of inhibiting microorganisms that are

resistant to common antibiotics (D.K. Rahhi, 2016).

According to Wasser et. al (2005), the number of mushrooms

on earth is estimated at 140,000 yet only 1400 (10%) are known.

In essence, pharmacological potentials of about 90% of mushrooms

on earth are yet to be exploited. A large number of the unknown

species of mushrooms whose health promoting properties are

unknown reside in Africa and probably in Nigeria. This is

because there are little or no data about them. Most available

data on these mushrooms are on their nutritional compositions

(1,2,22). Most of these mushrooms are still obtained in the

wild. Diverse species of mushrooms are found in Nigeria. Those

that are common belongs to the following species; Termitomyces,

Plerotus, Lentinus, Lenzites, Trametes, Ganoderma etc. The

present study was aimed at assessing antimicrobial and free

radical scavenging properties of four wild mushrooms,

Termitomyces clypeatus, Termitomyces robustus, Lentinus subnudus

and Lenzites species, obtained from Ado Ekiti, Nigeria.