IV. INVESTIGATION OF C . DIPNTHERIAE
147
the principle of double diffusion and specific toxin-antitoxin precipitation
(Oudin, 1946, 1948). Elek (1948) and Ouchterlony (1948) almost simultaneously described a inethod whereby this reaction takes place in gel, culturing the bacteria directly on the medium in which antidiphtheria1 serum
has been incorporated. Frobisher et al. (1951) recommended improved
conditions for this test.
When the antitoxin is introduced on a paper strip sunk in the depth of
the agar medium, precipitation lines develop within 1 8 4 8 h between the
culture streak and the antitoxin lines (Fig. 39).
When the antitoxin is introduced in a groove in the medium (Maximescu and Drggoi, 1971), the sensitivity of the test is increased (Fig. 40).
(ii) Tissue culture test. Sensitivity of tissue cultures to diphtherial toxin
has been known for a long time. Levaditi and Muttermilch (1913a-d) as
well as Burrows and Suzuki (1918) demonstrated that growth of chicken
embryo fibroblasts was inhibited by diphtherial toxin. This effect was
specifically neutralised by the respective antitoxin. Penso and Vicari
(1957), La Placca (1957) and Souza and Evans (1957) were able to perform
thorough studies on the cytotoxic activity of diphtherial toxin and also
demonstrated the possibility of neutralising this activity by specific
antitoxin. Calalb et al. (1965) compared the sensitivity of their test performed on Macaccus rhesus kidney cell cultures with all other tests used for
determining the toxigenicity of C. diphtheriue, i.e. EOF plates and in vivo
methods-guinea pig and rabbit tests-and concluded that tissue cultures
were well suitable for the demonstration of diphtherial toxin.
The technique uses trypsinised monkey kidney secondary cell cultures
suspended in Hanks's solution at a density of 50 000 cells/ml.
The cell suspension is distributed in 2 ml quantities, in Jena or Pyrex
tubes (160/16 mm) maintained in a slanting position at 37°C for 2-3 days,
until a continuous cell monolayer forms. The maintenance medium used
is modified by Dubreuil and Pavilanis (1958) and contains 1% calf serum.
The broth culture of the strain under study is inoculated in 0.2 ml
quantities in two to four tissue culture tubes. A tube with 1 A.U. antidiphtherial serum added is used as a control. After incubation at 37"C, the
toxigenicity is appreciated by direct daily microscopic examination during
the first four days following inoculation, according to the cytotoxic effect of
diphtherial toxin on the tissue culture. The amount of toxin produced in an
overnight broth culture shows characteristic cytopathologic effects within
24-48 h. The first cytotoxic changes show up in the arrangement of the
cells. Groups of more refractory cells delimited by different sized lacunae
appear. Hence holes in the cell monolayer, and cell detachment are observed.
The tissue culture layer disappears almost completely within 24-48 h
(Figs 41 and 42).
147
the principle of double diffusion and specific toxin-antitoxin precipitation
(Oudin, 1946, 1948). Elek (1948) and Ouchterlony (1948) almost simultaneously described a inethod whereby this reaction takes place in gel, culturing the bacteria directly on the medium in which antidiphtheria1 serum
has been incorporated. Frobisher et al. (1951) recommended improved
conditions for this test.
When the antitoxin is introduced on a paper strip sunk in the depth of
the agar medium, precipitation lines develop within 1 8 4 8 h between the
culture streak and the antitoxin lines (Fig. 39).
When the antitoxin is introduced in a groove in the medium (Maximescu and Drggoi, 1971), the sensitivity of the test is increased (Fig. 40).
(ii) Tissue culture test. Sensitivity of tissue cultures to diphtherial toxin
has been known for a long time. Levaditi and Muttermilch (1913a-d) as
well as Burrows and Suzuki (1918) demonstrated that growth of chicken
embryo fibroblasts was inhibited by diphtherial toxin. This effect was
specifically neutralised by the respective antitoxin. Penso and Vicari
(1957), La Placca (1957) and Souza and Evans (1957) were able to perform
thorough studies on the cytotoxic activity of diphtherial toxin and also
demonstrated the possibility of neutralising this activity by specific
antitoxin. Calalb et al. (1965) compared the sensitivity of their test performed on Macaccus rhesus kidney cell cultures with all other tests used for
determining the toxigenicity of C. diphtheriue, i.e. EOF plates and in vivo
methods-guinea pig and rabbit tests-and concluded that tissue cultures
were well suitable for the demonstration of diphtherial toxin.
The technique uses trypsinised monkey kidney secondary cell cultures
suspended in Hanks's solution at a density of 50 000 cells/ml.
The cell suspension is distributed in 2 ml quantities, in Jena or Pyrex
tubes (160/16 mm) maintained in a slanting position at 37°C for 2-3 days,
until a continuous cell monolayer forms. The maintenance medium used
is modified by Dubreuil and Pavilanis (1958) and contains 1% calf serum.
The broth culture of the strain under study is inoculated in 0.2 ml
quantities in two to four tissue culture tubes. A tube with 1 A.U. antidiphtherial serum added is used as a control. After incubation at 37"C, the
toxigenicity is appreciated by direct daily microscopic examination during
the first four days following inoculation, according to the cytotoxic effect of
diphtherial toxin on the tissue culture. The amount of toxin produced in an
overnight broth culture shows characteristic cytopathologic effects within
24-48 h. The first cytotoxic changes show up in the arrangement of the
cells. Groups of more refractory cells delimited by different sized lacunae
appear. Hence holes in the cell monolayer, and cell detachment are observed.
The tissue culture layer disappears almost completely within 24-48 h
(Figs 41 and 42).