The document describes several methods for enumerating and identifying microorganisms in foods: 1) Total plate count, coliform test, and tests for mesophilic bacteria, staphylococci, and pathogenic bacteria like Salmonella and Shigella are discussed. 2) Culture-based techniques like streak plating, spread plating, and pour plating on agar plates are used to determine microbial numbers. 3) The coliform test involves presumptive, confirmation, and completed stages to identify coliform bacteria. Testing for specific microorganisms like Salmonella involves enrichment and plating followed by screening and confirmation tests.

1. CHAPTER 5
METHODS OF ENUMERATING MICROORGANISMS OF
FOODS
• Methods of enumerating microorganisms of foods are
mainly bacteriological, since foods are most often spoiled
because of bacterial growth.
• However, yeast, molds and virus sometimes cause food
spoiled.
• There are several ways to enumerate microorganisms:
a) Total plate count
b) Coliform test
c) Testing for mesophilic bacteria
d) Testing for staphylococci
e) Testing for pathogenic bacteria: eg - Salmonella,
Shigella
• A variety of culture-based techniques can be used to
determine the number of microbes in a sample.
• A major assumption of such methods is that, under
suitable condition, an individual viable microbial cell will
be able to multiply and grow to give a visible change in the
growth medium eg: a colony on agar based medium or
turbidity (cloudiness) in a liquid medium.

2. TOTAL PLATE COUNT
• Many culture methods make use of a solidified medium
within a petri plate.
• The three most important procedures involving in plate
counts are:
1) Streak dilution plate
2) Spread plate
3) Pour plate
a) Streak dilution plate
• Streaking a plate for single colonies is one of the most
important basic skills in microbiology.
• A sterile inoculation loop is used to streak the organism
over the surface of the medium (Figure 13.1)
• The aim is to achieve single colonies at some point on the
plate.
• Single colonies containing cells of a single species and
derived from a single parental cell.

3. Procedures:
• Keep the lid of the petri plate as close to the base as possible to
reduce aerial contamination.
• Allow the loop to glide over the surface of the medium. Hold
the handle at the balance point, sweeping movements, as the
agar surface is easily damaged and torn.
• Do not breath directly onto the exposed agar surface and
replace the lid as soon as possible.
b) Spread plate
• This method is used with cells in suspension, either in a
liquid growth medium or in appropriate sterile diluents.
• It is one method of quantifying the number of viable cells
(or colony forming units) in a sample after appropriate
dilution.
• An L-shaped glass spreader is sterilized by the end of the
spreader in a beaker containing a small amount of 70% v/v
alcohol, allowing the excess to drain from the spreader and
then igniting the remainder in a Bunsen flame.
• After cooling, the spreader is used to distribute a
known volume of cell suspension across the plate (Figure
13.2)

4. 3) Pour plate
• This procedure also uses ceils in suspension, but required
molten agar, usually in screw-capped bottles containing
sufficient medium to prepare a single Petri plate (i.e. 15-20
ml), maintained in a water bath at 45°C - 50°C.
• A known volume of cell suspension is mixed with this
molten agar, distributing the cells throughout the medium.
• Then is then poured without delay into an empty sterile Petri
plate and incubated, giving widely spaced colonies (Figure
13.3)
• Colonies are formed within the medium, they are far smaller
than those of the surface streak method, allowing higher cell
numbers to be counted.
Disadvantages:
1) Typical colony morphology seen in surface grown cultures
will not be observed for those colonies that develop within
the agar medium.
2) Some of the suspension will be left behind in the screw-
capped bottle

7. Count the accurate result which contain 30 - 300 colonies
↓
Count the mean
↓
Calculate the colony count per ml of that particular dilution by
dividing by the volume (in ml) of liquid transferred to each plate
(v)
Example: Count per ml (unit in CFU) = C/V x M
C= Mean of colony eg: 5.5 colonies V= Volume of
transferred to plate eg: 0.05ml M= Multiplication factor
eg: for a dilution of 10'7, the multiplication factor would
be 107
or in other words: mean colony count of 5.5 colonies per plate for
volume of 0.05ml at a dilution of 10'7, the count would be:
5.5 / 0.05 x 10^ 1.1 xlO^FUmI-1

8. COLIFORM TEST
What is coliform bacteria ?
• Coliform bactria is a gram negative nonspore forming
bacilli usually found in the human and animal intestine.
They ferment lactose to acid and gas
• Coliform test been carried out to test the presence of
enteropathogenic bacteria such as Salmonella, Shigella and
others
• The test normally using coliform bacteria as indicator Why
coliform bacteria?
• Because they are able to survive for extensive periods of
time in the environment, relatively easy to cultivate in the
laboratory and numerous. E.coli is the most important
indicator organism within the group
The ideal indicator organism should:
a) Be present whenever the pathogens concerned are
present
b) Be present only when there is a real danger of pathogen
being present
c) Occur in grater numbers than the pathogen to provide a
safety margin
d) Survive in the environment as long as the potential
pathogens

9. e) Be easy to detect with a high degree of realibility
Test for conforms involves 3 stages:
a) Presumptive tests
b) Confirmation tests
c) Completed tests
a) Presumptive test
Example in solid sample like food
Use plating method
• Food is added to petri dishes (in duplicated)
• Then molten violet red bile agar is added, mix, then allowed to
harden
• The plate then incubates at 35°C for 18-24 hours.
• Positive result: dark red colonies with a surrounding zone of
precipitated bile at least 0.5mm in diameter
Example for drinking water:

10. Most Protable Number (MPN) method
• In this method, samples were inoculated in 10ml (101),
1ml(100) and 0.1ml (10-1) into lactose both tubes.
• The tubes are incubated-and coliform organisms are
identified by their production of gas from lactose.
• Referring to an MPN table, a statistical range of the
number of coliform bacteria is determined by observing
how many broth tube showed gas.
• However presumptive test for coliforms does not constitute
absolute identification of these organisms. All positive
result should be confirmed by further testing which is
confirmation test.
B) Confirmation test
• This test being carried out because gas formation in lactose
broth at 37°C is characteristic not only of fecal
Salmonella, Shigella and E.coli strains but also of non
fecal coliform like Enterobacter aerogenes and some
Klebsiella species.
• As a result, in this second stage test (confirmation), the
presence of enteric bacteria is confirmed by reculture the
positive result:
Positive result from plating method:

11. • Those colonies from violet red bile agar are transfer to a
separate tube of brilliant green lactose bile broth and then
incubate for 48 hours under 35° C before examined for the
gas presence.
d) Completed test
• Positive tube from Brilliant Green Lactose bile broth
cultures are streaked and stabbed on slant of nutrient agar
• After incubation for 18-24 hours at 35 °C, the slant is
examined for the growth on the surface and in the stabbed
portion of the slant
• Gram stain smear was made from agar slant:
• Positive result showed: Gram negative, non-sporing rods
TEST FOR MESOPHILLIC BACTERIA
• MesophHIic bacteria is a bacteria that can grow at the
optimum temperature (between 20 °C- 50°C).
• However many mesophiles have an optimal temperature of
about 37° C, corresponds to human body temperature.
• Many of the normal resident microorganism of the human
body, such as Escherichia coli are mesophiles.
TESTING METHOD FOR MESOPHILIC BACTERIA

12. • Aliquots from serially diluted sample/suspensions are
either pour or surface plated using non selective media
such as Plate Count Agar (PCA), tryptic soy agar, nutrients
agar and others.
• However, PCA is recommended for colony forming units
(CFU) determination.
• The temperature and time of plate incubation required for
the colonies to develop differ with the microbial groups
being enumerated.
• For mesophilic bacteria, normally incubation temperature
is 37ºC for 24-48 hours.
TESTING FOR STAPHYLOCOCCI
Why Staphylococci?
• Because certain strains of Staphylococc like S.aeureus can
grow on food and produce heat-stable enteroxins, which
cause food poisoning.
Characteristics of Staphylococci
1) Gram positive, cocci arranged in clusters.
2) Biochemical test: Oxidase (negative)
Catalase(positive)
3) Some strains {S.aereus) are coagulase test (positive) but
some are coagulase (negative)

13. • Several methods involving direct plating and or / enrichment
techniques have been proposed for the detection of
coagulase-positive Staphylococci in foods.
Testing methods for Staphvlococci
• Medium used for Staphylococci Baird-Parker agar
medium.
• In this medium, its content all the nutrients that
Staphylococci required
Presumptive test (more 100 Staphylococci)
• When greater than 100 Staphviococci aureus / gram are
expected, direct plating of a diluted sample on
Baird-Parker medium followed by incubation for 30 hour
at 37° C is recommended.
• Result: Staphylococci grow on this medium as a black
colony surrounded by a clear zone
Confirmatory test
These tests include:
1) The utilization of glucose anaerobically, which separates
the Staphylococci aureus from other species of
Staphylococci.'
2) The ability to produce coagulase, which separates
Staphylococci aureus from other species of Staphylococci
How to carry out the coagulase test?

14. • The tests involve inoculation samples into blood serum
and incubation for certain period under certain
temperature to determine coagulation of the serum.
3) Also, S. aureus is separated from other Staphylococci based
on its property of utilizing mannitol anaerobically.
Presumptive test (low Staphvlococci aureus)
When count of less than 100 / Staphylococci aureus are
expected, the most probable number (MPN) enrichment
technique is recommended.
How to carry out the test?
• Using 0.1g, 1g and 10g samples of the food to be analyzed.
• Growth in anaerobic tellurite glycine mannitol
pyruvate broth and incubated at 37°C is determined.
• The positives tube are plated on Baird-Parker agar based
medium and typical colonies are examined for the
producing coagulase as described.
TESTING FOR PATHOGENIC BACTERIA
Pathogenic bacteria is a bacteria which have ability to
cause infection to the organisms.
Example of pathogenic bacteria like Salmonella, Shigella
and others.
Salmonella and Shigella are common pathogenic bacteria
that cause food poisoning.

15. Detection of Salmonella in food
The detection method of Salmonella involves several stages:
a) Enrichment of samples
b) Plating of enrichment cultures
c) Screening
d) Confirmation test
Enrichment of sample
• The purpose to do enrichment of samples is to supply all
the nutrients that required by Salmonella to grow.
How to carry out the enrichment method?
• The food sample are weighed and put into screw-capped
jars.
• Nutrients broth was added into jars.
•Then the lids of the jars are now tightened and the jars are
shaken vigorously after which they are incubated for 24
hours at 35°C.
Plating of enrichment cultures
After incubation a loopful of enrichment culture is streaked
onto a plate of:
1) Salmonella-Shigella agar
2) Brilliant green agar

16. The plates are then incubated for 22-24 hours at 35°C
Salmonella-Shigella agar: Salmonella colonies are usually
colorless, transparent and usually larger than 5 mm in
diameter and may have black centers.
Brilliant green agar: Salmonella colonies are pink to deep
red in color.
Screening
• From Salmonella-Shigella and Brilliant green agar, at
least 5 typical colonies are selected and transferred in
each case to a triple sugar iron agar slant.
• The slant being streaked and the butt stabbed.
• The slant are incubated overnight at 35° C and then
examined.
• Result: For Salmonella positive, cultures the slant should
be alkaline (purplish), the butt showed show acid
(yellow).
Confirmation test
• Triple sugar iron agar positive cultures are now examined
for their reaction to polyvalent antiserum.
• Result: Positive result showed a sign of agglutination
with antiserum.

17. Testing for Shigella
The testing methods for Shigella involved
a) Enrichment of sample
b) Plating the enrichment culture
c) Screening test
d) Confirmation test
Enrichment test
The enrichment procedure for Shigella and the same are those
for Salmonella
Plating the enrichment culture
After enrichment, the cultures were streaked on:
1) Salmonella-Shigella agar
2) Me Con key agar
The plate are incubated for 24hours at 35° C.
Result in broth agar: Typical colonies are colorless and transparent

18. Screening
Typical colonies are transferred into
1) Triple sugar iron slants, incubated at 35°C, at 24 to 48 hours.
Result: On this medium the slant is alkaline (purple) and
the butt acid (yellow)
2) Lysine iron agar in which the slant is streaked and the butt
stabbed
Result: The slant is alkaline (purple) and file butt of the
lysine iron agar is acid (yellow)