Conventional, Molecular, and Biosensor-Based Detection Methods ◾ 541
Indirect Estimation
Dilution to Extinction in Nonselective Broths
This method consists of serial dilution of a sample and transfer of an aliquot (usually 1.0 mL or 0.1 mL)
to a final 5 mL or 10 mL of a nonselective broth, such as tryptic soy broth. This is followed by incubation of the tubes at a specific temperature for a specific period of time, which depends on the specific
microbial group being investigated. The tubes are then examined for the presence or absence of growth
(from the turbidity of the broth). Using the highest sample dilution that gave growth and assuming
that this tube had one to nine viable cells of the group of interest, microbial numbers per milliliter or
gram sample are estimated. The estimated numbers, however, can vary widely from the actual numbers. This method is not used much for the microbiological estimation in food. 4
Most Probable Number (MPN) in Selective Broth
In this method, aliquots from a serially diluted sample are inoculated in a broth (in tubes) having
one or more selective agents that facilitate growth of selected microbial groups present in a food.
Generally, three or five broth tubes in each dilution and a minimum of three consecutive dilutions
are used. After incubation at a recommended temperature and time, the broth tubes in each dilution are scored for the presence and absence of growth. From the number of tubes showing growth
in each of the three successive dilutions, the number of viable cells of the specific microbial group
can be estimated from the available statistically calculated tables. This method also gives wide
variation. MPN methods are used quite often for the estimation of coliforms and fecal coliforms
in foods and water using brilliant green lactose bile broth and E-C broth, respectively. 3,4 A commercial automated MPN analysis system (TEMPO ® ) is now available for food quality assurance.
Dye Reduction Test
This method is based on the principle that some dyes, such as methylene blue and resazurin, are
colored in oxidized states but colorless under reduced conditions. This change can occur as a result
of microbial metabolism and growth. It is assumed that the rate of reduction during incubation
of a specific concentration of methylene blue added to a food is directly proportional to the initial
microbial load in the food. However, as microbial groups in a mixed culture in a food differ in the
rate of metabolism and growth and ability to reduce the environment, this method is not considered very accurate and effective with different foods. This method is generally used to determine
the microbiological quality of raw milk.
Enumeration of Injured Microbial Groups by Selective Media 7
Sublethally injured coliforms and pathogenic bacterial cells, when enumerated by selective agar
media, may die as a result of their developed sensitivity to selective agents in the media and thus
cannot be detected (see Chapter 10). They are first allowed to repair the injury in nonselective
media (broth or agar) for a short period, which enables the cells to regain resistance to the selective agents. Following repair, they can be exposed to selective media. For the enumeration of
coliforms in food that may contain injured coliforms, the diluted sample can first be pour-plated
in nonselective PCA and incubated for one to two hours at 25°C–35°C, thus enabling the repair
of the cells. Double-strength violet red bile agar can then be overlaid on the plates and the plates
incubated at 35°C for 24 hours for selective growth of coliforms.
Indirect Estimation
Dilution to Extinction in Nonselective Broths
This method consists of serial dilution of a sample and transfer of an aliquot (usually 1.0 mL or 0.1 mL)
to a final 5 mL or 10 mL of a nonselective broth, such as tryptic soy broth. This is followed by incubation of the tubes at a specific temperature for a specific period of time, which depends on the specific
microbial group being investigated. The tubes are then examined for the presence or absence of growth
(from the turbidity of the broth). Using the highest sample dilution that gave growth and assuming
that this tube had one to nine viable cells of the group of interest, microbial numbers per milliliter or
gram sample are estimated. The estimated numbers, however, can vary widely from the actual numbers. This method is not used much for the microbiological estimation in food. 4
Most Probable Number (MPN) in Selective Broth
In this method, aliquots from a serially diluted sample are inoculated in a broth (in tubes) having
one or more selective agents that facilitate growth of selected microbial groups present in a food.
Generally, three or five broth tubes in each dilution and a minimum of three consecutive dilutions
are used. After incubation at a recommended temperature and time, the broth tubes in each dilution are scored for the presence and absence of growth. From the number of tubes showing growth
in each of the three successive dilutions, the number of viable cells of the specific microbial group
can be estimated from the available statistically calculated tables. This method also gives wide
variation. MPN methods are used quite often for the estimation of coliforms and fecal coliforms
in foods and water using brilliant green lactose bile broth and E-C broth, respectively. 3,4 A commercial automated MPN analysis system (TEMPO ® ) is now available for food quality assurance.
Dye Reduction Test
This method is based on the principle that some dyes, such as methylene blue and resazurin, are
colored in oxidized states but colorless under reduced conditions. This change can occur as a result
of microbial metabolism and growth. It is assumed that the rate of reduction during incubation
of a specific concentration of methylene blue added to a food is directly proportional to the initial
microbial load in the food. However, as microbial groups in a mixed culture in a food differ in the
rate of metabolism and growth and ability to reduce the environment, this method is not considered very accurate and effective with different foods. This method is generally used to determine
the microbiological quality of raw milk.
Enumeration of Injured Microbial Groups by Selective Media 7
Sublethally injured coliforms and pathogenic bacterial cells, when enumerated by selective agar
media, may die as a result of their developed sensitivity to selective agents in the media and thus
cannot be detected (see Chapter 10). They are first allowed to repair the injury in nonselective
media (broth or agar) for a short period, which enables the cells to regain resistance to the selective agents. Following repair, they can be exposed to selective media. For the enumeration of
coliforms in food that may contain injured coliforms, the diluted sample can first be pour-plated
in nonselective PCA and incubated for one to two hours at 25°C–35°C, thus enabling the repair
of the cells. Double-strength violet red bile agar can then be overlaid on the plates and the plates
incubated at 35°C for 24 hours for selective growth of coliforms.