For Abstract:

1. Aim : To isolate the Coagulate-positive Staphylococcus aureus bacteria on the door handles of St. George’s University buses.

2. Background :

Method:

This study will be conducted on the St. George’s University campus, using sterile swabs to

obtain samples from the door handles of frequently travelled buses on the school grounds. These

samples will be transferred to the laboratory so that the bacterial population can be inoculated

and further investigated.

Mannitol salt agar (MSA) will be used to grow the bacteria as it is both selective and differential

and will allow Staphylococcus aureus to be identified from other bacterial species. In the

laboratory, the microorganisms from the sample will be transferred to the nutrient agar by rolling

the swab gently on a portion of MSA. The aseptic technique will then be used to streak the

bacteria from the original area which will allow single, pure colonies of bacteria to be isolated

from the sample. Columbia agar and blood agar should be prepared in addition to the Mannitol

salt agar. The Columbia agar, which is a general purpose agar, will be used as a control to

evaluate the other bacteria present in the sample. Mannitol Salt agar only supports the growth of

Staphylococci bacteria due to its concentration of Sodium Chloride (NaCl). Columbia agar, on

the other hand, will allow any fastidious bacteria present in the sample to grow and can be used

to compare and contrast the colony morphology of the other bacteria to that of the Staphylocci

bacteria on the MSA. The blood agar plate will be used as part of several confirmatory tests to

isolate Staphylococcus aureus from other Staphylococci strains.

After inoculation, the MSA and Columbia agar plates will be incubated at 37o Celsius for 24

hours, ensuring that sufficient Oxygen is available for growth of the bacteria. After incubation,

the colonies on the MSA plate will be observed and Staphylococcus aureus will be identified by

its unique appearance (colony morphology and color) on the Mannitol Salt agar. If other

Staphylococci colonies show identical characteristics to Staphylococcus aureus, further

conclusive tests should be performed.

Conclusive tests for Staphylococcus aureus:

Certain strains of Staphylococci bacteria may show similar morphology to the Staphylococcus

aureus on Mannitol Salt agar. To effectively determine which colony belongs to Staphylococcus

aureus, additional tests need to be performed on the analogous bacterial colonies. Two such tests

can be used for the identification of Staphylococcus aureus: DNase (deoxyribonuclease) test and

the coagulase test. Both are incredibly useful and effective tests for distinguishing

Staphylococcus aureus from other bacteria and may be used independently or in sequence, the

latter, according to David et al. (2010), being much more effective.

Coagulase Test

The coagulase test is one of the effective and commonly used tests to identify Staphylococcus

aureus, being 98.1% specific and 98.7% sensitive (Tiwari et al., 2008). There are two types of

coagulase tests that can be used: The tube coagulase test and the slide coagulase test. Both tests

will be described as possible procedures that can be utilized in this laboratory determination of

Staphylococcus aureus.

1. Tube Coagulase Test

This coagulase test is more widely used than the slide coagulase test to identify Staphylococcus

aureus. The first step of this procedure is to isolate the apparent Staphylococcus aureus colonies

on the Mannitol salt agar. To do this, a single colony showing the morphology of Staphylococcus

aureus must be transferred from the MSA and inoculated in a liquid nutrient broth. After

incubation of the bacteria, three separate test tubes will be prepared using 1ml of 1/10 of diluted

rabbit plasma for each tube. Two of the test tubes will be used as control for the experiment. One

tube will be labeled “negative control” and filled with 0.2 mL of sterile nutrient broth. The other

test tube will be labeled “positive control” and in this tube, 0.2 mL of pure, known

Staphylococcus aureus will be added. The final tube will be used as the “test” and bacteria from

the broth culture will be added to this tube. The test tubes will be incubated at 37o Celsius,

observing the suspensions every half hour over a period of 4 hours. After four hours, the test

tubes will be observed for positive and negative results.

2. Slide Coagulase Test

The slide coagulase test is another type of coagulase test that can be performed. Like the tube

coagulase test, the colonies must first be isolated from the Mannitol Salt agar, this time using a

blood agar plate. After incubation for at least 24 hours, the bacteria on the blood agar plate will

be transferred to slide, thus the name (slide coagulation test) of the procedure. To a sterile slide,

two drops of water should be placed on opposite ends, dedicating one side for “control” and the

other for the “test”. To both water droplets on the slide, bacteria from a single colony on the

blood agar plate should be added and mixed thoroughly using a sterile inoculating loop. To the

test side only, a drop of citrated plasma should be added to the water and bacteria suspension and

the slide should be observed for any agglutination.

DNase Test

The DNase (deoxyribonuclease) test identifies Staphylococcus aureus by analyzing the activity

of a particular enzyme in certain bacteria which degrade Deoxyribonucleic acid. DNA, Sodium

chloride, acid (usually Hydrochloric acid) and nutrients which support growth are all present on

the DNase agar and all aid in detecting the activity of the enzyme.

The colonies to be tested must be retrieved from a blood agar plate. To do this, the colonies from

the Mannitol Salt agar plate which show the characteristics of Staphylococcus aureus will be

cultured beforehand at ideal conditions for growth (37 o C for 24 hours with availability to

Oxygen). The colonies on the blood agar plate will then be inoculated onto the DNase agar using

a sterile inoculating loop and streaked using the aseptic technique. A positive control and a

negative control will be included at this step. The negative control will include a strain of

Staphylococci bacteria that does not exhibit DNase activity, such as the Staphylococcus

epidermidis. The positive control will be a pure culture of known Staphylococcus aureus, the

bacteria that will be identified. All of the plates will be incubated at 37 o C for 24 hours. After 24

hours, the plates will be flooded with Hydrochloric acid and observed after 2 minutes to detect

the Staphylococcus aureus bacteria.

Expected Findings

Things that might be helpful to include:

Notes:

The other tests should only be done if there are multiple colonies with the same

morphology and colour as Staph. aureus

Although not commonly done, both the slide coagulase test and tube coagulase test can

be done to identify Staphylococcus aureus as it gives positive results for both tests,

unlike other Staphylococci strains.

Results for MSA: Staphylococcus aureus -> Medium-sized yellow colonies, surrounded by yellow zones

Results for DNase: After application and penetration of hydrochloric acid into the medium, DNase positive organisms such as Staphylococcus aureus will be surrounded by clear zones of depolymerized DNA while the medium further away from the inoculation band will be opaque and whitish due to polymerized DNA. Colonies of DNase negative organisms will not show any clearing around the colonies

Results for Coagulase tests: (look it up)

Expected Findings:-

After innoculation using Mannitol salt agar plates:-

Plate No:- Streak Plates

# of colonies Size of colonies Colour of

colonies

Spreading of

colonies

Translucent/

opaque

1

2

3

4

5

6 (control)

Repeat using Blood Agar plates

Results:-

1. For MSA : Staphylococcus aureus -> Medium-sized yellow colonies, surrounded by

yellow zones

Coagulase test after incubation:-

Specimen

Nos:-

Slide Test

Observations

Tube Test

Observations

Positive Negative “test” “negative control” “positive control”

positive negative positiv

e

negative positive negative

1

2

3

4

5

6 (control)

For “slide” test: - Use blood agar plates

For “tube” test: - Use mannitol salt agar plates

Results:-

1. Tube test:-

(postive) – Clotting of the rabbit plasma

( negative) – No clotting

2. Slide test:-

(positive)- forms clumps that will form a suspension in coagulate plasma

(negative)- when colonies mix homogeneously in solution

DNase test using after incubation :-

Samples after being

immersed in Hydrochloric

Acid

Observations Positive Negative

1

2

3

4

5

Positive control

Negative control

Transfer colonies from blood agar plate onto the DNase agar to be innoculated

Results:-

1. Positive test - organisms will be surrounded by clear zones of depolymerized DNA

while the medium further away from the inoculation band will be opaque and whitish

due to polymerized DNA.

2. Negative test : - Organisms will not show any clearing around the colonies.

Note :- The other tests should only be done if there are multiple colonies with the same morphology and colour as Staph. Aureus. Although not commonly done, both the slide coagulase test and tube coagulase test can be done to identify Staphylococcus aureus as it gives positive results for both tests, unlike other Staphylococci strains.

Limitations:-

Firstly, when using BD mannitol salt agar, plates should be incubated for 48 to 72 hours

ideally for all the staphylococcal species to be seen. Secondly, other staphylococcus species

besides S. aureus may dislay a positive reaction to mannitol. In order to confirm the presence of

S. aureus, other tests need to be performed. Thirdly not all species examined may have a

biochemical nature that matches the pattern of a known genus and species. Fourthly, there is a

possibility that auto agglutination may occur when doing the conclusive test. Fifthly, in order to

produce accurate results, slide tests need to be read quickly. Generally, the tube test produces

more accurate results than the slide test. The Coagulase Test is rarely used for identifying

unknowns. Lastly, mannitol salt agar only supports the growth of staph due to its concentration

of Sodium chloride.

Challenges:-

Obtaining single colonies when inoculating samples.

Reducing the risk of contamination (eg. Remembering to constantly sterilize

inoculating loop).

Pictures:

FIGURE 3. STAPHYLOCOCCUS AUREUS ON MANNITOL SALT AGAR1`~

FIGURE 3. DNASE TEST SHOWING DNASE POSITIVE AND NEGATIVE BACTERIA

FIGURE 5. SLIDE COAGULASE TEST SHOWING POSITIVE AND

NEGATIVE RESULTS

FIGURE 4. TEST COAGULASE TEST SHOWING POSITIVE AND NEGATIVE REULTS