St.

Xavier’s College
Department of Microbiology
MB36152P
Assignment (Total Marks 5)

The answers should be in your own words and not a direct copy paste from the internet. The
assignments will be scanned in the plagiarism detector. Return the completed assignment to
sarkar.kasturi@gmail.com on or before 1.07.2020.

1. Doctor prescribed for a second time ELISA test, as negative result appeared for
antibodies against dengue virus in the first time in a patient suspected with dengue
fever for 5 days. Discuss on the possible reasons. 5

Or

2. During an experiment to isolate normal flora of your skin, you do not find any growth
on the blood agar plate. Give a possible explanation for this. 5

Or

3. During an experiment to isolate normal flora of your skin, you do not find any growth
on the mannitol salt agar plate. Give a possible explanation for this. 5
 DEPARTMENT OF MICROBIOLOGY
ST. XAVIER’S COLLEGE (AUTONOMOUS), KOLKATA
30, MOTHER TERESA SARANI, KOLKATA – 700016

NAME: SOUNAK DAS

ROLL NO.: 3-16-17-0629
REGN. NO.: A01-1112-0877-17
SUBJECT: MB36152P
MODULE: 15
SEMESTER: VI
3. During an experiment to isolate normal flora of your skin, you do not find
any growth on the mannitol salt agar plate. Give a possible explanation for
this. 5

A. Mannitol Salt Agar (MSA): -

Mannitol Salt Agar is a selective media that is used to isolate and identify
Staphylococcus aureus in bacteriological samples. It is used to detect and
enumerate S. aureus in clinical samples, food & dairy products, cosmetics,
pools and water sources.
Components of the media are:
• Proteose peptone – 10.0 gm/l
• Beef extract – 1.0 gm/l
• Sodium chloride – 75.0 gm/l
• D- mannitol – 10.0 gm/l
• Phenol red – 0.025 gm/l
• Agar – 15.0 gm/l
If the media is prepared, the final pH adjusted should be 7.4 ± 0.2 at 25oC.
In this media, peptone and beef extract are primary protein source. Peptone
in this media is obtained from casein (animal source). It provides C and N
along with P, S and other essential minerals. Beef extracts are dehydrated
cow extracts that acts as source of protein and vitamin. High salt
concentration in this media is the most essential component as it only allows
the growth of microbe that can tolerate such conditions. Only
Staphylococcus spp. and other halophiles can grow in these conditions. D-
mannitol is also an essential component in this media as it helps in
differentiation. Staphylococcus aureus contains an enzyme coagulase that
can degrade the mannitol. Thus, it is fermented and acid is produced in the
media. Phenol red is an acid base indicator that helps in detection and
differentiation. It is orange-red at pH 8.4 and yellow at pH 6.8 generally. The
acid produced due to carbohydrate fermentation by S. aureus changes the
colour of the indicator thereby giving yellow coloured colonies. The
solidifying agent used in this media is Agar.
 Fig. Mannitol Salt Agar plate showing (1) Micrococcus spp., (2) Staphylococcus
epidermidis and (3) Staphylococcus aureus colonies.
(Source: https://en.wikipedia.org/wiki/Mannitol_salt_agar#/media/File:Chapmanes.jpg)

A wide variety of microbes are found in our skin. These includes

Staphylococcus aureus, S. epidermidis, Micrococcus spp., Streptococcus spp.,
Diphtheroid (Coryneform), Propionibacterium acnes and other gram-
negative bacilli. When a skin sample is taken and incubated in Mannitol Salt
Agar (MSA) Media, most of the microbe should not be able to grow due to
high salt concentration. It has been found that only Staphylococcus aureus
and S. epidermidis are able to grow efficiently in this media. Occasionally,
growth of Micrococcus spp. and few other microbes are observed. However,
it is the S. aureus that by utilising mannitol changes the colour of the media.
Thus, surrounding area and colonies of S. aureus appears yellow in colour.
This helps in identification and detection of S. aureus in skin sample.
Thus, absence of any growth in Mannitol Salt Agar plates confirms that the
skin of the person from which sample was taken lacks Staphylococcus
aureus, S. epidermidis and Micrococcus spp. primarily. However, it may also
be noted that improper sample collection, defective media plates and
incorrect incubation time can show negative growth results.