226 Modern Food Microbiology
microscope (oil immersion objective). DMCs are most widely used in the dairy industry for assessing
the microbial quality of raw milk and other dairy products, and the specific method employed is that
originally developed by R.S. Breed (Breed count). Briefly, the method consists of adding 0.01 ml of a
sample to a 1-cm
2 area on a microscope slide, and following fixing, defatting of sample, and staining,
the organisms or clumps of organisms are enumerated. The latter involves the use of a calibrated
microscope (for further details, see reference 73). The method lends itself to the rapid microbiological
examination of other food products, such as dried and frozen foods.
Among the advantages of DMC are that it is rapid and simple, cell morphology can be assessed,
and it lends itself to fluorescent probes for improved efficiency. Among its disadvantages are that
it is a microscopic method and therefore fatiguing to the analyst, both viable and nonviable cells
are enumerated, food particles are not always distinguishable from microorganisms, microbial cells
are not uniformly distributed relative to single cells and clumps, some cells do not take the stain
well and may not be counted, and DMC counts are invariably higher than counts by SPC. In spite
of its drawbacks, it remains the fastest way to make an assessment of microbial cells in a food
product.
A slide method to detect and enumerate viable cells has been developed.
11 The method employs the
use of the tetrazolium salt ( p-iodophenyl-3- p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Cells
are exposed to filter-sterilized INT for 10 minutes at 37
◦ C in a water bath followed by filtration on
0.45-µm membranes. Following drying of membranes for 10 minutes at 50
◦ C, the special membranes
are mounted in cottonseed oil and viewed with coverslip in place. The method was found to be
workable for pure cultures of bacteria and yeasts, but it underestimated APC by 1–1.5 log cycles
when compared using milk. By use of fluorescence microscopy and Viablue (modified aniline blue
fluorochrome), viable yeast cells could be differentiated from nonviable cells.
60,67 Viable cells can
be determined by staining with acridine orange (0.01%) followed by epifluorescence microscopy and
enumeration of those that fluoresce orange. This is the gist of the acridine orange direct count (AODC)
method.
Howard Mold Counts
This is a microscope slide method developed by B.J. Howard in 1911 primarily for the purpose of
monitoring tomato products. The method requires the use of a special chamber (slide) designed to
enumerate mold mycelia. It is not valid on tomato products that have been comminuted. Similar to the
Howard mold count is a method for quantifying Geotrichum candidum in canned beverages and fruits,
and this method, as well as the Howard mold count method, is fully described by AOAC.
89 The DEFT
method has been shown to correlate well with the Howard mold count method on autoclaved and
unautoclaved tomato concentrate, and it could be used as an alternative to the Howard mold count.
100
MICROBIOLOGICAL EXAMINATION OF SURFACES
The need to maintain food contact surfaces in a hygienic state is of obvious importance. The
primary problem that has to be overcome when examining surfaces or utensils for microorganisms is
the removal of a significant percentage of the resident biota. Although a given method may not recover
all organisms, its consistent use in specified areas of a food-processing plant can still provide valuable
information as long as it is realized that not all organisms are being recovered. The most commonly
used methods for surface assessment in food operations are presented below.
microscope (oil immersion objective). DMCs are most widely used in the dairy industry for assessing
the microbial quality of raw milk and other dairy products, and the specific method employed is that
originally developed by R.S. Breed (Breed count). Briefly, the method consists of adding 0.01 ml of a
sample to a 1-cm
2 area on a microscope slide, and following fixing, defatting of sample, and staining,
the organisms or clumps of organisms are enumerated. The latter involves the use of a calibrated
microscope (for further details, see reference 73). The method lends itself to the rapid microbiological
examination of other food products, such as dried and frozen foods.
Among the advantages of DMC are that it is rapid and simple, cell morphology can be assessed,
and it lends itself to fluorescent probes for improved efficiency. Among its disadvantages are that
it is a microscopic method and therefore fatiguing to the analyst, both viable and nonviable cells
are enumerated, food particles are not always distinguishable from microorganisms, microbial cells
are not uniformly distributed relative to single cells and clumps, some cells do not take the stain
well and may not be counted, and DMC counts are invariably higher than counts by SPC. In spite
of its drawbacks, it remains the fastest way to make an assessment of microbial cells in a food
product.
A slide method to detect and enumerate viable cells has been developed.
11 The method employs the
use of the tetrazolium salt ( p-iodophenyl-3- p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Cells
are exposed to filter-sterilized INT for 10 minutes at 37
◦ C in a water bath followed by filtration on
0.45-µm membranes. Following drying of membranes for 10 minutes at 50
◦ C, the special membranes
are mounted in cottonseed oil and viewed with coverslip in place. The method was found to be
workable for pure cultures of bacteria and yeasts, but it underestimated APC by 1–1.5 log cycles
when compared using milk. By use of fluorescence microscopy and Viablue (modified aniline blue
fluorochrome), viable yeast cells could be differentiated from nonviable cells.
60,67 Viable cells can
be determined by staining with acridine orange (0.01%) followed by epifluorescence microscopy and
enumeration of those that fluoresce orange. This is the gist of the acridine orange direct count (AODC)
method.
Howard Mold Counts
This is a microscope slide method developed by B.J. Howard in 1911 primarily for the purpose of
monitoring tomato products. The method requires the use of a special chamber (slide) designed to
enumerate mold mycelia. It is not valid on tomato products that have been comminuted. Similar to the
Howard mold count is a method for quantifying Geotrichum candidum in canned beverages and fruits,
and this method, as well as the Howard mold count method, is fully described by AOAC.
89 The DEFT
method has been shown to correlate well with the Howard mold count method on autoclaved and
unautoclaved tomato concentrate, and it could be used as an alternative to the Howard mold count.
100
MICROBIOLOGICAL EXAMINATION OF SURFACES
The need to maintain food contact surfaces in a hygienic state is of obvious importance. The
primary problem that has to be overcome when examining surfaces or utensils for microorganisms is
the removal of a significant percentage of the resident biota. Although a given method may not recover
all organisms, its consistent use in specified areas of a food-processing plant can still provide valuable
information as long as it is realized that not all organisms are being recovered. The most commonly
used methods for surface assessment in food operations are presented below.