Fundamental Food Microbiology, Third Edition - Fuad Fathir

560 FUNDAMENTAL FOOD MICROBIOLOGY
compounds, they allow selective growth and colony formation of several closely
related species. The differential agents then help differentiate these species or
groups from one another by their specific colony characteristics. 1–3
B. Indirect Estimation
1. Dilution to Extinction in Nonselective Broths
The method consists of serially diluting a sample and transfer of an aliquot (usually
1.0 ml or 0.1 ml) to a final 5 or 10 ml of a nonselective broth, such as tryptic soy
broth. This is followed by incubating 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 and absence of growth (from the
turbidity of the broth). With 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. 1–3
2. 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 incubating at 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 to estimate coliforms and
fecal coliforms in foods and water by using brilliant green lactose bile broth and E-
C broth, respectively. 1–3
3. Dye Reduction Test
The 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 because 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, because 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. 1–3