Fundamental Food Microbiology, Third Edition - Fuad Fathir

DETECTION OF MICROORGANISMS IN FOOD AND FOOD ENVIRONMENT 565
hybridization of the DNA probe with cell DNA or rRNA; capture of the hybridized
DNA containing the probe; and measurement of either radioactivity or color production,
depending on the probe (i.e., if it is isotopic or nonisotopic). The methods
are specific and relatively more rapid than traditional methods.
2. Polymerase Chain Reaction (PCR)
The PCR technique helps amplify a segment of DNA located between two DNA
segments of known nucleotide sequence. It is thus possible to obtain large numbers
of copies of a specific DNA segment from a small sample (50 ng), which in turn
facilitates its detection by gel electrophoresis (nonisotopic) or by Southern hybridization
(both isotopic and nonisotopic). The method is very sensitive and may not
need preenrichment or enrichment steps. However, to eliminate confusion between
dead and viable cells of a pathogen as the source of DNA, a short preenrichment
step to increase the viable cell number of the target pathogen may be necessary. The
method has been developed to identify such pathogens as Lis. monocytogenes,
pathogenic Esc. coli, and Vib. vulnificus.
C. Measurement of Microbial Level
Several indirect methods to estimate microbial levels or loads in a food have been
developed. They are relatively more rapid than the traditional CFU enumeration
method and some are also automated.
1. Electrical Impedance Measurement
This method is based on the theory that as microorganisms grow in a liquid medium,
they metabolize the uncharged or weakly charged substrate to produce highly
charged small products (e.g., amino acids and lactic acid). This causes a change in
the electrical impedance (resistance to flow of an alternating current) and conductance
of the medium. By measuring these two parameters, bacterial growth in a
medium as a function of time at a given temperature can be monitored. Impedance
detection time (IDT) is the time required by microorganisms initially present in a
food to reach �10 6 /ml. It is inversely proportional to the initial microbial load: a
higher IDT means a lower initial load. From the IDT and the slope of the curve, the
initial load as well as generation time of a microbial population can be calculated.
Several automated systems are now commercially available that describe the technique
as well as methods to interpret the data. By using selective conditions, the
population of a specific bacterial group in a food can also be determined by this
method.
2. Bioluminescence Methods
The bioluminescence method measures the ATP content in a sample as an indirect
measurement of microbial load. 1,6 As only viable cells retain ATP, the amount of
ATP is regarded as directly related to the microbial load in the sample. Using the
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