RESULT:

Table 1: Count of Typical and Suspect of S. aureus

Dilution Count of Typical S. aureus Colonies Count of Suspect S. aureus Colonies

factor Plate 1 Plate 2 Plate 3 Total Plate 1 Plate 2 Plate 3 Total
10-1 0 0 1 1 TNTC TNTC TNTC TNTC
10-2 0 0 1 1 EST EST EST 2223
221 X 4 716 623
= 884
10-3 0 0 0 0 85 31 25 141
CFU/g
1 1 1 1
10-1 : 1 X X = 1 X 102 CFU/g 10-3: 141 X X
10−1 0.1 10−3 0.1

= 1.41 X 106 CFU/g

Table 2: Reaction on MSA and Appearance on BHI Agar of S. aureus

Isolate number Reaction on MSA Appearance on BHI Confirmed S. aureus
Agar Agar CFU/g

Typical

1 Colonies surrounded Colonies round and 1 X 102 CFU/g + 1.41

by yellow agar yellow pigmented
X 106 CFU/g =
2 Colonies surrounded Colonies round and
1.41 X 106 CFU/g
by yellow agar yellow pigmented
Suspect

1 Colonies surrounded Colonies round and

by yellow agar yellow pigmented
2 Colonies surrounded Colonies round and
by yellow agar yellow pigmented

Positive control Colonies surrounded Colonies round and N/A

by yellow agar yellow pigmented

Negative control No growth Colonies round and

off-white
 Table 3: Coagulase reaction of Two Confirmed Staphylococcus aureus Isolate and Positive
and Negative control
Isolate Coagulase

Confirmed 1 Positive

Confirmed 2 Positive

Positive control Positive

Negative control negative

DISCUSSION:

In this practical, the sample used is Nasi Kerabu, and the agar used are, Baird-Parker
Agar, Brain Heart Infusion Agar (BHI) and Mannitol Salt Agar (MSA). Baird-Parker Agar is
a type of agar used for the selective isolation of gram-positive Staphylococci species. It
contains lithium chloride and tellurite to inhibit the growth of alternative microbial flora, while
the included pyruvate and glycine promote the growth of Staphylococci. Staphylococcus
colonies show up black in colour with clear zones produced around them.

Mannitol Salt Agar (MSA) helps to demonstrate the ability of a bacterium to grow in a
7.5% salt environment. Species of staphylococci are able to tolerate this salt concentration but
other pathogenic bacteria may not. This concentration inhibits the growth of most other gram-
positive and gram-negative bacteria (Tankeshwar, 2013). An expected colony characteristics
growth on this agar for Staphylococcus aureus are, yellow colonies and may have yellow halo
around colonies. Then, Brain heart infusion (BHI) Agar is a growth medium for microorganism
growth. It is a nutrient-rich medium. It is a nutrient-rich medium, and can be used to culture a
variety of fastidious organisms such as streptococci, pneumococci and meningococci, which
can be otherwise challenging to grow.

We have conducted the Coagulase test in order to differentiate Staphylococcus aureus

(positive) from Coagulase Negative Staphylococcus (CONS). This is because Coagulase is an
enzyme produced by S. aureus that converts fibrinogen in plasma to fibrin, which is from
soluble stated to insoluble. From the result above, it shows positive for Coagulase except for
negative control test. Coagulase-positive staphylococci are responsible for approximately 75
per cent of the food-borne illnesses. This is because the presence of enterotoxin that contributed
to the poisoning.

Thermonuclease test is based on the principle of Staphylococcus aureus contains a heat-

stable thermonuclease and coagulase negative staphylococcus does not. This is a rapid test to
differentiate between the two organisms. The positive test show a pink zone of clearing at the
edge of the well with a darker blue ring at the outer periphery of the zone, which is, indicates
thermonuclease activity. Hence, for negative result shows no zone or a small clear zone around
the well.

Reference:

Tankeshwar, (2013). Mannitol Salt Agar (MSA): Composition, uses and colony characteristics.
Retrieved from: https://microbeonline.com/mannitol-salt-agar-msa-composition-uses-
and-colony-characteristics/

Rafner, H.B., & Stretton C.W. 1985. Thermonuclease test for same day identification of S.
aureus in blood cultures. J. Clin. Microbiol. 21:995-996.

Victor, R., Lachica, F., Jang, S. S., & Hoeprich, P. D. (1979). Thermonuclease seroinhibition
test for distinguishing Staphylococcus aureus from other coagulase-positive
staphylococci. Journal of Clinical Microbiology, 9(1), 141–143.

Tankeshwar. “Coagulase Test: Principle, Procedure and Interpretation -.” Access on 26 Apr.
2018. From microbeonline.com/diagnostic-tests-biochemical-tests-coagulase-
test/#comments.