A SIMPLE METHOD OF SUPPLYING CARBON DIOXIDE
IN JARS FOR BACTERIOLOGIC CULTURES
LUTHER THOMPSON
Section on Clinical Pathology, The Mayo Clinic, Rochester, Minnesota
In July, 1934, McLeod et al. called attention to the fact that
carbon dioxide has a stimulating effect on the growth of Neisseria
gonorrhoeae. Previously, Huddleson had used carbon dioxide to
advantage in culturing Brucella abortus. The value of McLeod's
method of culturing Neisseria gonorrhoeae has been confirmed by
Leahy and Carpenter, who have used a method similar to that
described by Huddleson for introducing carbon dioxide into the
jar in which the cultures are incubated. The apparatus necessary for Huddleson's method consists of a cylinder of carbon
dioxide, a reducing valve, a graduated receptacle for collecting the
carbon dioxide, and suitable connections with a water supply for
forcing the carbon dioxide from the measuring cylinder into the
culture jar.
METHOD
The method to be described is very simple and requires no special apparatus.
The carbon dioxide is derived from sodium bicarbonate which has been mixed
with sulphuric acid. Two solutions are prepared: one, a molar solution of
sodium bicarbonate, which is made by dissolving 84 grams of sodium bicarbonate in water and making the volume up to one liter; the other, a solution
of sulphuric acid, which is made by diluting concentrated sulphuric acid so that
1 cc. of acid is added to 29 cc. of water. The acid so made is sufficient to react
with the bicarbonate solution part for part and still leave a slight excess of
acid. Sulphuric acid, being non-volatile, does not affect the cultures. Each
cubic centimeter of the bicarbonate solution should furnish 22.4 cc. of carbon
dioxide.
The picture (fig. 1) shows how the jar is used. An ordinary 3 quart museum
jar, like the one in the illustration, has proved very satisfactory. A rubber
ring is used for a seal, but no other precautions have to be taken to prevent
loss of carbon dioxide from the jar. The plates that are to be incubated are
placed in the jar, as shown; then, a sufficient quantity of the bicarbonate solu313
314
LUTHER THOMPSON
tion to produce approximately a 10 per cent concentration of carbon dioxide
within the jar is placed in the small wide mouth bottle which is similar to the
one shown in the picture. The cover is put in place and an amount of the
sulphuric acid solution equal to the amount of bicarbonate solution that is
employed is added with a pipet, through a hole in the cover. The stopper is
restored to the hole as promptly as possible, and the jar is placed in the incubator.
FIG. 1. CARBON DIOXIDE JAK
Excellent growth of Neisseria gonorrhoeae has been obtained in
twenty-four hours with this method. No difficulty has been
experienced so far, in a limited number of cases, in culturing
Neisseria gonorrhoeae in cases in which it can be demonstrated in
smears. It has been possible to culture the organism in cases in
which the smear was negative or doubtful. It appears that
carbon dioxide is very helpful in promoting growth and that
Neisseria gonorrhoeae can be grown as readily in an atmosphere of
METHOD OF SUPPLYING CARBON DIOXIDE IN JARS
315
carbon dioxide as can streptococci on the ordinary blood agar
plate under the usual conditions. It is believed that the method
of culturing and identifying Neisseria gonorrhoeae by the oxidase
reaction, as described by McLeod, is adapted to routine clinical
use, and that a simple method of getting the proper amount of
carbon dioxide, such as is here described, will do much to encourage its use.
In two instances it was possible to secure a profuse growth of
Neisseria intracellularis (the meningococcus) from spinal fluids
in less than twenty-four hours by using the same medium as for
Neisseria gonorrhoeae and by incubating in the jar as described
above. Evidently carbon dioxide has a markedly stimulating
effect upon this organism also. In the two cases in which Neisseria intracellularis was found in the spinal fluid, it was also
possible to demonstrate it with ease by culturing from the nasopharynx. It seems probable that this method would be an excellent one for the detection of meningococcus carriers.
REFERENCES
(1) HUDDLESON, I. P . : Brucella infections in animals and man. New York,
Oxford University Press, p. 11, 1934.
(2) LEAHY, ALICE D. AND CAEPENTEE, C. M.: The isolation of Neisseria
gonorrhoeae.
Jour. Bacterid. 29: 36. 1935.
(3) MCLEOD, J. W., COATES, J. C , HAPPOLD, F. C , PRIESTLEY, D. P. AND
WHEATLEY, B.: Cultivation of the gonococcus as a method in the
diagnosis of gonorrhoea with special reference to the oxydase reaction and to the value of air reinforced in its carbon dioxide content.
Jour. Path, and Bacteriol. 39: 221-231. 1934.