Introduction
The raw, semi-processed and processed foods as well as the ingredients used in the
preparation of processed foods may contain several microorganisms. The microbial
examination of foods is generally done to know the presence or absence of specific
microorganisms, types of microorganisms, number of microorganisms and their products in
foods. The information on the microorganisms associated with food will help in;
Estimating shelf life (suitability for human consumption) by determining microbiological
quality of a food or constituent of food.
Identifying the cause of spoilage or presence of pathogens, when food is implicated in food
borne illness.
Methods of enumeration
Several methods are available for the enumeration of microorganisms from food. These can
be broadly divided in to direct methods or indirect enumeration methods based on whether
the microorganisms are counted directly or the products released by them is estimated.
I. Direct enumeration methods
a. Direct counting methods
Direct microscopic count (DMC)
Direct counting on membrane filters
b. Culture based methods
Plate count method
Pour plate method
Spread plate method
Most probable number (MPN) technique
II. Indirect enumeration methods
a. Alternative Methods (chemical and physical methods)
Dye reduction test
Electrical methods
ATP determination
b. Rapid Methods
Immunological methods
DNA/RNA methodology
Study of microorganisms in foods by conventional methods
Enumeration of microorganisms in foods is traditionally performed by directly counting all
microorganisms present in foods or by allowing them to develop in to colonies and then
counting.
3.2.1 Direct counting methods
Microorganisms present in the food can be counted by observing the food sample directly
or retaining the microorganisms on a filter paper by filtering the sample and then observing
under microscope.
A. Direct microscopic count (DMC)
DMC involves detecting the presence of microorganisms in food by microscopic observation.
It is a simple method and easy to perform.
DMC is performed by making a smear of food specimen/cultures on to microscopic slides,
staining with appropriate dye and viewing and counting all cells using microscope under oil
immersion objective. This method can be used only when microorganisms are present in
large numbers (106/ml). It is commonly used in dairy industry for assessing microbial quality
of raw milk and other dairy products.
Advantages
Rapid and easy enumeration
Can be employed to any foods (Ex: dried/frozen foods)
Simple to perform
Cell morphology can be assessed
Efficiency can be increased by using florescent probes
Disadvantages
Requires tiresome counting under microscope causing fatigue to analyst
Counts both viable and non-viable cells
Food particles may interfere with counting and mistaken for microorganisms
Cells may not be distributed uniformly (single cells/ clumps)
Some cells may not take up stain and missed while counting
DMC counts are always higher than standard plate count
Requires dilution of sample
B. Direct counting on membrane filters
Membrane fillers with pore size smaller (0.45 um) than bacteria retain bacteria and the
retained bacteria can be counted using microscope.
Procedure involved
Concentrating/collecting bacteria on polycarbonate filters by filtering known volume of
homogenized sample.
Staining and counting of retained bacteria.
Placing the membrane on a nutrient agar media or absorbent pad saturated with culture
media of choice, and incubating
Following growth , colonies are counted
Advantages
Well suited for samples containing low numbers of bacteria
Facilities concertinaing bacteria by filtering large volume of sample
Only small volume of food samples can be used for a single membrane
Efficiency of membrane filter method can be increased by staining with florescent dyes (Ex.
acridine orange) and observing under epiflorescence microscope (DEFT: Direct
Epiflorescence Filter technique). Viable cells fluoresce green and are counted. Non viable
cells appear orange. Acridine orange is an metachromatic fluorochrome which binds to
double stranded DNA of viable bacterial cells .
Can be used to enumerate microorganisms from a variety of foods (fresh fish, meat, fish/
meat products, water samples etc).
3.2.2 Culture based methods
Culture methods involve examination of microorganisms in food by encouraging them to
multiply in a liquid or solid media. On solid agar media bacteria develop as colonies and
counting such viable colonies gives microbial load in foods.
Enumerating microorganisms by culture based methods can be done by using plate count
methods or MPN technique.
Culture media
A wide variety of media with varied composition capable of supporting the growth are
available for the cultivation of different microorganisms.
The composition of the media varies depending on;
Group/type of microorganism to be studied
Overall purpose of the study
Whether to grow wide range of microorganisms or specific types
Resusitation of damaged but viable cells
Type of diagnostic information required
Ex: General purpose media: Plate count agar
Lactose broth: For Escherichia coli
Seective media: Baird parker agarfor Staphylococcus
Bismuth sulphite agar for Salmonella
TCBS for Vibrios
A. Plate count method
This method is variously referred to as total plate count (TPC), standard plate count (SPC) or
aerobic plate count (APC). It is most widely used conventional method for determining
viable cells or colony forming units (CFU) in foods.
SPC involves blending/ homogenizing the sample, serially diluting in appropriate diluent,
plating in or on suitable agar media, incubating at appropriate temperature for a given time,
and counting visible colonies as CFU.
Principle involved
SPC is based on the principle that each viable bacterial cells multiples and grows in to a
visible colony. Thus, counting number of colonies gives an idea about bacterial cells present
in a sample. Counts determined by taking average of replicate plates showing 30-300
colonies.
Factors affecting SPC
Sampling method employed.
Distribution of microorganisms in food.
Nature of food biota
Nutritional adequacy of plating media
Incubation temperature and time
Type of diluent used
Presence of other competing organisms etc.
Plating on selective media for specific organisms is limited by degree of inhibition and
effectiveness of selective/ differential agents employed.
SPC can be performed by pour plate method or spread plate method (surface plating
method)
a). Pour plate method
Appropriate dilution of the sample (1 ml) is mixed with agar medium, allowed to set,
incubated at appropriate temperature and colonies developed are counted. Here colonies
develop both on surface and subsurface of agar plate.
Proper mixing of sample with agar medium is necessary so as to get isolated colonies which
can be done by 2 ways.
One ml of appropriate sample dilution is added to petri-plate and about 15 ml of agar
medium is added and mixed by rotating the plate in clockwise and anticlockwise direction.
One ml of appropriate sample dilution is added to test tube containing about 15 ml of
molten agar medium, mixed by rolling the tube between the palm and poured to petriplates, allowed to set and incubated.
b). Spread plate method
Diluted sample (0.1 ml) is spread on the surface of pre-poured, hardened agar plates using
glass rod, incubated at appropriate temperature and colony developing on surface counted.
Advantages
Suitable for heat sensitive psychrotrophs in food as they do not come in contact with
molten agar.
Enables providing colony features useful in presumptive identification especially on selective
media.
Favours strict aerobes on surface, but microaerophils grow slowly
Disadvantage
Problem of spreaders and colony crowding makes the enumeration difficult.
B. Most probable number (MPN) technique
MPN is suited for enumerating the presence of low numbers of microorganisms in foods.
This involves inoculating replicate tubes of appropriate liquid media (3 or 5 tube) with three
different sample sizes/ dilutions of the material to be studied and incubating at appropriate
temperature. Then the absence or presence of growth is observed and MPN table consulted
to get probable number of organisms in the sample. MPN numbers are generally higher
than SPC.
Advantages
Relatively a simple method and assay to perform
Results are comparable from one laboratory to another
Specific group of organism determined by use of specific media.
Suitable for detecting organisms present in low numbers
Method of choice for coliform detection
Disadvantages
Requires use of large number of glassware and large volume of sample
Can not observe colony morphology
Lack of precision
Study of microorganisms by alternate and rapid methods
The types and number of microorganisms present in food sample can be determined either
by measuring the metabolites released by the microorganisms and constituents of microbial
cells employing physical /chemical methods, or by using rapid methods such as enzyme
linked immunosorbant assay (ELISA) or polymerase chain reaction (PCR).ck of precision.
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