CHAPTER TWO

METHODOLOGY

2.1. MATERIALS

2.1.1. Glassware

These include microscopic slides, pipettes, McCartney bottle, measuring cylinders, conical
flasks, bacteriological test tubes, beakers.

2.1.2. Consumables

They include Paper tape, cotton wool, petri dishes, microtiter plates, filter paper.

2.1.3. Equipment

These include oven, autoclave, incubator, weighing balance, microscope and fridge.

2.1.4. Reagents

These include 70% ethanol, iodine, safranin dye, crystal violet, distilled water, immersion oil.

2.1.5. Media Used

They include nutrient agar, mannitol salt agar, nutrient broth, Mueller Hinton agar, DNase agar
and glycerol.
2.2 METHODS

2.2.1 Sterilization

Sterilization is the process of eliminating all microorganisms from a surface or object, whereas
disinfection is the process of lowering the microbial population to a level deemed safe for the
general public health.

Laboratory work bench, using cotton wool soaked in 70% ethanol to disinfect light switches,
door handles, workbenches, and other surfaces in the lab. To sanitize the inoculating loops, they
were heated to a red-hot temperature using a Bunsen burner flame. For the purposes of
conducting bacteriological processes, keeping isolates, and pouring media, an aseptic
environment was maintained using a Bunsen burner flame. Media and glassware were
autoclaved for 15 minutes at 121°C and 15psi to disinfect them.

2.2.2. Media Preparations

The required quantity of the medium was weighed into jar according to the manufacturer’s
instruction and appropriate volume of distilled water was added. The solution was homogenized
using hot plates and sterilized in the autoclave at 1210C and 15psi for 15 minutes. The media
were allowed to cool to 450C and pour aseptically, the plates were allowed to solidify and kept
in the oven at 370C overnight for the sterility test of the media.

2.2.3. Sample Collection

Archived samples of Staphylococci species, collected from giant rats in Ile-Ife town were
analyzed.

Gram positive bacteria appeared purple against the background while Gram negative bacteria
appeared punk. Staphylococci appear round, purple and irregular clusters under light microscope,
USA 300 and M19 was used as the positive control.
[Link]. Biochemical Test

Biochemical tests are designed to identify and categorise microorganisms according to their
metabolic characteristics, the roles of their enzymes, and their ability to perform particular
biochemical reactions.

[Link].1. Gram Staining

The purpose of the test is to distinguish bacteria as either Gram-positive or Gram-negative based
on how their cells absorb color. Gram-positive bacteria appear violet because they have a thick
peptidoglycan layer in their cell walls that holds onto the primary stain, crystal violet. In contrast,
Gram-negative bacteria have a thinner peptidoglycan layer that cannot retain the crystal violet;
they lose the stain when treated with a decolorizer like ethanol. These bacteria then take on a red
or pink color from the counterstain, safranin.

Reagents

1. Crystal Violet ( Primary Stain )

2. Safranin ( Secondary Stain )

3. Decolorizer ( E.g Ethanol)

4. Iodine Solution/ Gram’s Iodine ( mordant that fixes crystal violet to their cell wall)

5. Water ( Preferably a washing bottle )

Procedures

a. A smear of the suspension was prepared on a clean slide using a loopful of culture that was
less than 24 hours old.

b. The smear was allowed to air dry and then was heat-fixed.

c. Crystal violet was applied and left on the smear for 30 seconds to 1 minute, then it was
gently rinsed with water.

d. The smear was flooded with Gram’s iodine for 1 minute and then gently washed with water.
e. The slide was treated with 95% alcohol (or acetone) for about 30 seconds to decolorize,
followed by a gentle rinse with water.

f. Safranin was applied for about 1 minute, then the slide was rinsed with water.

g. The slide was allowed to air dry and then was observed under a microscope.

USA 300 was used as the Gram-positive control and appeared as violet-colored clusters.

[Link].2. Catalase Test

This test is used to detect the presence of the catalase enzyme in bacteria, which breaks hydrogen
peroxide down into water and oxygen. It is commonly employed to differentiate between
Staphylococcus and Streptococcus species.

Procedures

A drop of hydrogen peroxide was placed on a clean, grease-free microscope slide, and a single
colony from an 18–24-hour-old pure culture grown on nutrient agar was added. The formation of
bubbles, indicating oxygen release, showed that the isolate produced the catalase enzyme. The
absence of bubbling indicated that the isolate lacked catalase activity. USA 300 was used as the
positive control.

[Link].3. Coagulate Test

The coagulase test is used to detect coagulase-positive staphylococci, such as Staphylococcus

Procedures

I． 0.5 ml portion of plasma was dispensed into small test tubes.

II．single colony from a 24-hour culture was introduced into 0.5 ml of plasma to create a

milky suspension.
 III．0.5 ml of plasma was dispensed into a separate tube without any microbial inoculation

to serve as the negative control.

IV．USA 300 was added to a tube and used as the positive control.

V．Tubes were incubated at 37°C for 18–24 hours.

Any level of clot formation is considered a positive result. In many cases, the plasma forms a
firm gel that stays in place when the tube is tilted or inverted, though clots may sometimes
appear as floating masses within the fluid. USA 300 was used as the positive control, while a
tube containing 0.5 ml of inoculated plasma served as the negative control.

[Link].4. DNase Test

The test is designed to detect bacteria that produce the enzyme deoxyribonuclease (DNase),
which enables them to degrade DNA. It is commonly used to distinguish Staphylococcus aureus
from other Staphylococcus species.

Procedures

A single colony from an 18–24-hour-old culture on nutrient agar is streaked onto DNase agar
and incubated at 37°C overnight. After incubation, the plate is flooded with 1N HCl, and the
excess acid is poured off. The plate is then examined for the presence or absence of a clear zone
around the bacterial growth. A clear zone indicates that the microorganism produces DNase,
while the absence of a clear zone suggests it does not produce the enzyme. Staphylococcus
aureus is a strong DNase producer. USA 300 is used as the positive control.

[Link].5. Oxidase Test

The oxidase test is used to identify bacteria that produce the enzyme cytochrome c oxidase,
which facilitates the transfer of electrons from cytochrome to oxygen, leading to the oxidation of
the reagent tetramethyl-p-phenylenediamine into a colored compound. This test is commonly
used to identify Staphylococcus species, as they are oxidase-negative. It also helps distinguish
Staphylococcus from other Gram-positive bacteria that are oxidase-positive, such as
Pseudomonas aeruginosa.
Procedures

Single colony of pure 18-24 hours old culture on nutrient agar plate was rubbed on filter paper
that as already get stained with oxidase reagent and observed for any color change. Change in
color of the rubbed colony to blue indicates that the microorganism produces cytochrome c
oxidase enzyme and the absence of color change shows that the microorganism dos not produce
cytochrome c oxidase enzyme.