Topic 5

~Methods of
enumerating
microorganisms of
food~
Prepared by:
Ilyanie Hj. Yaacob
 Syllabus content
Food sampling: total plate count, MPN, detection of
pathogenic bacteria.
Preparation for food samples for analysis

Food safety indicator

~Measurement
of growth~
 Measurement of growth
Foods are most often spoiled due to bacterial growth -
yeast, molds and virus could also cause food spoiled.
Enumeration is the determination of the number of
individual viable microbes in a sample - the measurement is
expressed as the number of viable organisms/ml of culture.
Several methods of measuring are available, as in the table
below.
Direct methods Indirect methods
Serial dilution & plate Turbidity
counts
Direct microscopic count Metabolic activity
Most probable number Dry weight
Filtration
 Measurement of growth direct methods

1) Serial dilution and plate counts.

Many culture methods make use of a solidified medium
within a petri plate.
There are three most important way used to obtain pure
cultures:

i streak plate method

ii spread plate method

iii pour plate method

 Measurement of growth direct methods

During all three procedures, some

cautions must be taken:

i Keep the lid of the petri plate

as close to the base as possible
to reduce aerial contamination.

ii - Do not breath directly onto the

exposed agar surface and replace
the lid as soon as possible.
 Measurement of growth direct methods

i - Streak plate method.

In this method, a sterile inoculation loop is used to streak the
organism over the surface of the medium.
The aim is to achieve single colonies at some point of the
plate.
Single colony = visible cluster of microorganisms growing on
the surface of or within a solid medium, presumably cultured
from asinglecell.
During streaking, the loop is allowed to glide over the surface
of the medium.
A person must hold the handle at the balance point and do
the sweeping movements, as the agar surface is easily
 Measurement of growth direct methods

ii - Spread plate method.

This method is used with cells in suspension (liquid growth
medium or sterile diluents/diluting agent).
In this method, an L-shaped glass spreader is used it is
sterilized using alcohol and bunsen flame.
The spreader is used to distribute a known volume of cell
suspension across the plate.
 Measurement of growth direct methods

iii Pour plate method.

This procedure also uses cells in suspension, but required
molten agar maintained in a water bath at 45C - 50C.
Colonies are formed within the medium, they are far smaller
than those of the surface streak method, allowing higher cell
numbers to be counted.
However, typical colony morphology seen in surface grown
cultures will not be observed for those colonies that develop
within the agar medium.
 Measurement of growth direct methods

Wherever a single living

bacterium is deposited on an
agar plate, it will divide to form
a colony.
Each bacterium represents a
colony-forming unit.
Colonies present can be count
by putting the plate under the
magnifying lens of a colony
counter.
 Measurement of growth direct methods

Only plates that contain 30-300 colonies should be used to

calculate the original concentration.
Less than 30 = counts are not considered to be statistically
reliable.
More than 300 = most likely colony formation will be
suppressed, leading to underestimation of the actual bacterial
numbers.

TMTC = too many to count @ TNTC = too numerous to

count
Thus, it is usually necessary to dilute the original bacterial
culture before transferring the culture onto the plate serial
dilution.
Measurement of growth direct methods
Measurement of growth direct methods
Measurement of growth direct methods
 Measurement of growth direct methods

The formula:

Number of colony-forming units/ml = C x M (answers in

CFU/ml).

C: no. of colony V: volume transferred to plate M:multiplication

factor.
 Measurement of growth direct methods

Example of calculation:

1 ml of a sample was mixed with 99 ml of buffer. 1 ml of this

was plated using the pour plate method in nutrient agar. After
incubation, 241 colonies were present on the plate. How many
colony-forming units were present per ml of the original
sample ?

No. of CFU/ml = (241 colonies/1ml) x 102 = 2.41 x 104

colony-forming units/ml.
 Measurement of growth direct methods

Example of calculation:

An overnight culture of Escherichia coli is used as a sample.

1ml of this culture is added to a bottle containing 99ml of
buffer. This dilution is mixed well, and 1ml of this is mixed in 9
ml of buffer. This second dilution is diluted by three successive
1/10 dilutions. The last (fifth) dilution is plated, i.e. 0.1 ml is
plated on nutrient agar. After incubating the plate, 56 colonies
are counted. How many colony-forming units were present per
ml of E. coli culture?
 Measurement of growth direct methods

Dilution factor:

No. of CFU/ml = (56 colonies/0.1ml) x 106 = 5.6 x 108

colony-forming units/ml.
 Measurement of growth direct methods
2) Direct microscopic counts.
In this method, a known volume of
bacterial suspension is inserted into
Petroff-Hausser counting chamber
(hemocytometer) using a calibrated
pipette.
The microorganisms are counted in
specific calibrated areas.
An accurate count requires that the
bacteria be homogenously distributed
throughout the culture.
 Measurement of growth direct methods

3) Most probable number (MPN).

MPN method is normally used when the number of bacteria
to be counted is too low thus may not be detected using
standard plate count method.
In MPN method, samples are serially diluted so that the
inocula will sometimes, but not always, contain viable
organisms.
 Measurement of growth direct methods

Samples were inoculated into

three, five or ten tube of broth.
The tubes are incubated and
organisms are identified by their
production of gas and/or by
becoming cloudy.
Referring to an MPN table, a
statistical range of the number of
organisms is determined by
observing the number of positive
tubes.
Measurement of growth direct methods
 Measurement of growth direct methods

4) Filtration.
In this method, a known
volume of water or air is
drawn through a filter with
pores too small to allow
passage of bacteria.
When the filter is then
placed on a solid medium,
each colony that grows
represents originally one
organism collected by the
filter.
 Measurement of growth indirect method

1) Turbidity.
Turbidity or a cloudy appearance
in a culture tube indicates the
presence of organisms.
Estimates of growth can be
measured using photoelectric
device, eg. Spectrophotometer.
Sample with very high cell
densities must be diluted to
ensure accurate readings.
 Measurement of growth indirect method

However, a few problems arise:

a) cultures contain fewer than 1million cells/ml will display little

or no turbidity even when growth is occurring.

b) turbidity can also be produced by high concentration of dead

cells in a culture.
 Measurement of growth indirect method

2) Metabolic activity.
Measuring the metabolic products of a population can be used
to estimate the bacterial growth.
The rate of the products formed reflects the mass of bacteria
present.
Eg. Gas production, acid production, use of glucose, use of
oxygen.
 Measurement of growth indirect method

3) Dry weight.
To calculate the dry weight of cells, it must be separated from
the medium through certain processes, eg. filtration or
centrifugation.
The cells are dried and the mass is weighed.
~Example of
test~
 Example of test - coliform test
Coliform bacteria is a gram-negative nonspore forming
bacilli usually found in the human and animal intestine -
they ferment lactose with the production of acid and gas.
Coliform bacteriaare a commonly
usedbacterialindicatorof sanitary quality of foods and
water in coliform test - E.coli is the most important indicator
organism within the group.
Coliform test is carried out to test the presence of
enteropathogenic bacteria such as Salmonella, Shigella and
others.
 Example of test - coliform test
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 reliability.

 Example of test - coliform test
The test involves three stages:

1 presumptive tests

2 confirmation tests

3 completed tests
 Example of test - coliform test
1 Presumptive tests.
There are two standard methods of counting bacteria:

i) Standard plate count (SPC) method normally used for

samples that have a relatively large number of bacteria.

ii) Most probable number (MPN) method normally used

when the number of bacteria to be counted is too low thus
may not be detected using SPC method.
 Example of test - coliform test

Using SPC method, if the sample is

solid (e.g. food), it will be suspended
in liquid and is added to petri dishes
in duplicated.
Then molten violet red bile
agar/MacConkey agar is added, mix,
then allowed to harden.
The plate then incubates at 35C for
18-24 hours.
Positive result shows dark red
colonies with a surrounding zone of
precipitated bile at least 0.5mm in
 Example of test - coliform test
In MPN method, samples were inoculated
into three, five or ten tube of lactose
broth.
The tubes are incubated and coliform
organisms are identified by their
production of gas and acids (positive
result).
Referring to an MPN table, a statistical
range of the number of coliform bacteria
is determined by observing the number
of positive tubes.
 Example of test - coliform test
2 Confirmation test.
Presumptive test for coliforms does not constitute absolute
identification of these organisms - all positive result should
be confirmed by confirmation test.
For example, gas formation in lactose broth at 37C 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.
 Example of test - coliform test
All positive result from SPC and
MPN methods are inoculated into
Brilliant Green Lactose Bile broth.
The presence of bile and brilliant
green in BGLB broth make this
medium selective for Gram-
negative bacteria.
The tubes are then incubated for
48 hours under 37C before
examined for the gas presence
(positive result).
 Example of test - coliform test
3 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.

 Example of test - testing for
mesophilic bacteria
Mesophilic bacteria is a bacteria that can grow at the
temperature between 20 C- 50C.
However many mesophiles have an optimal temperature of
about 37 C, corresponds to human body temperature.
Example of test - testing for mesophilic bacteria

The procedures:
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 thus for mesophilic bacteria, normally
incubation temperature is 37C for 24-48 hours.
 Example of test - testing for Staphylococci

Staphylococci are tested because certain strains such as

S.aeureus can grow on food and produce heat-stable
enteroxins, which cause food poisoning.
Characteristics of Staphylococci:

- Gram positive, cocci arranged in clusters.

- Biochemical test: Oxidase (negative) Catalase(positive)

- Some strains are coagulase positive but some are coagulase

negative.
Several methods is proposed for the detection of coagulase-
positive Staphylococci in foods.
Baird-Parker agar medium is used as its contain all the
nutrients that Staphylococci required.
 Example of test - testing for Staphylococci

1- Presumptive test (more than

100 Staphylococci).
When greater than 100 S.
aureus/g are expected, direct
plating of a diluted sample on
Baird-Parker medium followed
by incubation for 30 hour at 37
C is recommended.
Positive result showed
Staphylococci grow on this
medium as a black colony
surrounded by a clear zone.
 Example of test - testing for Staphylococci

Confirmation test.
These tests include:

1) The utilization of glucose anaerobically, which separates

the S. aureus from other species of Staphylococci.

2) The ability to produce coagulase, which separates S.

aureus from other species of Staphylococci - the tests
involve inoculation samples into blood serum and
incubation for certain period under certain temperature to
determine coagulation of the serum.

3) Separation of S. aureus from other Staphylococci based

on its property of utilizing mannitol anaerobically.
 Example of test - testing for Staphylococci

Presumptive test (less than 100 Staphylococci).

When count of less than 100 S. aureus are expected, the
MPN enrichment technique is recommended.
0.1g, 1g and 10g samples of the food will be analyzed using
anaerobic tellurite glycine mannitol pyruvate broth.
The samples are incubated at 37C.

The positives tube are plated on Baird-Parker medium and

typical colonies are examined for the producing coagulase
as described.
 Example of test - testing for
pathogenic bacteria
Pathogenic bacteria is a bacteria which have ability to
cause infection to the organisms.
Salmonella and Shigella are common pathogenic bacteria
that cause food poisoning.
 Example of test - testing for
pathogenic bacteria
Detection of Salmonella.
The detection method of Salmonella involves:

1) Enrichment of samples

2) Plating of enrichment cultures

3) Screening

4) Confirmation test
 Example of test - testing for
pathogenic bacteria
1) Enrichment of sample.
The purpose to do enrichment of samples is to supply all
the nutrients that required by Salmonella to grow.
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 35C.
 Example of test - testing for
pathogenic bacteria
2) Plating of enrichment cultures.
After incubation, a loopful of enrichment culture is streaked
onto a plate of:

- Salmonella-Shigella agar

- Brilliant green agar

The plates are then incubated for 22-24 hours at 35C.

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.
 Example of test - testing for
pathogenic bacteria
3) 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 - for Salmonella positive, the slant should be
alkaline (purplish) and the butt showed show acid (yellow).
 Example of test - testing for
pathogenic bacteria
4) Confirmation test.
Triple sugar iron agar positive cultures are now examined
for their reaction to polyvalent antiserum.
Positive result showed a sign of agglutination with
antiserum.
 Example of test - testing for
pathogenic bacteria
Detection of Shigella.
The detection method of Shigella involves:

1) Enrichment of samples

2) Plating of enrichment cultures

3) Screening

4) Confirmation test
 Example of test - testing for
pathogenic bacteria
1) Enrichment of sample.
The procedures are the same as Salmonella.
 Example of test - testing for
pathogenic bacteria
2) Plating of enrichment cultures.
After incubation, a loopful of enrichment culture is streaked
onto a plate of:

- Salmonella-Shigella agar

- McConkey agar
The plates are then incubated for 22-24 hours at 35C.

Result in both agar: Shigella colonies are usually colorless,

transparent.
 Example of test - testing for
pathogenic bacteria
3) Screening.
From both agar, at least 5 typical colonies are selected and
transferred in each case to a triple sugar iron agar slant or
lysine agar.
The slant being streaked and the butt stabbed.

The slant are incubated overnight at 35 C and then

examined - for Shigella positive, the slant should be alkaline
(purplish) and the butt showed show acid (yellow).
 Example of test - testing for
pathogenic bacteria
4) Confirmation test.
Slide agglutination tests with antisera for serogroup &
serotype confirm the identification.