J. Howard Mueller

A Protein-Free Medium for Primary Isolation of the Gonococcus and Meningococcus:

It is desired to report the development of a transparent, reproducible medium, containing no heat-labile material such as serum, which is sufficiently stable to withstand autoclave sterilization and storage, and which will support satisfactory primary growth of either meningococci or gonococci. The latter were used as the test organisms during the experimental work, since a constant source of primary cultures of the meningococcus is ordinarily not available. The similarity between the two organisms, and the reputedly greater difficulty expected in the cultivation of the gonococcus, indicated that a medium suitable for this organism should prove equally satisfactory for the meningococcus. As a basis for comparison, the Difco “Proteose No. 3 Chocolate Agar” has been used. This medium has been adopted as a standard in certain laboratories for the diagnosis of gonorrhoea 1 and one of us (J.H.) was thoroughly familiar with its use. The plan as formulated in beginning the experiments was essentially that followed with considerable success with other organisms, namely, to select the most suitable empirical medium available and attempt to break it down to its essential component parts. The experience of the senior author in 1918 with the Gordon and Hine 2 pea meal extract agar for meningococcus carrier detection had shown that this medium, when carefully prepared, was eminently satisfactory. It proved, upon trial, to be equally so for primary cultivation of the gonococcus, and it was therefore chosen as the starting point in the investigation. It consists essentially of a tryptic digest of meat, agar, and a 5% NaCl extract of pea flour, the latter presumably supplying some essential protein material. The fractionation of the pea extract proved surprisingly simple, since it could be accomplished by mechanical means.

A new sulphur-containing amino acid isolated from casein

In a report on a study of the cultural requirements of streptococci made last year before this society, the writer stated that a compound containing sulphur had been isolated from casein, which was apparently not related to cystine, and which seemed to be required for the growth of the test organisms. Although subsequent work has shown that this sulphur compound, when pure, is apparently not concerned in the growth of streptococci, it seemed desirable to make a study of the substance, both because of the uncertainty of the nature of non-cystine protein sulphur, and also in order to be able to effect a separation of this compound from the bacterial growth inducing factor in the amino acid fraction under investigation. While there are still many points to be cleared up in connection with the substance, perhaps enough information has been obtained to warrant a preliminary report. There have been a number of difficulties met with in the work. The yield is very small, and probably not by any means quantitative, and further, no insoluble compounds suitable for separation have so far been found, so that purification has consisted largely in methods for the removal of impurities. In order to obtain sufficient material for analyses, thirty pounc-ls of cormnercial Argentine casein were used. Briefly, the method consists in hydrolysis with sulphuric acid, neutralization with sodium carbonate, and piecipitation with mercuric sulphate solution. From the washed precipitate, freed from electrolytes, a considerable quantity of other material is removed by a second precipitation with nlercuric sulphate, the sulphur compound this time remaining in the filtrate. Further purification is effected by precipitation of the filtrate, after removing electrolytes, by silver sulphate and barium hydroxide, and the compound itself is obtained from the silver filtrate, freed, of course, from Ag and Ba, by fractional crystallization, finally from dilute acetone.

Substitution of β-Alanine, Nicotinic Acid, and Pimelic Acid for Meat Extract in Growth of Diphtheria Bacillus

Conclusion Minute amounts of nicotinic acid, β-alanine, and pimelic acid, present together, permit about two-thirds the maximal growth of the diphtheria bacillus obtained from whole tissue extracts when added to a suitable control medium. The pimelic acid appears to be the least essential of the three. Differences appear between strains, even of the Park 8 organism, which may well be multiplied when further strains are examined.

Tetanus Toxin Production on a Simplified Medium

The routine preparation of tetanus toxin for the purpose of antitoxin production, or for conversion into toxoid, appears to offer difficulties similar to those formerly encountered in the case of diphtheria toxin, although perhaps in lesser degree. Active immunization against tetanus by means of toxoid, now coming into general use, demands that an attempt be made to place the manufacture of this important biological product on as satisfactory a basis as it has now been possible to reach in the case of diphtheria toxin. 1 , 2 The traditional explanation for failures and irregularities with the latter, namely the types of peptones and meat infusions used in the medium, has been shown to be completely erroneous, although still held to be applicable in dealing with tetanus. In this country, Witte, or the very similar Berna peptone is generally held to be indispensable, together with veal infusion. Elsewhere, enzymatic digests are employed, often with the addition of even more complex materials. The writers have undertaken a study of the nutritional requirements of the tetanus bacillus and a parallel study of factors influencing the production of its toxin. Since an exact definition of the substances essential to growth may well prove to be a time-consuming matter, it seems worth while to report at this time the production of potent toxin on a simplified, but by no means chemically defined medium, and to indicate that the concentration of iron in the medium, as in the case of diphtheria, is at least one of the controlling factors. As a basis for experiment we have employed the casein acid hy-drolysate medium described by one of us2 for the production of diphtheria toxin. This medium itself fails to support growth of any of the tetanus strains investigated, as was to be expected, but when it is enriched by the addition of tryptophane, glucose and 1 % of a commercial liver extract, growth occurs with all the strains which have been tried.

Growth-determining substances in bacteriological culture media

Some months ago a report was made of a series of experiments based on the observation that, while a peptone-free meat infusion broth would produce abundant growth of hemolytic streptococci, short boiling with charcoal removed this property entirely. The addition of commercial peptone or of a sulphuric acid hydrolysate of certain proteins, such as casein or meat, reactivated the charcoal-treated infusion and heavy cultures of streptococci could be obtained on the mixture, while neither one alone gave the slightest trace of growth. It was shown that the activating material in the protein hydrolysate was precipitated by mercuric sulphate, and that it had not been possible to identify it with any of the amino acids known to be precipitated by this reagent either alone or in combination. It is the purpose of the present communication to describe the further purification of this activating material. Much of the work has been done using a commercial preparation called “aminoids” in place of an acid hydrolysate of casein. This consists of an enzyme digest of milk proteins continued until the product is biuret free. It has been used simply as an economy of time since in handling large quantities the acid hydrolysis is somewhat cumbersome. Every step in the separation, however, has been checked on an acid hydrolysate and it is believed that there is no essential difference in the factors involved. In attempting to separate the active material from the mercuric sulphate precipitate, fractional precipitation with the same reagent was tried. This led to the discovery that there were two factors in the precipitate, both of which were necessary to reactivate the charcoal-treated infusion. One of these must be carried down by adsorption in the original mercuric sulphate precipitate, or else its solubility is influenced by the presence of other substances, for it is not reprecipitated, to any extent, from the mixture by the addition of mercuric sulphate.

Amino Acids Required by the Diphtheria Bacillus for Growth

The writer 1 described experiments indicating that a strain of diphtheria bacillus under investigation required for its growth, among other things, a substance associated with the “proline” fraction of amino acids obtained by Dakins butyl alcohol extraction method. Further work with this material has shown that not a single factor, but 3 separate substances were involved in the accelerating effect there described. It was learned to begin with, that the action was not entirely limited to the “proline” fraction, but that the “monoamino” fraction even more frequently exerted it, and occasionally the 2 added together gave better results. Before any material progress was made in separating and recognizing these factors, it became necessary to introduce a quantitative method for the estimation of relative amounts of growth. The method finally used was a micro kjeldahl determination of the amount of nitrogen in the bacterial growth on a measured amount of material for a definite time. The details of this procedure will be described elsewhere. Careful examination of the effect produced by the proline fraction showed that proline itself was evidently not concerned, but that the acceleration in growth was apparently produced by minute quantities of ethyl alcohol used as solvent in handling the fraction. Pure ethyl alcohol exerted the same effect, and maximum acceleration of growth with our particular strain of organism is reached with a concentration of about 0.5%. Larger quantities of course become inhibitory. In place of ethyl alcohol, sodium acetate, glycerol, or maltose all appear to enhance the growth when added to the same control solutions.

THE RELATION OF THE CARRIER TO EPIDEMIC MENINGITIS

Excerpt By way of orientation, it may be well to review briefly the facts, so far as they appear to be known, regarding the elements involved in the spread of epidemic meningococcal infection. We s...

Nutritional Factors Concerned with Colony Development of C. Diphtheriae

Summary Rapid development of single colonies of a number of strains of C. diphtheriac on a “synthetic medium” agar occurs only in the presence of certain constituents of the blood serum of the horse and ox. Human and swine sera are ineffective. Activity is not impaired by removal of the protein by coagulation. The active material does not dialyze and evidently consists of at least two substances, one of which is soluble in acetone. Cows milk is rich in these agents, which will be further characterized in subsequent reports.

“Residue antigen” from a strain of Friedlander bacillus.

The work of Zinsser and Parker, 1 Heidelberger and Avery 2 and one of us (Mueller) 3 has indicated that most bacteria produce carbohydrate gums possessing the property of reacting specifically with antibodies formed in response to injections of the original bacteria. It is, therefore, of interest to study a number of these gums, called by Zinsser “residue antigens,” from different bacterial sources. Only by such study can it be determined to what extent it will be necessary to alter the present conception of the chemical basis of immunological specificity. Since the Friedlander Bacillus grows readily and abundantly on simple media, and is known to produce considerable quantities of a complex carbohydrate presumably related to the capsular material, 4 ∗ this organism was chosen as being particularly adapted to the work. An old stock strain has been used, the source of which is unknown. It may be mentioned here that among several strains examined, there are distinct serological differences, as might be expected from the unsatisfactory cultural classification of this group. Work upon other strains is being continued by one of us, (Smith) and it is possible that a classification of the group based on relationship of the residue antigens produced may be developed as an incident to the purely chemical study of these substances. Cultures were made in broth, prepared from casein hydrolyzed for six hours with four times the weight of hydrochloric acid. After removal of the excess acid, and neutralization, there is added an inorganic salt mixture and glucose. One hundred liters of broth were used for the preparation here reported. After a weeks growth in the incubator, the flasks were Arndded for one hour, passed through a Sharples centrifuge to remove the bacteria, concentrated to about one tenth on the water bath and precipitated by adding two volumes of alcohol.

A preliminary report on the chemical composition of residue antigen.

In a number of preparations of residue antigen obtained from various micro-organisms, chemical analyses have been carried out to find whether or not the presence of proteins, etc., could be definitely excluded. To this end, total nitrogen has been determined, together with amino-nitrogen before and after acid hydrolysis of the preparation. The bacteria were grown in large agar plates, washed off with saline and treated in the manner previously described in detail, 1 and the resulting material dried in the dessicator. Specific precipitin tests were given by saline solutions of the dry material with immune rabbit sera, but insufficient quantities were available to attempt to correlate the dilution at which the test became negative, with the chemical composition. For analysis, the entire specimen was carefully weighed, brought into solution in a few cc. of water, and diluted to a definite volume, usually in a 10 cc. volumetric flask, aliquot portions being taken for separate determinations. Total nitrogen was determined by the micro-kjeldahl method, titrating with 0.02 N solutions, and the amino-nitrogen determinations were carried out by the micro-Van Slyke method. Phosphorus was determined by Tisdalls 2 method, after wet ashing. In those preparations in which sufficient material was obtained for duplicate analyses, agreement was fairly uniform. However, we do not believe that absolute accuracy can be claimed for all the determinations, particularly those of amino-nitrogen after hydrolysis, because of the small quantity available for use, and the relatively low content of nitrogen. The values for total nitrogen are probably the most accurate. It will be observed that there is a rather considerable variation in each type of nitrogen in the different lots. Considering the results as a whole, one sees that the low and variable total nitrogens point to the presence of a nitrogenous substance associated with a non-nitrogenous compound.