Culture, Microscopic, and Sampling Methods
229
surfaces.
41 In a recent study, the swab, RODAC, and adhesive tape (Mylar) methods were compared for
the examination of pork carcasses, and the correlation between adhesive tape and RODAC was better
than that between adhesive tape and swab or between RODAC and swab.
25 Plastic strips attached to
pads containing culture media have been used to monitor microorganisms on bottles.
27
Swab/Agar Slant
The swab/agar slant method described in 1962 by N.-H. Hansen has been used with success by some
European workers. The method involves sampling with cotton swabs that are transferred directly to
slants. Following incubation, slants are grouped into one-half log 10 units based on estimated numbers
of developed colonies. The average number of colonies is determined by plotting the distribution on
probability paper. A somewhat similar method, the swab/agar plate, was proposed by ∅lgaard.
90 It
requires a template, a comparator disc, and a reference table, making it a bit more complicated than
the other methods noted.
Ultrasonic Devices
Ultrasonic devices have been used to assess the microbiological contamination of surfaces, but the
surfaces to be examined must be small in size and removable so that they can be placed inside a
container immersed in diluent. Once the container is placed in an ultrasonic apparatus, the energy
generated effects the release of microorganisms into the diluent. A more practical use of ultrasonic
energy may be the removal of bacteria from cotton swabs in the swab-rinse method.
102
Spray Gun
A spray gun method was devised by Clark
22,23 based on the impingement of a spray of washing
solution against a circumscribed area of surface and the subsequent plating of the washing solution.
Although the device is portable, a source of air pressure is necessary. It was shown to be much more
effective than the swab method in removing bacteria from meat surfaces.
METABOLICALLY INJURED ORGANISMS
When microorganisms are subjected to environmental stresses such as sublethal heat and freezing,
many of the individual cells undergo metabolic injury, resulting in their inability to form colonies on
selective media that uninjured cells can tolerate. Whether a culture has suffered metabolic injury can be
determined by plating aliquots separately on a nonselective and a selective medium and enumerating
the colonies that develop after suitable incubation. The colonies that develop on the nonselective
medium represent both injured and uninjured cells, whereas only the uninjured cells develop on the
selective medium. The difference between the number of colonies on the two media is a measure of
the number of injured cells in the original culture or population. This principle is illustrated in Figure
10–2 by data from Tomlins et al.
127 on sublethal heat injury of S. aureus. These investigators subjected
the organism to 52
◦ C for 15 minutes in a phosphate buffer at pH 7.2 to inflict cell injury. The plating
of cells at zero time and up to 15 minutes of heating on nonselective trypticase soy agar (TSA) and
selective TSA + 7.0% NaCl (stress medium; TSAS) revealed only a slight reduction in numbers on
TSA, whereas the numbers on TSAS were reduced considerably, indicating a high degree of injury
229
surfaces.
41 In a recent study, the swab, RODAC, and adhesive tape (Mylar) methods were compared for
the examination of pork carcasses, and the correlation between adhesive tape and RODAC was better
than that between adhesive tape and swab or between RODAC and swab.
25 Plastic strips attached to
pads containing culture media have been used to monitor microorganisms on bottles.
27
Swab/Agar Slant
The swab/agar slant method described in 1962 by N.-H. Hansen has been used with success by some
European workers. The method involves sampling with cotton swabs that are transferred directly to
slants. Following incubation, slants are grouped into one-half log 10 units based on estimated numbers
of developed colonies. The average number of colonies is determined by plotting the distribution on
probability paper. A somewhat similar method, the swab/agar plate, was proposed by ∅lgaard.
90 It
requires a template, a comparator disc, and a reference table, making it a bit more complicated than
the other methods noted.
Ultrasonic Devices
Ultrasonic devices have been used to assess the microbiological contamination of surfaces, but the
surfaces to be examined must be small in size and removable so that they can be placed inside a
container immersed in diluent. Once the container is placed in an ultrasonic apparatus, the energy
generated effects the release of microorganisms into the diluent. A more practical use of ultrasonic
energy may be the removal of bacteria from cotton swabs in the swab-rinse method.
102
Spray Gun
A spray gun method was devised by Clark
22,23 based on the impingement of a spray of washing
solution against a circumscribed area of surface and the subsequent plating of the washing solution.
Although the device is portable, a source of air pressure is necessary. It was shown to be much more
effective than the swab method in removing bacteria from meat surfaces.
METABOLICALLY INJURED ORGANISMS
When microorganisms are subjected to environmental stresses such as sublethal heat and freezing,
many of the individual cells undergo metabolic injury, resulting in their inability to form colonies on
selective media that uninjured cells can tolerate. Whether a culture has suffered metabolic injury can be
determined by plating aliquots separately on a nonselective and a selective medium and enumerating
the colonies that develop after suitable incubation. The colonies that develop on the nonselective
medium represent both injured and uninjured cells, whereas only the uninjured cells develop on the
selective medium. The difference between the number of colonies on the two media is a measure of
the number of injured cells in the original culture or population. This principle is illustrated in Figure
10–2 by data from Tomlins et al.
127 on sublethal heat injury of S. aureus. These investigators subjected
the organism to 52
◦ C for 15 minutes in a phosphate buffer at pH 7.2 to inflict cell injury. The plating
of cells at zero time and up to 15 minutes of heating on nonselective trypticase soy agar (TSA) and
selective TSA + 7.0% NaCl (stress medium; TSAS) revealed only a slight reduction in numbers on
TSA, whereas the numbers on TSAS were reduced considerably, indicating a high degree of injury