Chapter 12
Bioassay and Related Methods
After establishing the presence of pathogens or toxins in foods or food products, the next important
concern is whether the organisms/toxins are biologically active. For this purpose, experimental animals
are employed where feasible. When it is not feasible to use whole animals or animal systems, a variety
of tissue culture systems have been developed that, by a variety of responses, provide information
on the biological activity of pathogens or their toxic products. These bioassay and related tests are
the methods of choice for some foodborne pathogens, and some of the principal ones are listed in
Table 12–1.
WHOLE-ANIMAL ASSAYS
Mouse Lethality
This method was first employed for foodborne pathogens around 1920 and continues to be an
important bioassay method. To test for botulinal toxins in foods, appropriate extracts are made and
portions are treated with trypsin (for toxins of nonproteolytic Clostridium botulinum strains). Pairs
of mice are injected intraperitoneally (IP) with 0.5 ml of trypsin-treated and untreated preparations.
Untreated preparations that have been heated for 10 minutes at 100
◦ C are injected into a pair of mice.
All injected mice are observed for 72 hours for symptoms of botulism or death. Mice injected with the
heat preparations should not die because the botulinal toxins are heat labile. Specificity in this test can
be achieved by protecting mice with known botulinal antitoxin, and in a similar manner, the specific
serologic type of botulinal toxin can be determined (see Chapter 24 for toxin types).
Mouse lethality may be employed for other toxins. Stark and Duncan
45 used the method for Clostridium perfringens enterotoxin. Mice were injected IP with enterotoxin preparations and observed for
up to 72 hours for lethality. The mouse-lethal dose was expressed as the reciprocal of the highest
dilution that was lethal to the mice within 72 hours. Genigeorgis et al.
18 employed the method by
use of intravenous (IV) injections. C. perfringens enterotoxin preparations were diluted in phosphate
buffer, pH 6.7, to achieve a concentration of 5–12 µg/ml. From each dilution prepared, 0.25 ml was
injected IV into six male mice weighing 12–20 g, the number of deaths were recorded, and the LD 50
was calculated. The mouse is the most widely used animal for virulence assessment of Listeria spp.
The LD 50 for L. monocytogenes in normal adult mice is 10
5 –10
6 , and for 15-g infant mice, as few as
50 cells may be lethal (see Chapter 25).
285
Bioassay and Related Methods
After establishing the presence of pathogens or toxins in foods or food products, the next important
concern is whether the organisms/toxins are biologically active. For this purpose, experimental animals
are employed where feasible. When it is not feasible to use whole animals or animal systems, a variety
of tissue culture systems have been developed that, by a variety of responses, provide information
on the biological activity of pathogens or their toxic products. These bioassay and related tests are
the methods of choice for some foodborne pathogens, and some of the principal ones are listed in
Table 12–1.
WHOLE-ANIMAL ASSAYS
Mouse Lethality
This method was first employed for foodborne pathogens around 1920 and continues to be an
important bioassay method. To test for botulinal toxins in foods, appropriate extracts are made and
portions are treated with trypsin (for toxins of nonproteolytic Clostridium botulinum strains). Pairs
of mice are injected intraperitoneally (IP) with 0.5 ml of trypsin-treated and untreated preparations.
Untreated preparations that have been heated for 10 minutes at 100
◦ C are injected into a pair of mice.
All injected mice are observed for 72 hours for symptoms of botulism or death. Mice injected with the
heat preparations should not die because the botulinal toxins are heat labile. Specificity in this test can
be achieved by protecting mice with known botulinal antitoxin, and in a similar manner, the specific
serologic type of botulinal toxin can be determined (see Chapter 24 for toxin types).
Mouse lethality may be employed for other toxins. Stark and Duncan
45 used the method for Clostridium perfringens enterotoxin. Mice were injected IP with enterotoxin preparations and observed for
up to 72 hours for lethality. The mouse-lethal dose was expressed as the reciprocal of the highest
dilution that was lethal to the mice within 72 hours. Genigeorgis et al.
18 employed the method by
use of intravenous (IV) injections. C. perfringens enterotoxin preparations were diluted in phosphate
buffer, pH 6.7, to achieve a concentration of 5–12 µg/ml. From each dilution prepared, 0.25 ml was
injected IV into six male mice weighing 12–20 g, the number of deaths were recorded, and the LD 50
was calculated. The mouse is the most widely used animal for virulence assessment of Listeria spp.
The LD 50 for L. monocytogenes in normal adult mice is 10
5 –10
6 , and for 15-g infant mice, as few as
50 cells may be lethal (see Chapter 25).
285