IV. INVESTIGATION OF C. DIPHTHEHIAE
149
This test using Macaccus rhesus cells detects minute quantities of
diphtherial toxin (1/500 DLM); other tissue cultures (KB, HeLa, etc.)
show variations in the degree of sensitivity according to the type of the
tissue cells.
(iii) In vivo tests. Guinea pig test for C. diphtheriae: for each strain to be
tested, two guinea pigs are used: one of the animals is protected with 2000
A.U. antidiphtheria1 serum. Two to three hours later, both animals are
inoculated, subcutaneously with 3-5 ml of a 2-3 days broth culture. A
toxigenic strain kills the unprotected animal in 24-96 h, while protected
animals survive withtmt any sign of toxicity. Necropsy reveals specific
symptoms such as gelatinous haemorrhagic oedema at the site of inoculation, congestion of the abdominal wall; pleural and peritoneal serohaemorrhagic exudates and congestion of the adrenals. Features of diphtherial intoxication cannot always be found together.
Stains of low toxigenicity which produce less than 1 DLM of toxin usually
do not kill the animal, but can produce local necrosis; paralyses of posterior
limbs or death may occur later.
Non-toxigenic strains allow the guinea pigs to survive without any
pathological signs.
Guinea pig test in C. ulceram: like C. H$htheriae, not all C. ukerans
strains produce a toxin which can be neutralised by diphtherial antitoxin.
The guinea pig test is carried out as for C. diphtheriae. When two guinea
pigs of which one is protected with diphtherial antitoxin are inoculated
with a broth culture of C. ulcerans which produces diphtherial toxin,
both animals die. In the unprotected animal, death occurs in 1-2 days
with all signs specific for diphtherial toxin. I n the protected animal
death occurs later (3-4 days). The changes are: local haemorrhagic
gelatinous oedema, marked haemorrhagic congestion of all viscera, possibly
haemorrhagic exudates of the pleural and peritoneal cavities (characteristic of the pathogenicity of C. ulceram), but never the congestion of
the adrenals. C. ulceram strains, which do not produce diphtherial toxin,
kill both animals in the same way as in the protected animal.
Skin test in rabbit (Fraser’s test): this test is more sensitive than the
previous test as it detects 1/1000-1/2000 DLM of toxin. It is not obligatory
for routine work, but it may be used together with the guinea pig test, when
the Elek-Ouchterlony-Frobisher test plates do not present clear-cut
results.
An overnight broth culture is injected intradermally in 0.2 ml amounts
on one side of a depilated back of a rabbit (2500-3000 g weight). Approximately 10-12 strains may be tested per rabbit. Before inoculation, squares
are drawn with a special pencil on the depilated skin of the back of the
rabbit. Three and a half hours after injection, the rabbit is injected intra-
149
This test using Macaccus rhesus cells detects minute quantities of
diphtherial toxin (1/500 DLM); other tissue cultures (KB, HeLa, etc.)
show variations in the degree of sensitivity according to the type of the
tissue cells.
(iii) In vivo tests. Guinea pig test for C. diphtheriae: for each strain to be
tested, two guinea pigs are used: one of the animals is protected with 2000
A.U. antidiphtheria1 serum. Two to three hours later, both animals are
inoculated, subcutaneously with 3-5 ml of a 2-3 days broth culture. A
toxigenic strain kills the unprotected animal in 24-96 h, while protected
animals survive withtmt any sign of toxicity. Necropsy reveals specific
symptoms such as gelatinous haemorrhagic oedema at the site of inoculation, congestion of the abdominal wall; pleural and peritoneal serohaemorrhagic exudates and congestion of the adrenals. Features of diphtherial intoxication cannot always be found together.
Stains of low toxigenicity which produce less than 1 DLM of toxin usually
do not kill the animal, but can produce local necrosis; paralyses of posterior
limbs or death may occur later.
Non-toxigenic strains allow the guinea pigs to survive without any
pathological signs.
Guinea pig test in C. ulceram: like C. H$htheriae, not all C. ukerans
strains produce a toxin which can be neutralised by diphtherial antitoxin.
The guinea pig test is carried out as for C. diphtheriae. When two guinea
pigs of which one is protected with diphtherial antitoxin are inoculated
with a broth culture of C. ulcerans which produces diphtherial toxin,
both animals die. In the unprotected animal, death occurs in 1-2 days
with all signs specific for diphtherial toxin. I n the protected animal
death occurs later (3-4 days). The changes are: local haemorrhagic
gelatinous oedema, marked haemorrhagic congestion of all viscera, possibly
haemorrhagic exudates of the pleural and peritoneal cavities (characteristic of the pathogenicity of C. ulceram), but never the congestion of
the adrenals. C. ulceram strains, which do not produce diphtherial toxin,
kill both animals in the same way as in the protected animal.
Skin test in rabbit (Fraser’s test): this test is more sensitive than the
previous test as it detects 1/1000-1/2000 DLM of toxin. It is not obligatory
for routine work, but it may be used together with the guinea pig test, when
the Elek-Ouchterlony-Frobisher test plates do not present clear-cut
results.
An overnight broth culture is injected intradermally in 0.2 ml amounts
on one side of a depilated back of a rabbit (2500-3000 g weight). Approximately 10-12 strains may be tested per rabbit. Before inoculation, squares
are drawn with a special pencil on the depilated skin of the back of the
rabbit. Three and a half hours after injection, the rabbit is injected intra-