Methods of enumerating microorganisms in food

1. Methods of enumerating
microorganisms food
MIC 204

2. Ways to enumerate microorganisms
enumeration
Total plate
count
Coliform test
Testing for
mesophilic
bacteria
Testing for
staphylococci
Testing for
pathogenic
bacteria

3. Procedures involving in plate
counts
Streak dilution
plate
Spread plate
Pour plate

4. Streak dilution plate

5. Spread plate
 Method of quantifying the number of
viable cells (or colony forming units) in
a sample after appropriate dilution

7. Pour plate
 A known volume of cell suspension is
mixed with this molten agar distributing
the cells throughout the medium
Disadvantages:
1. Typical colony morphology seen in
surface cultures will not be observed for
those colonies that develop within the
agar medium
2. Some of the suspension will left behind
in the screw capped bottle

9. 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^7UmI-1

10. Coliform test
 Coliform bacteria – 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 use coliform bacteria as
indicator
 As a result, testing for coliform bacteria can be a
reasonable indication of whether other
pathogenic bacteria are present

11. Why?
Because:
 They are able to survive for extensive
periods of time in the environment
 Relatively easy to cultivate in the
laboratory and numerous

12. 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 greater numbers than the
pathogen to provide a safety margin
d) Survive in the environment as long as
the potential pathogens
e) Be easy to detect with a high degree of
realibility

13. Stages of coliforms test
a) Presumptive tests
b) Confirmation tests
c) Completed tests

14. 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
Violet red bile agar

15. Most Probable Number (MPN) method
 In this method, samples were inoculated in 10ml
(101), 1ml (100) and 0.1ml (10-1) into lactose
broth 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 tubes 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.
Example for drinking water:

16. 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:
 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.

17. c) 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
Brilliant green lactose bile broth

18. Test for mesophilic bacteria
 Mesophilic bacteria – bacteria that can
grow at the opt T (btw 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
microorganisms of the human body, such
as Escherichia coli are mesophiles.

19. Testing method for mesophilic
bacteria
 Pour plate aliquots serially diluted sample using
non selective media such as Plate Count Agar
(PCA), tryptic soy agar, NA 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.

20. Testing for staphylococci
Why Staphylococci?
 Because certain strains of Staphylococci 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)

21. Testing methods for
staphylococci
• Several methods involving direct plating
and or / enrichment techniques have
been proposed for the detection of
coagulase-positive Staphylococci in
foods.
 Medium used for Staphylococci is Baird-
Parker agar medium.
• In this medium, it contains
all the nutrients that
Staphylococci required
Baird- Parker agar

22. Presumptive test (more 100
Staphylococci)
• When greater than 100 Staphylococci
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

23. 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 tubes are plated on Baird-Parker
agar based medium and typical colonies are
examined for the producing coagulase as described.

24. 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?
• 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.

25. Testing for pathogenic
bacteria
 Pathogenic bacteria – bacteria which
have ability to cause infection to the
organisms.
 Example: Salmonella, Shigella and
others.
 These are common pathogenic
bacteria that cause food poisoning

26. 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

27. 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.

28. Plating of enrichment cultures
After incubation, a loop full of enrichment culture is
streaked onto a plate of:
1) Salmonella-Shigella agar
2) Brilliant green agar,
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.

30. 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).

31. 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.

32. 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

33. Plating the enrichment culture
After enrichment, the cultures were
streaked on:
1) Salmonella-Shigella agar
2) Mc Conkey agar

34. 1. Salmonella-Shigella agar
A. Klebsiella pneumoniae
B. Escherichia coli
Klebsiella pneumoniae & Escherichia coli are positive for acid production from
fermentation of the carbohydrate(s) present.
C. Salmonella sp.
D. Proteus mirabilis
Both Salmonella sp. & Proteus mirabilis product hydrogen sulfide.
E. Pseudomona aeruginosa
The Pseudomonas colonies are nearly colorless.

35. 2. Mc Conkey agar
 The plate is incubated for 24hours at
35° C.
 Result in agar: Typical
colonies are colorless
and transparent

36. 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)

37. Triple sugar iron slants Lysine iron agar
A. Salmonella sp.
B. Escherichia coli
C. Unknown
D. Proteus mirabilis