IV. INVESTIGATION OF C. DIPHfHERIAE
151
This test may also be used for C. ulcerans, although reactions are far
Phage and bacteriocin typing are described in Sections VI and X.
more intense.
X. PRACTICAL PROCEDURES OF PHAGE TYPING I N
C. DIPHTHERIAE
A basic description of the two phage typing schemes for C. diphtheriae
(original and additional) have already been given. The working principles
and practical considerations concerning the technique will be discussed
here.
A. Principles
Typing phages may be propagated in a tryptic digest broth such as Pope
Linggood's broth (see Saragea and Maximescu, 1964) supplied with
0.001% L-cystine for the original typing phages and in heart broth supplied
with O.OOl~o L-cystine for the additional typing phages.
The titre of a particular phage lysate depends upon the kind of phage
propagated and the technique used. For instance, C. diphtheriae phages,
even under the best conditions, never have routine tets dilutions (RTD)
above lOz-lO~, while C. hofmannii phages reach R T D titres of 108-1010
(RTD = the highest dilution still displaying confluent lysis on its propagating strain). The main problem associated with phage propagation is
not the preparation of high titre lysates but maintaining the phage with
its proper host range specificity. The lytic host range of every typing
phage preparation should be tested against the whole set of sensitive and
resistant strains from the respective phage-typing scheme. No typing
phage preparation should be used unless it conforms to the accepted
pattern. It is therefore advisable that for each established scheme, new
phage stocks should be obtained periodically from an international reference laboratory. Diphtheria phages are relatively stable for a period of
3-4 and even 6 months. Undiluted stock phage preparations may be stored
in the freezer and used as seed preparations for further propagation.
For long-term storage, diphtheria phage preparations may be lyophilised,
the phages keeping their host range for at least 10 years.
B. Phage-typing procedure
An overnight stock culture is grown in O . O O l ~ o cystinated nutrient
broth for 1+ h at 37°C. A plate of brain-heart agar, supplemented by bovine
serum, cystine and calcium chloride, is flooded with the culture under
151
This test may also be used for C. ulcerans, although reactions are far
Phage and bacteriocin typing are described in Sections VI and X.
more intense.
X. PRACTICAL PROCEDURES OF PHAGE TYPING I N
C. DIPHTHERIAE
A basic description of the two phage typing schemes for C. diphtheriae
(original and additional) have already been given. The working principles
and practical considerations concerning the technique will be discussed
here.
A. Principles
Typing phages may be propagated in a tryptic digest broth such as Pope
Linggood's broth (see Saragea and Maximescu, 1964) supplied with
0.001% L-cystine for the original typing phages and in heart broth supplied
with O.OOl~o L-cystine for the additional typing phages.
The titre of a particular phage lysate depends upon the kind of phage
propagated and the technique used. For instance, C. diphtheriae phages,
even under the best conditions, never have routine tets dilutions (RTD)
above lOz-lO~, while C. hofmannii phages reach R T D titres of 108-1010
(RTD = the highest dilution still displaying confluent lysis on its propagating strain). The main problem associated with phage propagation is
not the preparation of high titre lysates but maintaining the phage with
its proper host range specificity. The lytic host range of every typing
phage preparation should be tested against the whole set of sensitive and
resistant strains from the respective phage-typing scheme. No typing
phage preparation should be used unless it conforms to the accepted
pattern. It is therefore advisable that for each established scheme, new
phage stocks should be obtained periodically from an international reference laboratory. Diphtheria phages are relatively stable for a period of
3-4 and even 6 months. Undiluted stock phage preparations may be stored
in the freezer and used as seed preparations for further propagation.
For long-term storage, diphtheria phage preparations may be lyophilised,
the phages keeping their host range for at least 10 years.
B. Phage-typing procedure
An overnight stock culture is grown in O . O O l ~ o cystinated nutrient
broth for 1+ h at 37°C. A plate of brain-heart agar, supplemented by bovine
serum, cystine and calcium chloride, is flooded with the culture under