Culture, Microscopic, and Sampling Methods
223
MICROSCOPE COLONY COUNTS
Microscope colony count methods involve the counting of microcolonies that develop in agar layered
over microscope slides. The first was that of Frost, which consisted of spreading 0.1 ml of milk-agar
mixture over a 4-cm
2 area on a glass slide. Following incubation, drying, and staining, microcolonies
are counted with the aid of a microscope. In another method, 2 ml of melted agar are mixed with 2 ml
of warmed milk and, after mixing, 0.1 ml of the inoculated agar is spread over a 4-cm
2 area. Following
staining with thionin blue, the slide is viewed with the 16-mm objective of a wide-field microscope.
65
AGAR DROPLETS
In the agar droplet method of Sharpe and Kilsby,
115 the food homogenate is diluted in tubes of melted
agar (at 45
◦ C). For each food sample, three tubes of agar are used, the first tube being inoculated with
1 ml of food homogenate. After mixing, a sterile capillary pipette (ideally delivering 0.033 ml/drop)
is used to transfer a line of 5 × 0.1-ml droplets to the bottom of an empty Petri dish. With the same
capillary pipette, three drops (0.1 ml) from the first 9-ml tube are transferred to the second tube, and,
after mixing, another line of 5 × 0.1-ml droplets is placed next to the first. This step is repeated for the
third tube of agar. Petri plates containing the agar droplets are incubated for 24 hours, and colonies
are enumerated with the aid of a 10× viewer. Results using this method from pure cultures, meats,
and vegetables compared favorably to those obtained by conventional plate counts; droplet counts
from ground meat were slightly higher than plate counts. The method was about three times faster,
and 24-hour incubations gave counts equal to those obtained after 48 hours by the conventional plate
count. Dilution blanks are not required, and only one Petri dish per sample is needed.
DRY FILM AND RELATED METHODS
A rehydratable dry film method consisting of two plastic films attached together on one side and
coated with culture medium ingredients and a cold-water-soluble jelling agent was developed by the
3M Company and designated Petrifilm. The method can be used with nonselective ingredients to make
aerobic plate counts (APCs), and, with selective ingredients, certain specific groups can be detected.
Use of this method to date indicates that it is an acceptable alternative to SPC methods that employ
Petri dishes, and it has been approved by AOAC.
For use, 1 ml of diluent is placed between the two films and spread over the nutrient area by pressing
with a special flat-surface device. Following incubations, microcolonies appear red on the nonselective
film because of the presence of a tetrazolium dye in the nutrient phase. In addition to its use for APC,
Petrifilm methods exist for the detection and enumeration of specific groups, such as coliforms and
E. coli. For APC determination on 108 milk samples, this dry film method correlated highly with the
conventional plate count method and was shown to be a suitable alternative.
45 When compared to violet
red bile agar (VRBA) and MPN for coliform enumeration on 120 samples of raw milk, Petrifilm-VRB
compared favorably to VRBA counts, and both were comparable to MPN results.
84 A dry medium
EC (E. coli) count method has been developed; it employs the substrate for β-glucuronidase so that
E. coli is distinguished from other coliforms by the formation of a blue halo around colonies. When
compared to the classical confirmed MPN and VRBA on 319 food samples, the EC dry medium gave
comparable results.
75
Redigel is a plating medium that does not use agar as a solidifying agent. It is employed by inoculating
presterilized ingredients with food homogenates or diluents followed by mixing and holding to allow
223
MICROSCOPE COLONY COUNTS
Microscope colony count methods involve the counting of microcolonies that develop in agar layered
over microscope slides. The first was that of Frost, which consisted of spreading 0.1 ml of milk-agar
mixture over a 4-cm
2 area on a glass slide. Following incubation, drying, and staining, microcolonies
are counted with the aid of a microscope. In another method, 2 ml of melted agar are mixed with 2 ml
of warmed milk and, after mixing, 0.1 ml of the inoculated agar is spread over a 4-cm
2 area. Following
staining with thionin blue, the slide is viewed with the 16-mm objective of a wide-field microscope.
65
AGAR DROPLETS
In the agar droplet method of Sharpe and Kilsby,
115 the food homogenate is diluted in tubes of melted
agar (at 45
◦ C). For each food sample, three tubes of agar are used, the first tube being inoculated with
1 ml of food homogenate. After mixing, a sterile capillary pipette (ideally delivering 0.033 ml/drop)
is used to transfer a line of 5 × 0.1-ml droplets to the bottom of an empty Petri dish. With the same
capillary pipette, three drops (0.1 ml) from the first 9-ml tube are transferred to the second tube, and,
after mixing, another line of 5 × 0.1-ml droplets is placed next to the first. This step is repeated for the
third tube of agar. Petri plates containing the agar droplets are incubated for 24 hours, and colonies
are enumerated with the aid of a 10× viewer. Results using this method from pure cultures, meats,
and vegetables compared favorably to those obtained by conventional plate counts; droplet counts
from ground meat were slightly higher than plate counts. The method was about three times faster,
and 24-hour incubations gave counts equal to those obtained after 48 hours by the conventional plate
count. Dilution blanks are not required, and only one Petri dish per sample is needed.
DRY FILM AND RELATED METHODS
A rehydratable dry film method consisting of two plastic films attached together on one side and
coated with culture medium ingredients and a cold-water-soluble jelling agent was developed by the
3M Company and designated Petrifilm. The method can be used with nonselective ingredients to make
aerobic plate counts (APCs), and, with selective ingredients, certain specific groups can be detected.
Use of this method to date indicates that it is an acceptable alternative to SPC methods that employ
Petri dishes, and it has been approved by AOAC.
For use, 1 ml of diluent is placed between the two films and spread over the nutrient area by pressing
with a special flat-surface device. Following incubations, microcolonies appear red on the nonselective
film because of the presence of a tetrazolium dye in the nutrient phase. In addition to its use for APC,
Petrifilm methods exist for the detection and enumeration of specific groups, such as coliforms and
E. coli. For APC determination on 108 milk samples, this dry film method correlated highly with the
conventional plate count method and was shown to be a suitable alternative.
45 When compared to violet
red bile agar (VRBA) and MPN for coliform enumeration on 120 samples of raw milk, Petrifilm-VRB
compared favorably to VRBA counts, and both were comparable to MPN results.
84 A dry medium
EC (E. coli) count method has been developed; it employs the substrate for β-glucuronidase so that
E. coli is distinguished from other coliforms by the formation of a blue halo around colonies. When
compared to the classical confirmed MPN and VRBA on 319 food samples, the EC dry medium gave
comparable results.
75
Redigel is a plating medium that does not use agar as a solidifying agent. It is employed by inoculating
presterilized ingredients with food homogenates or diluents followed by mixing and holding to allow