Detection and Enumeration of

Microbes in Foods
By Dr Zaheer Ahmed
 We need to know how many and what type of
bacteria are present in food to:
 Ensure that the food is safe
 Provide an estimate of the shelf-life
 Provide a means of monitoring processing
procedures and within regulation
 to meet certain set standards
 to determine quality of the food
 for public health purposes
 METHOD
Traditional method Rapid Method
 Plate counts

Direct epifluorescent
Membrane filtration
 Most probable number
filter technique
 Direct microscopic count (DEFT)
 Dye reduction tests Electrical impedance
 Indicator Enzyme-linked
immunosorbent
assay(ELISA)

3
Remember this? Conventional/traditional
techniques
Sample preparation
Aseptic technique
Analyze sample as quickly as possible
If sample must be held, prevent further growth
SOPs for preparation
Homogenization
Standard sample size
Standard dilution and counting protocol
Collection, handling and sample
prepration
Collect sample properly in sterilize condition
Handling during way to lab
Storing
Homogenization
Stomacher is used for solid material such as meat and
fruits etc.
Food sample+diluent and stomacher
Analysis of samples
PLATE COUNT DEPENDS ON
 Diluent
 Food homogenate
 Dilution series
 Medium
 Plating method
 Incubate conditions

APC means condition grown for bacterial will be aerobic i.e.

in presence of oxygen

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 Serial Dilutions

• In order to form single colonies to count, food

samples require to be diluted and serial
dilutions prepared

Diluents used:
• 0.8 % NaCl (Saline)
• 0.1% Buffered Peptone Water
•
Standard Plate Count is carried out to estimate the
number of viable bacteria in a food sample.

A high SPC of the food product indicates poor

sanitary conditions of the food processing plant and a
low SPC indicates the good quality of the processed
food in terms of bacterial load, however, SPC does
not indicate the absence of pathogens in the finished
food product.

 SPC is based on the serial dilution of the food sample

in sterilized dilution blanks followed by plating on the
nutrient media.
The dilution of sample is required because usually
food samples contain a high number of bacteria,
which are difficult to count.

If undiluted sample is plated, a lawn will form on the

plate and the individual isolated colonies that can be
counted will not be formed.

After serially diluting the representative sample of

food, plating is carried out by pour plate or spread
plate technique.
In pour plate technique, the dilution is added to the
sterile petriplate followed by the addition of molten
nutrient agar.
 Typical Technique for Agar Colony Count

Food Sample + Diluent

(always 1:10 ratio e.g. 10 g sample+ 90 ml diluent)

Homogenize (Stomacher) for 2 minutes

Prepare Dilution Series

 Spread plate

Number of
colony forming units (cfus)
?????
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Plate count method

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 Spread Plate Technique

Spread using a
glass or plastic
hockey stick

0.1 ml
diluted
sample

Colonies form on the surface of agar

Count plates with 25-250 colonies
 Pour Plate Technique

1ml
diluted
sample

25ml Molten Mix by

Agar Medium Swirling
The diluent's and agar are mixed and plates are
incubated after solidification for the growth of
bacterial colonies.

The SPC of the food sample is reported as colony

forming units per ml (cfu/ml).

The term colony forming unit is used because the

bacterial colony obtained on the agar plate may not be
produced by a single bacteria but bacterial clumps
may also give rise to the colony hence cfu/ ml and not
cells/ ml is used for reporting the results.
Pour plate

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Calculation and Results

For calculating SPC only those plates having the

colony numbers from 25 to 250 are considered. Plates
having more than 250 colonies are designated as Too
Numerous To Count (TNTC).

Alternatively in such cases count may be reported as

estimated count. Number of colonies on valid plates
(colonies ranging from 25-250) are counted. =
The average obtained from the duplicates at a single dilution is
multiplied by the dilution factor to obtain the colony forming
units per ml (cfu/ml) of the food sample e.g. if plates at 10-4
dilution are having 50 and 60 colonies the cfu/ml will be
calculated as:
Precautions

1) The sample taken for analysis should be representative

of the whole [Link] a sample taken for analysis is small
it will give erroneous results.

2) Blending of the sample should be done carefully so as to

prepare a homogeneous suspension.

3) Dilutions should be mixed properly before each transfer.

4) Molten agar should not be very hot (40-42OC is optimum

temperature) at the time of pouring.
5) Plates should be incubated in an inverted position so as to
avoid moisture condensation and consequently lawn formation
on the surface of agar.

6) Diluent taken may differ with the type of food product.

Sodium citrate (2%) may be used for fatty foods such as
cheese. Normal saline, buffers or even distilled water may also
be used as diluent.
APPLICATION OF PLATE COUNT

Check quality of RM & final products

Check condition hygiene
Estimate storage life of products
Determine
Production
Transport
Storage
Determine pathogens

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 SELECTION OF MEDIA IN FOOD MICROBIOLOGY
Medium Use

Plate count agar Aerobic mesophilic count

MacConkey broth MPN of coliforms in water

Brilliant green/Lactose/Bile broth MPN of coliforms in food

Braid Parker agar Staphylococcus aureus

Thiosulfate/Bile/Citrate/agar Vibrio sp.

Adam and Moss (2003)

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 Spread Plate Technique

Advantages Disadvantages
 Identify colony  More likely for bacteria to
morphology clump
 Easier to count  Motile bacteria can spread
 Bacteria not exposed to over plate
heat from molten agar Cells with low O2 tolerance
won’t grow
 Pour Plate Technique

Advantages Disadvantages
 Bacteria dispersed  Cannot identify colony
 Larger volume used (more morphology
sensitive)  Counting more difficult
 Cells with low O2 tolerance  Heat from agar may
can grow damage cells
 More rapid than spread
plate
 Easy to perform
Example not from your book
 How to Calculate cfu per g or ml

• Basic calculation is used for all plate

techniques (Spread, Pour,, etc.)

• Calculations differ with respect to weight of

sample examined (10 g, 11 g etc.) volumes
plated (0.1, 1, or 0.02 ml etc.) and dilutions
used (100 , 10-1 , etc..)
 How to Calculate cfu per g or ml

Examples: (These below refer to spread plate

counts, i.e volume used on plates is 0.1 ml)

You have been asked to find the CFU/g of bacteria

in a sample of ground beef (Calculate CFU/g)
 How to Calculate cfu per g or ml

 You used 10 g of beef sample

 You added 90 ml of diluent to the 10 g beef
sample and homogenized.
 This makes your first dilution:
10/10 + 90 = 10/100 = 1/10 = 10-1
 You then prepared 3 subsequent serial dilutions (after
the first) and plated out, on duplicate plates (2 plates
per dilution) using the spread plate technique
 After incubation you counted the following number of
colonies
 How to Calculate cfu per g or ml

Plate 10-1 10-2 10-3 10-4

(original
sample)
1 TNTC 248 22 1

2 TNTC 200 23 2

Average - 224 TFTC TFTC

 How to Calculate cfu per g or ml

1st Dilution (10-1) = TNTC therefore, will not be

considered

2nd Dilution (10-2) = 224; there are 25-250 colonies.

You will use this dilution to calculate the CFU/g in
original sample

• CFU/g = average number of colonies on duplicate

plates x 1 / dilution factor
 How to Calculate cfu per g or ml

• CFU/g = average number of colonies on duplicate

plates x 1 / dilution factor

Average number of colonies on duplicate plates:

(Count on plate 1 + count on plate 2) / 2
= 224

Dilution Factor = Initial Dilution x subsequent dilution

(s) x amount plated
 How to Calculate cfu per g or ml

Initial Dilution= Amount of food used

amount of food used + volume of the diluent

Initial Dilution = 10/10 + 90 = 10/100 = 10-1

Subsequent Dilution = Volume of dilution transferred

Volume of dilution transferred + Volume of Diluent

Subsequent Dilution = 1/1 + 9 = 1/10 = 0.1 = 10-1

 How to Calculate cfu per g or ml

Note: Even though you made 2 other dilutions, they

are not used in the calculation because the dilutions
you use in the calculations are up to and including
the dilution where 25-250 colonies fall, this occurs at
the first dilution after the original

Dilution Factor = Initial Dilution x subsequent dilution

(s) x amount plated

Amount plated = 0.1 ml since spread plates were

made
 How to Calculate cfu per g or ml

Dilution Factor = Initial Dilution x subsequent dilution

(s) x amount plated

Dilution Factor = 10-1 x 10-1 x 0.l

= 0.1 x 0.1 x 0.1
= 0.001 = 10-3
• CFU/g = average number of colonies on duplicate
plates x 1 / dilution factor
• CFU/g = 224 x 1/10-3
• CFU/g = 224 x 103 = 2.24 x 105
The roll tube method is basically the same as the pour
plate method, but screw cap tubes are used in place of
petri plates.

Test tubes containing 2-4 ml of plate count agar or other
appropriate medium are sterilized, and the melt agar is
allowed to cool to 45 C.

A 0.1ml of the aliquot of the appropriate dilution is added

and the tube is immersed in cold water in a horizontal
position and rolled.
The agar will solidify forming the thin layer on the inner wall
of the tube. The tubes are then incubated upside down to
prevent any condensation that may form from causing
smearing of the colonies.

Advantage of this method is saving with the use of less amount

of agar.
Spiral Plate Count
This method can be said to be a variation of Standard
Plate Count.
In this a very small volume of inoculum is plated on
the surface of the plate and this gives a high dilution
(10,000:1) of inoculum on a single plate.
 A mechanical device called Spiral Plating Machine is
used for this purpose.
It dispenses the inoculum on the surface of rotating
hardened agar plate.
The plate is then incubated at the suitable incubation
temperature depending on the bacterial culture plated
on the plate.
After incubation the isolated colonies are
counted using a counting grid that covers the
area of agar plate and is divided into four
concentric circles and eight wedges or octants.
The method is best suited for the liquid
samples such as milk, since food particles may
cause blocking of the dispenser.
Advnatage/Disadvantage
 The advantage includes the use of fewer plates, fewer
dilution bottles and less agar and the ability to process
a greater number os samples.
 Disadvantage include limitation of the types of
samples that can be processed and difficulty in
counting colonies.
Membrane Filter Technique:

Fluid samples (water or diluent) can passed through the

membrane filters to collect [Link] with 0.45um
are typically used.

The filter can either be placed on to plate containing culture

medium of choice or used in direct microscopic count assay
(DMC).

This membrane filter technique is useful for examination of

water or other diluted sample that can easily pass through
the filter.
 Filtration
0.45 μm
Liquid food
• Low number of
MO.
Large volume of
food
• Count
• Sterilize

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[Link]
Therefore even low numbers of microbes are
detected, since large volume (1 liter or more) can be
passed through a single filter

The method can also be used to determine microbial

numbers in air. Small amount of viscous liquid sample
such as milk or juice, can be processed without
clogging the membrane.
A number of the commercial method based on the
membrane filter technique are available.

The direct epiflourescent technique use fluorescent

dye to stain bacteria that are then counted using
fluorescence microscope.

The Direct Epifluorescent Filter Technique

(DEFT) is a rapid method for enumerating
bacteria. Used widely in the dairy industry for
milk and milk products, it has also been applied
to beverages, foods, clinical specimens and in
environmental research.
The technique involves capturing bacterial cells on the
surface of polycarbonate membrane filters, staining with a
fluorochrome such as acridine orange, and visualisation
using epifluorescence microscopy.

Enumeration of bacteria is greatly facilitated by image

analysis and Perceptive Instruments has great expertise on
DEFT and similar direct epifluorescence microscopy
techniques.

 The number of cells per gram or millimetre is calculated by

multiplying the average number perfield by the microscope
factor.

This method has also been used for estimating the numbers of
bacteria in milk, meat and poultry products and on food contact
surfaces.
 Membrane epiflurescent

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[Link]/.../ 4681/[Link]
A rapid technique for the detection of pathogens in food products
Membrane epiflurescent

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Direct Epifluorescence Microscopy
 Live imaging direct from the microscope

 Automatic count and sizing of fluorescent cells

 Discrimination between visible and non-visible cells

 Data transfer directly to Excel or Oracle databases

 Electronic signature and GLP compliance

 Wide range of proven applications

The hydrophobic grid membrane filter method is also
used to estimate microbial numbers associated with a
variety of foods.
 For this method specially designed filter containing
1,600 grid that restrict microbial growth and colony
size are used.
Generally 1 ml of a 1:10 sample homogenate is passed
through the filter. Grid contain that contain colonies
are counted and a most probable number is
determined.
 DMC
Direct microscopic count is carried out to estimate the
number of bacterial cells or the somatic cells in raw and
processed milk.

The direct microscopic count can be related to the quality

of milk. Presence of a large number of bacterial and
somatic cells in milk may point to the mastitic condition of
the milching animal.

Also, if there is reason to suspect that a food has caused

food poisoning or has undergone microbial spoilage, the
original product or a low serial dilution of it should be
used to prepare a slide for direct microscopic examination.
The DMC of sample is carried out by examining stained
smear of a measure volume of sample say milk (0.01 ml)
spread over a known area of glass slide (1.0 cm2 ) and
counting the number of cells or clumps of cells in a
microscopic field.
The diameter of a field is measured with a stage
micrometer for calculation of microscopic factor (MF).
The total number of bacteria per ml is calculated by
multiplying average number of cells with microscopic
factor.
The method is simple and rapid and facilitates
simultaneous enumeration and observation of cells. It can
be conveniently used for screening and grading of foods
especially, liquid foods.
MICROSCOPIC COUNT

58
 However, major disadvantage is that both dead as
well as cells are taken into account and hence less
suitable for heat treated foods.
 Direct Microscopic counts
Advantages Disadvantages

Rapid  Count dead and live cells

Simple  Difficult to distinguish
 Minimum and inexpensive between food particles and
equipment bacteria
 Identify morphology  Need at least 104 cfu/ml to
see sufficient cells to count
 Low volume used (low
accuracy)
Flourescence dyes are now available that can be used
to differentially stain live and dead bacteria. Using
such stains, the total bacteria count and a live-dead
count can be obtained.

 Since stains are intensely flourescent, they can be

used in samples that may contain low numbers of
bacteria. The disadvantage are that the fluorescent
signals fade overtime and food constituents may also
stain or fluorescent, interferring with the ability to
count bacterial cells.
Metabolism Based Methods
In general microbiology metabolism of
microorganisms is used to determine starch hydrolysis,
sugar fermentation, production of hydrogen sulfide and
indole, or nitrate reduction.

Measurement of microbial metabolism or production of

metabolic products in foods can be used to estimate the
bacterial populatoion.

Today many of these test have been miniaturized and

are he bases of number of rapid tests.
Microorganisms obtain energy through chemical
reactions, i.e. oxidation-reduction reactions, where the
energy source becomes oxidized while another
compound is reduced..

Oxidation reaction consist of electron transfer and can

therefore be measured electronically with a
potentiometer.

The redox potenttial can also be determined with

indicators and dyes. The addition of such compounds
to metabolizing bacteria results in the transfer of
electron to the indicator and subsequent change in
color
Several oxidation reduction indicators or dyes, including
methylene blue, resazurin, and tetrazolium, can be used.

The rate at which the indicator shows a change in color is

related to metabolic rate of microbial culture. Basically greater
the number of bacteria, the faster the color change occurs. Test
using these compounds are often referred as dye reduction
tests.
Dye Reduction Tests
 These tests are used to determine the microbiological
quality of milk.
 These are indirect tests where the number of micro-
organisms is not counted as in SPC; instead the time
required for reduction of dyes (methylene blue or
Rezaurin) istaken into account and correlated with the
milk quality.
 These dyes act as oxidation-reduction (O-R) indicators
and get decolorized due to the consumption of oxygen
and decrease in the O-R potential of milk as a result of
bacterial growth.
Methylene Blue Reduction (MBR) test
 Methylene blue is a dye, which remains blue in its
oxidized state and turns colourless on its reduction.
 This characteristic is put to use for estimation of
bacterial load of milk and milk products.
 When bacteria grow in milk they release hydrogen
during respiration, which is simultaneously accepted by
methylene blue.
 As a result, it is reduced to colourless or leuco
compound.
 The majority of bacteria, both aerobic and facultative
present in milk indulge in lowering of oxidation-
reduction potential of milk to such an extent that dye
gets decolorized.
 Hence greater the number of viable cells, shorter is the
time taken to reduce the dye.
 The result of this test is expressed in terms of time
required for the colour of methylene blue to disappear
as Methylene Blue Reduction (MBR) time at
incubation temperature of 37oC.
 This test renders very useful information on general
bacteriological quality of milk in a short period and
requires minimum apparatus.
 Limitations of this technique include its suitability
only for unheated milk and no indication of the type
of organisms.
 The methylene blue reduction time also depends on
the type of bacteria occurring in milk.
 Coliforms reduce this dye at a higher rate as compared
topsychrotrophic and thermoduric bacteria occurring
in milk.
 This is so because the incubation temperature favours
the growth of mesophilic bacteria (coliforms). High
numbers of lecucocytes if present also affect reduction
time of methylene blue in milk samples.
Requirements
Milk sample, methylene blue solution (0.4 mg/100 ml of
sterile distilled water), sterile tubes and stoppers, sterile
pipettes, water bath.
Procedure
1. Take 10 ml milk in a test tube and add 1 ml of methylene
blue solution to it.
2. Insert stoppers on the tubes and mix the contents by
inverting the test tubes gently.
3. Incubate the test tubes in a covered water bath at 37oC and
check the tubes for decolourization after every 30 min. of
incubation.
4. Remove the decolorized tubes after each reading and also
invert gently the remaining tubes.
5. Monitor the tubes for decolourization till 8 h.
Interpretation
The quality of milk is graded according to the time taken
for decolorization and is as follows:
 Class 1. Excellent, not decolorized in 8 h

 Class 2. Good, decolorized in less than 8 h but not less

than 6h.

 Class 3. Fair, decolorized in less than 6 hours but not

less than 2 h.

 Class 4. Poor, decolorized in less than 2 h

Surface Testing
The swab test method is most widely used method for
microbiological examination of surfaces in food
processing facilities.
The basic of this test methods include the swabbing a
given area with a moistened cotton or calcium
alginate swab.
 The area to be examined can be defined through the
use of templates that have predefined openings. The
template should be sterilized before use.
After the define area is swabbed, the swab is returned
to test tube containing the suitable diluents and
agitated to dislodge the bacteria
To facilitate the process the calcium alginate swabs are
used and disolved with the addition of
hexametaphosphate to the diluent
SPC generally used to calculate the bacteria
Direct Contact with agar: Other method for examing the
food contact surfaces include the direct contact of agar
with a test surface and the use of sticky film or tape.

In the replicate organism direct area contact method,

specially designed petir plates are used so that medium,
when poured, produced a raised agar surfaces. This agar
can make the direct contact with the surfaces. Selective
media can be used to check present of variety of
microbes.
This method is not suitable for heavy xontaminated or
rough surfaces.

Sterile sticky tape is commercially available for

sampling of surface from equipment to beef carcasses.
The tape and dispenser are similar in appearance to a
standard roll of adhesive tape used for sealing
packages.
The tape is withdrawn from the roll and held over a
premeasured are located on the tape dispenser.

 The sticky surface of the tape is then passed against

the area to be tested, and then tape is pressed on the
surface of agar plate. It not suitable for wooden
surfaces.
A sponge system has been employed for examination
of animal carcasses and food contact surfaces. Similar
to other contact system, a moistened sponge is wiped
across the tst area and then placed in to container
containing appropiate diluent. The container is agitaed
t dislodge the bacteria. And SPC method is used to
count the bacteria.
Thanks a lot for attention