Culture, Microscopic, and Sampling Methods
225
DYE REDUCTION
Two dyes are commonly employed in this procedure to estimate the number of viable organisms in
suitable products: methylene blue and resazurin. To conduct a dye-reduction test, properly prepared
supernatants of foods are added to standard solutions of either dye for reduction from blue to white
for methylene blue; and from slate blue to pink or white for resazurin. The time for dye reduction to
occur is inversely proportional to the number of organisms in the sample.
Methylene blue and resazurin reduction by 100 cultures was studied in milk; with two exceptions,
a good agreement was found between numbers of bacteria and time needed for reduction of the two
dyes.
43 In a study of resazurin reduction as a rapid method for assessing ground beef spoilage, reduction
to the colorless state, odor scores, and SPC correlated significantly.
111 One of the problems of using dye
reduction for some foods is the existence of inherent reductive substances. This is true of raw meats, and
Austin and Thomas
9 reported that resazurin reduction was less useful than with cooked meats. For the
latter, approximately 600 samples were successfully evaluated by resazurin reduction by adding 20 ml
of a 0.0001% resazurin solution to 100 g of sliced meat in a plastic pouch. Another way of getting around
the reductive compounds in fresh meats is to homogenize samples by Stomacher rather than by Waring
blender. By using Stomacher homogenates, raw meat was successfully evaluated by resazurin reduction
when Stomacher homogenates were added to a solution of resazurin in 10% skim milk.
54 Stomacher
homogenates contained less disrupted tissue and, consequently, lower concentrations of reductive
compounds. The method of Holley et al.
54 was evaluated further by Dodsworth and Kempton,
29 who
found that raw meat with an SPC >10
7 bacteria per gram could be detected within 2 hours. When
compared to nitroblue tetrazolium (NT) and indophenyl nitrophenyl tetrazolium (INT), resazurin
produced faster results.
104 With surface samples from sheep carcasses, resazurin was reduced in 30
minutes by 18,000 cfu/m
2 , NT in 600 minutes by 21,000 cfu/m
2 , and INT in 660 minutes by 18,000
cfu/m
2 .
108 Methylene blue reduction was compared to APC on 389 samples of frozen peas, and the
results were linear over the APC range of log 2–6 cfus. Average decolorization times were 8 and 11
hours for 10
5 and 10
4 cfu/g, respectively.
Dye-reduction tests have a long history of use in the dairy industry for assessing the overall microbial
quality of raw milk. Among their advantages are that they are simple, rapid, and inexpensive; and only
viable cells actively reduce the dyes. Disadvantages are that not all organisms reduce the dyes equally,
and they are not applicable to food specimens that contain reductive enzymes unless special steps are
employed. The use of fluorogenic and chromogenic substrates in food microbiology is discussed in
Chapter 11.
ROLL TUBES
Screw-capped tubes or bottles of varying sizes are used in this method. Predetermined amounts of
the melted and inoculated agar are added to the tube and the agar is made to solidify as a thin layer on
the inside of the vessel. Following appropriate incubation, colonies are counted by rotating the vessel.
It has been found to be an excellent method for enumerating fastidious anaerobes. For a review of the
method, see Anderson and Fung.
3
DIRECT MICROSCOPIC COUNT (DMC)
In its simplest form, the DMC consists of making smears of food specimens or cultures onto a
microscope slide, staining with an appropriate dye, and viewing and counting cells with the aid of a
225
DYE REDUCTION
Two dyes are commonly employed in this procedure to estimate the number of viable organisms in
suitable products: methylene blue and resazurin. To conduct a dye-reduction test, properly prepared
supernatants of foods are added to standard solutions of either dye for reduction from blue to white
for methylene blue; and from slate blue to pink or white for resazurin. The time for dye reduction to
occur is inversely proportional to the number of organisms in the sample.
Methylene blue and resazurin reduction by 100 cultures was studied in milk; with two exceptions,
a good agreement was found between numbers of bacteria and time needed for reduction of the two
dyes.
43 In a study of resazurin reduction as a rapid method for assessing ground beef spoilage, reduction
to the colorless state, odor scores, and SPC correlated significantly.
111 One of the problems of using dye
reduction for some foods is the existence of inherent reductive substances. This is true of raw meats, and
Austin and Thomas
9 reported that resazurin reduction was less useful than with cooked meats. For the
latter, approximately 600 samples were successfully evaluated by resazurin reduction by adding 20 ml
of a 0.0001% resazurin solution to 100 g of sliced meat in a plastic pouch. Another way of getting around
the reductive compounds in fresh meats is to homogenize samples by Stomacher rather than by Waring
blender. By using Stomacher homogenates, raw meat was successfully evaluated by resazurin reduction
when Stomacher homogenates were added to a solution of resazurin in 10% skim milk.
54 Stomacher
homogenates contained less disrupted tissue and, consequently, lower concentrations of reductive
compounds. The method of Holley et al.
54 was evaluated further by Dodsworth and Kempton,
29 who
found that raw meat with an SPC >10
7 bacteria per gram could be detected within 2 hours. When
compared to nitroblue tetrazolium (NT) and indophenyl nitrophenyl tetrazolium (INT), resazurin
produced faster results.
104 With surface samples from sheep carcasses, resazurin was reduced in 30
minutes by 18,000 cfu/m
2 , NT in 600 minutes by 21,000 cfu/m
2 , and INT in 660 minutes by 18,000
cfu/m
2 .
108 Methylene blue reduction was compared to APC on 389 samples of frozen peas, and the
results were linear over the APC range of log 2–6 cfus. Average decolorization times were 8 and 11
hours for 10
5 and 10
4 cfu/g, respectively.
Dye-reduction tests have a long history of use in the dairy industry for assessing the overall microbial
quality of raw milk. Among their advantages are that they are simple, rapid, and inexpensive; and only
viable cells actively reduce the dyes. Disadvantages are that not all organisms reduce the dyes equally,
and they are not applicable to food specimens that contain reductive enzymes unless special steps are
employed. The use of fluorogenic and chromogenic substrates in food microbiology is discussed in
Chapter 11.
ROLL TUBES
Screw-capped tubes or bottles of varying sizes are used in this method. Predetermined amounts of
the melted and inoculated agar are added to the tube and the agar is made to solidify as a thin layer on
the inside of the vessel. Following appropriate incubation, colonies are counted by rotating the vessel.
It has been found to be an excellent method for enumerating fastidious anaerobes. For a review of the
method, see Anderson and Fung.
3
DIRECT MICROSCOPIC COUNT (DMC)
In its simplest form, the DMC consists of making smears of food specimens or cultures onto a
microscope slide, staining with an appropriate dye, and viewing and counting cells with the aid of a