Experiment no.

1: Microbial Enumeration

:::: Post lab report ::::

Student name: Hitesh Chavada

Student no: 300780460

Subject : Food Microbiology

Subject code : BI-208-064

Instructor's Name : Katarzyna Mysiura-Miziniak

Date: 23 January, 2014

 Introduction:
The Standard Plate Count (spc) method is used for the determination of the viable
cells or colony forming units (cfu) in given food sample. This method popularly
known as Aerobic Plate Count or the Total Plate Count. Food sample should be
aseptically collected and then homogenized, serially diluted in appropriate diluents,
plated in or onto a suitable agar medium and incubated. This Standard Plate Count
method is used for the quantification of the aerobic and facultative anaerobic
mesophilic bacteria. For the SPC, TSA and Macconkey media is used.

Material and methods:

Please refer to BI-208 lab manual attached with this Post-lab report.
Result:
TSA Plates
Plate
Colony count
Is it in range?

Series of dilution
10-3
TNTC
No

10-4

10-5

127

17

YES

NO

1.3*106 cfu/ml

Calculation

Appearance of
colonies

MAC Plates
Plate
Colony count
Is it in range?

Series of dilution
10-3
1
No

10-4

10-5

No

Yes

0 cfu/ml EST
Calculation
No colonies observed
Appearance of
colonies

 QUESTIONS:
1. Compare your calculated TSA count and your calculated mac plate count.
Should the two types of media give the same number of colonies?
Ans: TSA and MAC,both are different kind of media. TSA media is general
media, in which all kind of bacteria means Gram +ve and Gram -ve can
grow .
MAC is selective media where only coliform bacteria can grow. It
isolates gram negative enteric bacteria.
Both media are giving different colony count. It is found that on TSA
more colony has been found than MAC comparatively.
2. Why did the colonies on the MAC plate turn pink?
Ans : compositions of Macconkey agar media are Lactose, Bile salt, Neutral red,
Crystal violet etc., only Gram negative bacteria can grow on MAC. Gram ve and
they take up lactose from the medium for their growth and produces acid which
decreases the pH of the media. Therefore, it will turn neutral red into the pink
colour.
3. Should everyone in the class get the same calculated number of organisms
from the sample? Explain your answer: a) yes.. because.. or b) no..because..
Ans: Everyone had different calculated number of microorganism however
working in the same environment and using the same food sample. It may be
due to the personal error and also the aseptic techniques with which everyone
works is different which would result in a small range of values of the results.
4. If I hadnt told you to plate out the 10 -3 to 10-5 dilutions, how would you
decide which dilution levels are necessary to achieve countable plates in this
procedure?
Ans: If the dilution has not given than two differen method can be used :
For Gram ve bacteria, Macfarlane method, based on turbidity, can be used :
by comparing the turbidity with the given lines and ranges.
For Gram +ve bacteria, Haemocytometer can be used. In the procedure the
1g sample is taken in 99ml of buffer so the dilution could be 10 2 and for the
further step 1ml of diluted sample added into 9ml of buffer which can be 1
into 10 dilutions. it is obvious to go for 10-3 to 10-5 dilutions.
5. Why agar plates are are inverted during the incubation period?
Ans: Plates are incubated in inverted position in incubator because aerobic
bacteria produces water vapours as they respire. If the condensed water droplets
fall on agar that would cause a spreading of colonies, ultimately leading to
drastic change in the actual number of colonies.

6. Does the Standard Plate Count method reveal the true total count of the
food product tsted? Why?
Ans: No, the standard plate count method is not revealing the true total count of
the food product tested because By we are assuming that each cell grows into
colony only, but many time that is not the true. Sometimes two or more small
colonies formed very close to each other on the plate. So, it is useful to
determine the approximate number of colonies.

Discussion:
-

Total aerobic count in the egg salad sandwich was found to be 1.3*10 6
cfu/ml for TSA plates and no colonies formed for MAC plates. Obvious
growth was found in all the plates according to all serial dilution
schedules. Some of the colonies were distributed at the bottom and some
of that embedded in the media. It is indicate that aerobic and facultative
anaerobic bacteria presents in the plates. The incubation time for both
TSA and MAC plates is 48 hours according to the laboratory manual but
due to the reduced incubation period of 24 hours, we observed very less
growth in TSA and no growth in MAC plates. The reason behind this for
getting out of range of colonies in 10-3 and 10-4 may be higher microbe
level or other contamination may have occurred during the practical
depends on the individual lab practice and the way of the handling.

References:

James Jay(2000); Modern Food Microbiology(Chapter 10)

Mary E. Allen (2005); MacConkey Agar Plates Protocols
Wilkie, Anne (2014); Food Microbiology, Lecture 2
Wilkie, Anne (2014); Microbial Enumeration, Food
Laboratory-1

Microbiology