11. SEROTYPING OF I.IST€CHIA .ldO,VOC YTOG'RNES AND RELATED SPECIES 33
A monocytosis-producing agent (MPA) of L. mcinocytogenes located in
the cell wall (Leeler and Gray, 1960) was extracted by Stanley (1949) from
dried and ground organisms with organic solvents. Amounts of extractable
lipids are highest after extraction with petrol-ether, but chloroform or
ether extracts are biologically more active. The isolated lipid MPA is
serologically inactive and of low tissue toxicity. A toxic component has
been isolated from whole cells of I,. moizocytogenes by aqueous ether
extraction and ethyl alcohol precipitation (Srivastava and Siddique, 1974).
This component was pyrogenic to rabbits, produced edema and erythema
in rabbit skin, and was lethal to chicken embryos. It was also immunogenic
to mice against a large homologous challenge dose.
111. ISOLATION PROCEDURES; CULTURAL AND
BIOCNEMICAL IDENTIFICATION
A. Isolation from Infected Material
When the organisms are numerous and not mixed with other bacteria,
cultivation on bacteriological media does not present any major difficulties.
Listeria are not fastidious and grow well on glucose-infusion agar, with or
without blood or serum added. They also multiply in infusion broth and
grow profusely in brain heart-, placenta- or liver broth. Tryptose is
preferable to most brands of peptone, and a 2% tryptose agar is an excellent
medium for cultivation, propagation and preservation of I,. mcinocytogenes.
The various techniques which have proved successful for isolation
generally stress the need for homogenisation, when the organisms are to be
grown from tissues. The bacteria are often incarcerated in the focal lesions
and frequently found intracellularly. The most effective method consists
of macerating suspected tissue in a mortar or Waring Blendor together
with a few millilitres of sterile distilled water or nutrient broth. Saline
should be avoided since it is said to harm the bacterium, especially if the
cells are low in number. A portion of the suspension is plated on sheep or
horse blood agar, tryptose agar and Trypaflavine Nalidixic Acid (TNA)
Agar (Bockemuhl et al., 1971). The remainder is stored at 4°C. Body fluids,
swabs etc. are plated, and a portion is stored at 4°C. The plates are incubated at 37°C for 18 to 24 h, that of T N A Agar for 48 h, and examined
with a scanning microscope, or a hand lens with the plate resting on a
laboratory tripod (Bearns and Girard, 1959) and with obliquely transmitted illumination as described by Henry (1933). When viewed in this
manner the colonies on tryptose agar are distinctively blue-green and so
characteristic that with little practice they can be identified quickly even
A monocytosis-producing agent (MPA) of L. mcinocytogenes located in
the cell wall (Leeler and Gray, 1960) was extracted by Stanley (1949) from
dried and ground organisms with organic solvents. Amounts of extractable
lipids are highest after extraction with petrol-ether, but chloroform or
ether extracts are biologically more active. The isolated lipid MPA is
serologically inactive and of low tissue toxicity. A toxic component has
been isolated from whole cells of I,. moizocytogenes by aqueous ether
extraction and ethyl alcohol precipitation (Srivastava and Siddique, 1974).
This component was pyrogenic to rabbits, produced edema and erythema
in rabbit skin, and was lethal to chicken embryos. It was also immunogenic
to mice against a large homologous challenge dose.
111. ISOLATION PROCEDURES; CULTURAL AND
BIOCNEMICAL IDENTIFICATION
A. Isolation from Infected Material
When the organisms are numerous and not mixed with other bacteria,
cultivation on bacteriological media does not present any major difficulties.
Listeria are not fastidious and grow well on glucose-infusion agar, with or
without blood or serum added. They also multiply in infusion broth and
grow profusely in brain heart-, placenta- or liver broth. Tryptose is
preferable to most brands of peptone, and a 2% tryptose agar is an excellent
medium for cultivation, propagation and preservation of I,. mcinocytogenes.
The various techniques which have proved successful for isolation
generally stress the need for homogenisation, when the organisms are to be
grown from tissues. The bacteria are often incarcerated in the focal lesions
and frequently found intracellularly. The most effective method consists
of macerating suspected tissue in a mortar or Waring Blendor together
with a few millilitres of sterile distilled water or nutrient broth. Saline
should be avoided since it is said to harm the bacterium, especially if the
cells are low in number. A portion of the suspension is plated on sheep or
horse blood agar, tryptose agar and Trypaflavine Nalidixic Acid (TNA)
Agar (Bockemuhl et al., 1971). The remainder is stored at 4°C. Body fluids,
swabs etc. are plated, and a portion is stored at 4°C. The plates are incubated at 37°C for 18 to 24 h, that of T N A Agar for 48 h, and examined
with a scanning microscope, or a hand lens with the plate resting on a
laboratory tripod (Bearns and Girard, 1959) and with obliquely transmitted illumination as described by Henry (1933). When viewed in this
manner the colonies on tryptose agar are distinctively blue-green and so
characteristic that with little practice they can be identified quickly even