I V . INVESTIGATION OF C . DIPHTHEHIAE
161
Bovine serum
20 ml
1% L-cystine
6 ml
10% lead acetate
1 ml
Aliquots of 2-3 ml are distributed in small tubes left to solidify vertically.
Inoculation should be made by stabbing. Starting 4-6 h after inoculation,
traces of black growth surrounded by a brown halo begin appearing when
the organism is a cystinase producer. The reaction becomes intensified
overnight. C. diphtheriae, C . ulcerans and C. ovis are the only high cystinase
producers in this genus. The medium keeps 1 month in the refrigerator.
8. Blake-Christensen medium for the urease test (recommended by Kauffmann, 1954)
Bacto-peptone Difco
I g
NaCl
5 g
KHzP04
2 g
Phenol red 1/500 solution
6 ml
Distilled water
900 ml
Dissolve by boiling all ingredients, except the Phenol red. Adjust to
pH = 6.8-6-9 and filter through paper. Autoclave at 120°C for 20 min.
Then add sterile 1 g glucose solution, 20 ml of a 20% urea solution which
has been sterilised by filtration and 6 ml Phenol red solution 1/500 (autoclaved 20 min at 110°C). The medium should be light yellow and clear.
Aliquots (2 ml) are distributed to small tubes and checked for sterility.
C . diphtheriae does not produce urease. Diphtheroids and C. ulcerans are
rich in urease.
9. Elek-Ouchterlony-Frobisher plates for the in vitro toxinogenicity test
(a) Base
Meat infusion
1000 ml
Protese peptone Difco
20 g
NaCl p.a.
5 g
Bacto agar (Difco)
11 g
Dissolve all ingredients at boiling point.
Adjust to pH = 7-6-74 with 10% NaOH solution.
Precipitate the medium by autoclaving 20 min at 120°C.
Filter through cotton, distribute in known quantities in flasks, autoclave
Meat infusion could be replaced by beef extract (Difco) (5 g to 1000 ml
20-30 min at 110°C.
distilled water).
161
Bovine serum
20 ml
1% L-cystine
6 ml
10% lead acetate
1 ml
Aliquots of 2-3 ml are distributed in small tubes left to solidify vertically.
Inoculation should be made by stabbing. Starting 4-6 h after inoculation,
traces of black growth surrounded by a brown halo begin appearing when
the organism is a cystinase producer. The reaction becomes intensified
overnight. C. diphtheriae, C . ulcerans and C. ovis are the only high cystinase
producers in this genus. The medium keeps 1 month in the refrigerator.
8. Blake-Christensen medium for the urease test (recommended by Kauffmann, 1954)
Bacto-peptone Difco
I g
NaCl
5 g
KHzP04
2 g
Phenol red 1/500 solution
6 ml
Distilled water
900 ml
Dissolve by boiling all ingredients, except the Phenol red. Adjust to
pH = 6.8-6-9 and filter through paper. Autoclave at 120°C for 20 min.
Then add sterile 1 g glucose solution, 20 ml of a 20% urea solution which
has been sterilised by filtration and 6 ml Phenol red solution 1/500 (autoclaved 20 min at 110°C). The medium should be light yellow and clear.
Aliquots (2 ml) are distributed to small tubes and checked for sterility.
C . diphtheriae does not produce urease. Diphtheroids and C. ulcerans are
rich in urease.
9. Elek-Ouchterlony-Frobisher plates for the in vitro toxinogenicity test
(a) Base
Meat infusion
1000 ml
Protese peptone Difco
20 g
NaCl p.a.
5 g
Bacto agar (Difco)
11 g
Dissolve all ingredients at boiling point.
Adjust to pH = 7-6-74 with 10% NaOH solution.
Precipitate the medium by autoclaving 20 min at 120°C.
Filter through cotton, distribute in known quantities in flasks, autoclave
Meat infusion could be replaced by beef extract (Difco) (5 g to 1000 ml
20-30 min at 110°C.
distilled water).