IV. INVESTIGATION OF C. DIPHTHERIAE
67
The exclusive use of Loeffler's medium for primary cultivation of
C. diphtheriae in many laboratories probably explains the delay and
difficulties encountered in the isolation and identification of C. diphtheriae
by many earlier workers.
(b) Oxygen requirements of different species within the genus vary widely.
C. diphtheriae develops freely and better under aerobic conditions. Diphtherial toxin is also better produced aerobically. Strains which have to be
primarily isolated under anaerobic conditions are now referred to the genus
Propionibacterium (e.g. C. acnes).
(c) Growthfactors. Most of the strains lack certain synthetic capacities in
connection with the formation of nicotinic and pantothenic acids. Thus,
nicotinic acid becomes an essential component for a suitable culture
medium as does pantothenic acid or in some cases beta-alanine from which
certain strains form their own pantothenate molecule.
Similarly, certain cultures require biotin, others pimelic acid, whereas
others are independent of these substances.
Growth and toxin production are favoured by the addition of amino
acids to a synthetic medium and of those aspartic acid and tryptophan are
apparently indispensable. Drew and Miller (1951) worked out a completely
defined medium for the growth and toxin production of strain PWs, the
highly toxigenic strain used throughout the world for commercial toxin
production. The medium contains beta-alanine, nicotinic acid, pimelic
acid, cystine, glycine, valine, leucine, methionine, biotin, glutamic acid,
tryptophan, ammonium ions and some salts such as magnesium, copper,
zinc, manganese and iron salts. For everyday use in research work the
casein hydrolysate medium of Mueller and Miller (1941) is more
practical. For research purposes Barksdale (1970) recommends this
medium supplemented with pantothenate, glutamate and tryptophan and
therefore calls it P G T medium. He also draws attention to the fact that
certain strains do not grow unless thiamine is also present.
Generally, for primary isolation, enrichment, selection of the organism,
as well as for routine diagnostic procedures and commercial manufacture of
diphtherial toxin, complex media are used, supplied with animal protein and
L-cystine. All the other necessary amino-acids are contained in the complex
media. The formulae of all media used in the routine standardised diagnostic scheme will be given in Section XIII.
(d) The optimum growth temperature is 37°C. For testing phage-host
bacteria relationships as well as for toxin production 35 "C is optimal.
67
The exclusive use of Loeffler's medium for primary cultivation of
C. diphtheriae in many laboratories probably explains the delay and
difficulties encountered in the isolation and identification of C. diphtheriae
by many earlier workers.
(b) Oxygen requirements of different species within the genus vary widely.
C. diphtheriae develops freely and better under aerobic conditions. Diphtherial toxin is also better produced aerobically. Strains which have to be
primarily isolated under anaerobic conditions are now referred to the genus
Propionibacterium (e.g. C. acnes).
(c) Growthfactors. Most of the strains lack certain synthetic capacities in
connection with the formation of nicotinic and pantothenic acids. Thus,
nicotinic acid becomes an essential component for a suitable culture
medium as does pantothenic acid or in some cases beta-alanine from which
certain strains form their own pantothenate molecule.
Similarly, certain cultures require biotin, others pimelic acid, whereas
others are independent of these substances.
Growth and toxin production are favoured by the addition of amino
acids to a synthetic medium and of those aspartic acid and tryptophan are
apparently indispensable. Drew and Miller (1951) worked out a completely
defined medium for the growth and toxin production of strain PWs, the
highly toxigenic strain used throughout the world for commercial toxin
production. The medium contains beta-alanine, nicotinic acid, pimelic
acid, cystine, glycine, valine, leucine, methionine, biotin, glutamic acid,
tryptophan, ammonium ions and some salts such as magnesium, copper,
zinc, manganese and iron salts. For everyday use in research work the
casein hydrolysate medium of Mueller and Miller (1941) is more
practical. For research purposes Barksdale (1970) recommends this
medium supplemented with pantothenate, glutamate and tryptophan and
therefore calls it P G T medium. He also draws attention to the fact that
certain strains do not grow unless thiamine is also present.
Generally, for primary isolation, enrichment, selection of the organism,
as well as for routine diagnostic procedures and commercial manufacture of
diphtherial toxin, complex media are used, supplied with animal protein and
L-cystine. All the other necessary amino-acids are contained in the complex
media. The formulae of all media used in the routine standardised diagnostic scheme will be given in Section XIII.
(d) The optimum growth temperature is 37°C. For testing phage-host
bacteria relationships as well as for toxin production 35 "C is optimal.