microbiology group assignment
DESCRIPTION
assignmentTRANSCRIPT
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