Food Microbiological
Analysis
Sampling and sample preparation
Qualitative and quantitative analysis
Objectives
At the end of this unit you must be able to:
• discuss methods available for microbiological analysis of food
• compare methods of analysis, indicating advantages and
disadvantages of each method
• detail procedures for collection and processing of food samples
calculate the microbial load of a sample
• recognize the difference between conventional and rapid
microbiological methods
Introduction
• Many types of microorganisms are found in foods.
• The ferment food, spoil food and cause diseases.
• Determining the types and numbers of microorganisms in a food is an
important.
• The aerobic plate count (APC) provides an estimation of the number of
microorganisms in a food.
• A rule of thumb is that as the microbial count increases, the quality of the
food decreases.
Sampling and sample preparation
• The methods used for sample collection and processing vary from food to
food and for specific microorganisms.
• A sample will only yield significant and meaningful information:
• if it represents the mass of material being examined,
• if the method of collection used protects against microbial contamination, and
• if the sample is handled in a manner that prevents changes in microbial numbers
between collection and analysis
• A representative sample must be collected using an aseptic technique.
• Care must be taken during sample collection to prevent the introduction of
microbes into the food sample.
• A representative sample must be collected using an aseptic technique.
• Instruments used for sample collection should be sterilized in the
laboratory rather than at the place of sampling.
• Sterile containers (plastic bags or widemouthed jars) should be labeled
with the name of the food, the date collected, and other information that
may be useful in the analysis of results and tracking of the sample.
• Once the sample is collected, it should be analyzed as quickly as
possible to prevent a change in the microbial population
• If this is not possible, the sample should be refrigerated and frozen
samples should be kept frozen.
• Products that are normally refrigerated should not be frozen, since
freezing may cause death or damage to some bacterial cells,
producing incorrect results.
• Before analysis, some preparation of the sample is generally required.
• For meaningful results, the sample should be processed to produce a
homogeneous suspension of bacteria so that they can be pipetted.
• If the sample is solid, the food is generally mixed with a sterile diluent, such
as Butterfield’s buffered phosphate or 0.1% peptone water.
• If an appropriate buffer is not used, the bacteria may multiply in the
dilution medium prior to being plated.
• To make a homogeneous suspension, the sample is added to a
• sterile plastic stomacher bag, diluent is added, and the sample is
processed in a stomacher (a device that has two paddles that move
rapidly back and forth against the sample).
• A typical-size food sample of 25 g is added to the sterile bag
containing 225 ml of sterile diluent and mixed (placed in the
stomacher), and a 1:10 dilution of the food and associated bacteria is
obtained.
Analysis of food samples
• Microbiological examination of food necessary to determine its
quality. This may be necessary to estimate its shelflife, its suitability
for human consumption or to confirm that it meets some established
microbiological criterion.
• The total mesophilic plate count is widely used as a broad indication
of microbiological quality, although it is unsuitable for this purpose in
fermented foods which contain large numbers of organisms as a
natural consequence of their preparation.
Analysis methods
• Qualitative analysis methods (Detection methods)
• Microscopy
• Cultural techniques
• Immunological methods
• Molecular Methods
• Quantitative analysis methods ( enumeration methods)
• Direct Microscopy
• Cultural techniques
• Plate counts
• Most probable number
• Membrane filter technique
• Other methods
• Dye reduction
• Electrical Methods
Direct Microscopy
• This method is used to count both living and dead cells.
• This is done by placing a suspension in a counting chamber such as Petroff-
Hausser for counting bacteria and Hemocytometer for the larger
eukaryotic microbes.
• These are special slides that have chambers of known depth (0.1 to 0.2
mm) with an etched grid on the chamber bottom.
• The number of microbes in a sample can be calculated by considering the
volume of the chamber and any sample dilution required.
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• The advantage of this method is that it is quick as no
incubation time is required.
• The disadvantages are that
• higher concentration of cells is required for accuracy
• it is difficulty or impossible to distinguish between dead
and alive cells.
• it is tedious
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Viable cell count
• This is used to count only viable cells.
• A viable cell is one able to divide and form offspring
and hence produce colony on solid media or turbidity
in liquid media.
• Viable cell count is done in three ways:
• Plate count/colony count,
• Membrane filter method and
• Most Probable Number (MPN) method.
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A. Plate count
• Plate count is done in two ways: the spread plate
method and the pour plate method.
• In the spread plate method, 0.1ml of diluted microbial
sample is spread over agar plate uniformly.
• The plates are incubated until colonies will occur.
• Colonies are then counted to give a colony forming unit
(cfu).
• The total number of viable cells in 1 ml of the original
sample is calculated by dividing the product of the
number of colonies counted and dilution factor by 0.1ml.
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Serial dilution
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• In the pour plate method, 0.1 ml to 1 ml of diluted
sample suspension is pipetted into a sterile plate.
• Melted agar medium at 45oC is added and mixed well
by swirling.
• The plate is incubated until colonies will develop.
• Both surface and subsurface colonies will develop.
• The number of colonies is counted to determine the
number of cells in a given volume of the original
suspension in the same way it is done in the spread
plate method.
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• In both methods, the greatest accuracy is obtained
when plates develop 30 to 300 colonies.
• The disadvantage of these methods is that it takes
time, usually about 24 hours.
• In addition, the pour plate method may kill some
heat sensitive microbes.
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B. Membrane filter method
• A known volume ( e.g. 100 ml) of microbial suspension,
usually water sample, is spread over membrane filter.
• The bacteria are filtered out and retained on the surface of
the filter.
• The membrane filter is then dried, placed on appropriate
plate and incubated.
• Colony count will be used to determine concentration of
microbes in the suspension.
• This method is applied frequently to detection and
enumeration of coliform bacteria.
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A membrane filter method
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C. The Most Probable Number (MPN) method
• Multiple serial dilutions are performed to reach a point of
extinction.
• That is, a dilution level at which not even a single cell is
deposited into one or more of the multiple tubes at that
dilution level test.
• The patterns of positive & negative results is then used to
estimate the concentration of bacteria in the original
sample, that is, the MPN of bacteria.
• Estimation is made by comparing the observed pattern
results with a table of statistical probabilities for obtaining
those results.
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