Culture, Microscopic, and Sampling Methods
221
appropriately. Following growth, the colonies are enumerated. Alternatively, a DMC can be made. In
this case, the organisms collected on the membrane are viewed and counted microscopically following
appropriate staining, washing, and treatment of the membrane to render it transparent. These methods
are especially suited for samples that contain low numbers of bacteria. Although relatively large
volumes of water can be passed through a membrane without clogging it, only small samples of dilute
homogenates from certain foods can be used for a single membrane.
The overall efficiency of membrane filter methods for determining microbial numbers by the DMC
has been improved by the introduction of fluorescent dyes. The use of fluorescent dyes and epifluorescent microscopes to enumerate bacteria in waters has been employed rather widely since the early
1970s. Cellulose filters were among the earliest used; however, polycarbonate Nucleopore filters offer
the advantage of retaining all bacteria on top of the filter. When lake and ocean waters were examined
using the two kinds of membranes, counts were twice as high with Nucleopore membranes as with
cellulose membranes.
52
Direct Epifluorescent Filter Technique
This membrane filter technique may be viewed as an improved modification of the basic method. The
direct epifluorescent filter technique (DEFT) employs fluorescent dyes and fluorescent microscopy,
52
and it has been evaluated by a number of investigators as a rapid method for microorganisms in
foods. Typically, a diluted food homogenate is filtered through a 5-µm nylon filter, and the filtrate is
collected and treated with 2 ml of Triton X-100 and 0.5 ml of trypsin. The latter reagents are used
to lyse somatic cells and to prevent clogging of filters. After incubation, the treated filtrate is passed
through a 0.6-µm Nucleopore polycarbonate membrane, and the filter is stained with acridine orange.
After drying, the stained cells are enumerated by epifluorescence microscopy, and the number of cells
per gram is calculated by multiplying the average number per field by the microscope factor. Results
can be obtained in 25–30 minutes, and numbers as low as around 6,000 cfu/g can be obtained from
meats and milk products.
DEFT has been employed on milk
97 and found to compare favorably with results obtained by
aerobic plate count (APC), and standard Breed DMC on raw milk that contained between 5 × 10
3 and
5 × 10
8 bacteria per milliliter. It has been adapted to the enumeration of viable Gram-negative and all
Gram-positive bacteria in milk in about 10 minutes.
108 As few as 5,700 bacteria per milliliter could be
detected in heat-treated milk and milk products in about 20 minutes.
98 In a collaborative study by six
laboratories that compared DEFT and APC, the correlation coefficient was generally above 0.9, but
the repeatability of DEFT was 1.5 times worse than APC, and reproducibility was only three times
that for APC.
96 Solid foods can be examined by DEFT after proper filtrations, and <60,000 organisms
per gram could be detected in one study.
99 DEFT has been employed successfully to estimate numbers
of microorganisms on meat and poultry
120 and on food contact surfaces.
53 For more information, see
reference 95.
Microcolony-DEFT
DEFT allows for the direct microscopic determination of cells; microcolony-DEFT is a variation
that allows one to determine viable cells only. Typically, food homogenates are filtered through DEFT
membranes, and the latter are then placed on the surface of appropriate culture media and incubated for
microcolony development. A 3-hour incubation can be used for Gram-negative bacteria and a 6-hour
221
appropriately. Following growth, the colonies are enumerated. Alternatively, a DMC can be made. In
this case, the organisms collected on the membrane are viewed and counted microscopically following
appropriate staining, washing, and treatment of the membrane to render it transparent. These methods
are especially suited for samples that contain low numbers of bacteria. Although relatively large
volumes of water can be passed through a membrane without clogging it, only small samples of dilute
homogenates from certain foods can be used for a single membrane.
The overall efficiency of membrane filter methods for determining microbial numbers by the DMC
has been improved by the introduction of fluorescent dyes. The use of fluorescent dyes and epifluorescent microscopes to enumerate bacteria in waters has been employed rather widely since the early
1970s. Cellulose filters were among the earliest used; however, polycarbonate Nucleopore filters offer
the advantage of retaining all bacteria on top of the filter. When lake and ocean waters were examined
using the two kinds of membranes, counts were twice as high with Nucleopore membranes as with
cellulose membranes.
52
Direct Epifluorescent Filter Technique
This membrane filter technique may be viewed as an improved modification of the basic method. The
direct epifluorescent filter technique (DEFT) employs fluorescent dyes and fluorescent microscopy,
52
and it has been evaluated by a number of investigators as a rapid method for microorganisms in
foods. Typically, a diluted food homogenate is filtered through a 5-µm nylon filter, and the filtrate is
collected and treated with 2 ml of Triton X-100 and 0.5 ml of trypsin. The latter reagents are used
to lyse somatic cells and to prevent clogging of filters. After incubation, the treated filtrate is passed
through a 0.6-µm Nucleopore polycarbonate membrane, and the filter is stained with acridine orange.
After drying, the stained cells are enumerated by epifluorescence microscopy, and the number of cells
per gram is calculated by multiplying the average number per field by the microscope factor. Results
can be obtained in 25–30 minutes, and numbers as low as around 6,000 cfu/g can be obtained from
meats and milk products.
DEFT has been employed on milk
97 and found to compare favorably with results obtained by
aerobic plate count (APC), and standard Breed DMC on raw milk that contained between 5 × 10
3 and
5 × 10
8 bacteria per milliliter. It has been adapted to the enumeration of viable Gram-negative and all
Gram-positive bacteria in milk in about 10 minutes.
108 As few as 5,700 bacteria per milliliter could be
detected in heat-treated milk and milk products in about 20 minutes.
98 In a collaborative study by six
laboratories that compared DEFT and APC, the correlation coefficient was generally above 0.9, but
the repeatability of DEFT was 1.5 times worse than APC, and reproducibility was only three times
that for APC.
96 Solid foods can be examined by DEFT after proper filtrations, and <60,000 organisms
per gram could be detected in one study.
99 DEFT has been employed successfully to estimate numbers
of microorganisms on meat and poultry
120 and on food contact surfaces.
53 For more information, see
reference 95.
Microcolony-DEFT
DEFT allows for the direct microscopic determination of cells; microcolony-DEFT is a variation
that allows one to determine viable cells only. Typically, food homogenates are filtered through DEFT
membranes, and the latter are then placed on the surface of appropriate culture media and incubated for
microcolony development. A 3-hour incubation can be used for Gram-negative bacteria and a 6-hour