542 ◾ Fundamental Food Microbiology
Dilution Scheme, Plating, Incubation, Selection of
Plates for Counting CFU, and Reporting Results
The standard and recommended method books listed above have detailed descriptions on these
steps. They should be followed as accurately as possible. The average of CFU from duplicate or
triplicate plates from a selected dilution should be used to report the counts of the specific microbial group per mL or g or cm 2 of the food sample. This data should be used in the proper interpretation of microbiological quality of the food.
Qualitative Methods to isolate Microorganisms in Foods
Isolation of Pathogens 3,8,9
The main objective of this method is to determine if a sample does or does not contain viable cells
or spores of a specific pathogen. Foods are tested for several pathogens, such as Salmonella, Esc. coli
O157:H7, Shiga-toxin producing Esc. coli (STEC), Lis. monocytogenes, Vibrio cholerae, and Shigella
spp., by the specific isolation procedure when necessary. For other pathogens, such as enteropathogenic Esc. coli, Yer. enterocolitica, and Cam. jejuni, isolation procedures are not generally used;
instead enumeration procedures are used.
An isolation procedure generally contains several steps, such as nonselective preenrichment
followed by selective enrichment and then testing on an agar medium containing selective and
differential agents. It is assumed that a food normally contains a low population of a pathogen
as compared to the associative microorganisms, and the pathogens could be in injured state. The
food sample (e.g., 25 g) is first preenriched in a nonselective broth (225 mL) and incubated for
the injured cells to repair and then multiply in order to reach moderately high numbers (along
with many other associated microorganisms). An aliquot is then transferred from the preenrichment broth to a selective enrichment broth and incubated. It is expected that, during incubation,
the specific pathogen and closely related microorganisms will selectively grow to a high number
while many of the associated microorganisms will not grow. A small amount (≈0.01 mL) of the
enrichment broth is then streaked on the surface of a prepoured selective-differential agar medium
plate, which is then incubated for a specified time for the colonies to develop. From the differential
colony characteristics, the presence of a specific pathogen can be tentatively established.
This is generally considered a presumptive test. For confirmation, the cells from the characteristic colonies are purified and examined by the recommended methods for biochemical reaction
profiles, serological reaction against a specific antibody, an immunochromatographic lateral flow
assay, or by the polymerase chain reaction (PCR) discussed below. Isolation of a pathogen using
the conventional methods can take 10–12 days, depending upon a particular species.
test for Bacterial toxins in Foods
Sta. aureus, Clo. botulinum, Bacillus cereus, Vibrio parahaemolyticus, and molds, while growing in
foods, are capable of producing toxins and causing intoxication or food poisoning among the consumers. Specific methods have been developed to test the presence of toxins in the suspected foods. 3,10
To detect Sta. aureus enterotoxin in foods, recommended methods are to initially extract the
toxin from 100 g of the food and then to concentrate the toxin in 0.2 mL sterile water or 0.2 M
saline. The presence of toxin in the concentrated extract is then assayed against a specific antibody
Dilution Scheme, Plating, Incubation, Selection of
Plates for Counting CFU, and Reporting Results
The standard and recommended method books listed above have detailed descriptions on these
steps. They should be followed as accurately as possible. The average of CFU from duplicate or
triplicate plates from a selected dilution should be used to report the counts of the specific microbial group per mL or g or cm 2 of the food sample. This data should be used in the proper interpretation of microbiological quality of the food.
Qualitative Methods to isolate Microorganisms in Foods
Isolation of Pathogens 3,8,9
The main objective of this method is to determine if a sample does or does not contain viable cells
or spores of a specific pathogen. Foods are tested for several pathogens, such as Salmonella, Esc. coli
O157:H7, Shiga-toxin producing Esc. coli (STEC), Lis. monocytogenes, Vibrio cholerae, and Shigella
spp., by the specific isolation procedure when necessary. For other pathogens, such as enteropathogenic Esc. coli, Yer. enterocolitica, and Cam. jejuni, isolation procedures are not generally used;
instead enumeration procedures are used.
An isolation procedure generally contains several steps, such as nonselective preenrichment
followed by selective enrichment and then testing on an agar medium containing selective and
differential agents. It is assumed that a food normally contains a low population of a pathogen
as compared to the associative microorganisms, and the pathogens could be in injured state. The
food sample (e.g., 25 g) is first preenriched in a nonselective broth (225 mL) and incubated for
the injured cells to repair and then multiply in order to reach moderately high numbers (along
with many other associated microorganisms). An aliquot is then transferred from the preenrichment broth to a selective enrichment broth and incubated. It is expected that, during incubation,
the specific pathogen and closely related microorganisms will selectively grow to a high number
while many of the associated microorganisms will not grow. A small amount (≈0.01 mL) of the
enrichment broth is then streaked on the surface of a prepoured selective-differential agar medium
plate, which is then incubated for a specified time for the colonies to develop. From the differential
colony characteristics, the presence of a specific pathogen can be tentatively established.
This is generally considered a presumptive test. For confirmation, the cells from the characteristic colonies are purified and examined by the recommended methods for biochemical reaction
profiles, serological reaction against a specific antibody, an immunochromatographic lateral flow
assay, or by the polymerase chain reaction (PCR) discussed below. Isolation of a pathogen using
the conventional methods can take 10–12 days, depending upon a particular species.
test for Bacterial toxins in Foods
Sta. aureus, Clo. botulinum, Bacillus cereus, Vibrio parahaemolyticus, and molds, while growing in
foods, are capable of producing toxins and causing intoxication or food poisoning among the consumers. Specific methods have been developed to test the presence of toxins in the suspected foods. 3,10
To detect Sta. aureus enterotoxin in foods, recommended methods are to initially extract the
toxin from 100 g of the food and then to concentrate the toxin in 0.2 mL sterile water or 0.2 M
saline. The presence of toxin in the concentrated extract is then assayed against a specific antibody