34
H. P. R. SEELIGER AND K. IIOHNE
in highly contaminated cultures (Gray et al., 1948; Gray, 1957; Hartwigk,
1958a) and distinguished from a number of common pathogens (Hartwigk,
1958a). Listeria colonies on T N A Agar are of a light green to blue-green
colour. The medium has to be clear and must be poured in a thin layer
(about 10 to 13 ml per 9 cm plate) in order to allow optimal evaluation
(Bockemuhl, 1971; Hohne, 1972).
If the initial culture fails to reveal L. monocytogenes, the tissue suspension,
fluids, swabs etc. which are kept in the refrigerator at 3 to 4°C should be
replated after 6 weeks and even after 3 months storage. Usually, a 6 weeks
period of refrigeration is sufficient for the growth to appear. Kampelmacher and van Noorle Jansen (1961) found it necessary to refrigerate
calf brain material as long as 6 months before the bacterium could be
detected.
The mechanism of the enhancing effect at 4°C is not fully understood.
When it was first described by Gray et al. in 1948, it was suggested that it
might involve an inhibitory factor in bovine brain. Attempts to demonstrate such a factor in brains from several different species have been unsuccessful (Murakimi and Kato, 1957). It has been shown repeatedly that
delayed growth of listeriae from infected tissue is not limited to bovine
brain, but that this is similar with all animal and human tissue and body
fluids. The growth at low temperatures may be connected with the psychrophylic nature of the organisms and with slow liberation from its intracellular position. Enhancement by cold storage has been used successfully to isolate the bacterium from such widely diversified sources as
pneumonic lungs of infants (MenEikova, 1956), vaginal swabs (Gray,
1960a), silage extracts (Gray, 1960b) and environmental specimens (Weiss
and Seeliger, 1975; Hohne et al., 1975).
Unless the organisms grow in pure culture, transfer suspected colonies
to fresh blood agar or tryptose agar plates for purification. The characteristic colonial forms are usually apparent after 24-48 h incubation at 37°C
and can be distinguished by the naked eye. Then smears are made and
Gram stained. If the morphology is characteristic the demonstration of
motility is of utmost importance.
For this purpose, a hanging drop preparation is made from a young
broth culture incubated at room temperature. I n addition semisolid
motility agar containing 0.2-0.4% agar is stabbed (about 1 cm). At 20-37°C
listeriae inoculated into the agar column swarm through the “motility
medium” and produce cloudiness. This can easily be observed by the
naked eye. About 0.5 cm below the surface of the agar a layer of increased
growth, like an umbrella, is found. I n this zone of reduced oxygen tension
Listeria shows a better development than under aerobic or strictly anaerobic
conditions.
H. P. R. SEELIGER AND K. IIOHNE
in highly contaminated cultures (Gray et al., 1948; Gray, 1957; Hartwigk,
1958a) and distinguished from a number of common pathogens (Hartwigk,
1958a). Listeria colonies on T N A Agar are of a light green to blue-green
colour. The medium has to be clear and must be poured in a thin layer
(about 10 to 13 ml per 9 cm plate) in order to allow optimal evaluation
(Bockemuhl, 1971; Hohne, 1972).
If the initial culture fails to reveal L. monocytogenes, the tissue suspension,
fluids, swabs etc. which are kept in the refrigerator at 3 to 4°C should be
replated after 6 weeks and even after 3 months storage. Usually, a 6 weeks
period of refrigeration is sufficient for the growth to appear. Kampelmacher and van Noorle Jansen (1961) found it necessary to refrigerate
calf brain material as long as 6 months before the bacterium could be
detected.
The mechanism of the enhancing effect at 4°C is not fully understood.
When it was first described by Gray et al. in 1948, it was suggested that it
might involve an inhibitory factor in bovine brain. Attempts to demonstrate such a factor in brains from several different species have been unsuccessful (Murakimi and Kato, 1957). It has been shown repeatedly that
delayed growth of listeriae from infected tissue is not limited to bovine
brain, but that this is similar with all animal and human tissue and body
fluids. The growth at low temperatures may be connected with the psychrophylic nature of the organisms and with slow liberation from its intracellular position. Enhancement by cold storage has been used successfully to isolate the bacterium from such widely diversified sources as
pneumonic lungs of infants (MenEikova, 1956), vaginal swabs (Gray,
1960a), silage extracts (Gray, 1960b) and environmental specimens (Weiss
and Seeliger, 1975; Hohne et al., 1975).
Unless the organisms grow in pure culture, transfer suspected colonies
to fresh blood agar or tryptose agar plates for purification. The characteristic colonial forms are usually apparent after 24-48 h incubation at 37°C
and can be distinguished by the naked eye. Then smears are made and
Gram stained. If the morphology is characteristic the demonstration of
motility is of utmost importance.
For this purpose, a hanging drop preparation is made from a young
broth culture incubated at room temperature. I n addition semisolid
motility agar containing 0.2-0.4% agar is stabbed (about 1 cm). At 20-37°C
listeriae inoculated into the agar column swarm through the “motility
medium” and produce cloudiness. This can easily be observed by the
naked eye. About 0.5 cm below the surface of the agar a layer of increased
growth, like an umbrella, is found. I n this zone of reduced oxygen tension
Listeria shows a better development than under aerobic or strictly anaerobic
conditions.