Felix Saunders

Nicotinic acid as an essential growth-substance for dysentery bacilli.

Recent evidence has shown that nicotinic acid is a compound of considerable biologic importance. Current interest in this compound can be attributed primarily to the work of Warburg, 1 Euler 2 and their associates, which demonstrated that the amide of nicotinic acid is a constituent of the coenzyme from horse blood and the cozymase of yeast. To those interested in the nutritive requirements of microörganisms this work has been of considerable significance as it supplied a clue to the chemical identity of one of the essential substances, or “growth-factors,” needed for development of certain of the more exacting bacteria. In studies of Staphylococcus aureus Knight 3 demonstrated that a combination of nicotinic acid and vitamin B1 (thiamin chloride) was effective in replacing a concentrate prepared from yeast. Neither substance would suffice in the absence of the other, but when both were supplied the staphylococcus developed in a culture-medium containing only known compounds. For growth of the diphtheria bacillus Mueller 4 found that nicotinic acid could replace one of several fractions obtained from liver. A combination of nicotinic acid, beta-alanine and, for some strains of the organism, pimelic acid was effective in promoting growth in the absence of tissue extract preparations. 5 Nicotinic acid without beta-alanine was relatively ineffective. 5 The work reported here deals with the growth-promoting effect of nicotinic acid upon dysentery bacilli. It is of interest for two reasons: first, through the use of nicotinic acid it is possible to cultivate dysentery bacilli in a solution of known chemical compounds and second, nicotinic acid alone is strikingly effective without the addition of any other “accessory” factor.

Inhibition of Respiration of Dysentery Bacilli by Sulfapyridine.

Summary It has been found that sulfapyridine in concentrations of 30 mg % inhibits the stimulation of respiration due to nicotin-amide. This inhibition is apparently competitive since the sulfapyridine must be added prior to the addition of the nicotinamide to obtain this effect. While percentage inhibition has varied in different experiments, sufficient experimental data have been obtained so as to leave no doubt as to the validity of the inhibition.

Quantitative response of the dysentery bacillus to nicotinamide and related compounds.

Summary By means of a titration method for estimation of bacterial growth it was found that growth is proportional to the quantity of nicotinamide present. Nicotinamide is more active than an equivalent amount of either pyridine-containing coenzyme. Hydrolysis increases the activity of the latter, indicating that the function of nicotinamide is not based entirely on synthesis to either of the known coenzymes. A method has been developed for determining nicotinamide and related substances in blood. The values obtained are higher if autoclaved blood is used.

Pyridine Derivatives and Other Compounds as Growth-Promoting Substances for Dysentery Bacilli.∗

Summary Vitamin B6, nicotinamide methiodide, pyrazine monocarboxylic acid and pyrazine 2,3-dicarboxylic acid showed no growth-promoting effect for dysentery bacilli when added to a basal synthetic medium. Thiazole 5-carboxylic acid and its amide exerted a low order of growth-promoting activity. The use of purified quinolinic acid in 1 × 10-4 molar concentration was uniformly followed by culture development. This may have been due to gradual conversion to nicotinic acid. Heating the quinolinic acid solution greatly increased its activity. The efficacy of quinolinic acid in curing pellagra may be due to contamination with small amounts of nicotinic acid.

Accessory Growth Factor Requirements of Some Members of the Pasteurella Group.

Most members of the Pasteurella group of bacteria develop satisfactorily in meat infusion-peptone media but fail to grow in simpler media made from hydrolyzed purified protein or in synthetic media. The substances in infusions of meat, other tissues or yeast which are needed for growth have not been previously identified. Accordingly we wish to report preliminary results of a study of the accessory growth factor requirements of some members of this group of organisms in which it will be shown that nicotinamide, pantothenic acid and, in some cases, the butyl factor for Clostridia are needed for prompt development.∗ Seventeen typical Pasteurella strains were used. These were stock laboratory cultures which had been secured from different sources. They were isolated originally from hemorrhagic septicemias in various species of animals. The results presented in this report apply only to the typical strains of animal origin and not to other species at times included in this genus. The basal medium consisted of a 0.5% solution of hydrolyzed purified gelatin to which was added a supplement of 8 amino acids, 0.3% glucose, 0.5% NaCl, 0.2% K2HPO4, 0.005% MgSO4 and 0.001% CaCl2. To this was added 1 cc of Hoagland salt mixture per liter of medium. The amino acid supplement consisted of 20 mg each of valine, tyrosine, tryptophane, cystine, methionine and histidine and 15 mg each of serine and threonine per liter. The medium was adjusted to pH 7.0 with N NaOH solution and tubed in 5 cc quantities. The accessory growth factors were sterilized by filtration and added aseptically to the basal medium. In the first tests a mixture of known substances was used on the assumption that perhaps some of them might be required by these organisms.

Sources of growth factors required by certain "fastidious" bacteria. Failure of ascorbic acid to replace growth-promoting principles.

In continuing the study of substances, apparently in the nature of accessory growth factors, which will permit the development of some of the more exacting pathogens in a synthetic medium, it seemed desirable to gain some information concerning the natural sources of such compounds before attempting further chemical purification. A number of different animal and plant tissues were selected as possible sources and tested for the presence of growth promoting principles. The tissues were finely ground, extracted with water and heated to boiling. The coagulum was removed by filtration and the filtrate treated with charcoal to adsorb the growth factors. The charcoal was then extracted in a continuous extractor with hot ethyl alcohol to remove the growth factors, the alcohol evaporated off in vacuo and the residue taken up in a measured amount of distilled water. The extracts were sterilized in the autoclave. The potency of these extracts was tested by adding them in several different dilutions to tubes of synthetic medium and noting the growth which followed inoculation with organisms which ordinarily refused to develop in the synthetic medium. Ten different cultures were used: Streptococcus hemolyticus (from scarlet fever), Streptococcus lactis, Staphylococcus aureus, Staphylococcus albus, Corynebacterium diphtheriae, Brucella abortus, Pasteurella avicida, Salmonella pullorum, Eberthella typhosa, and Shigella dysenteriae. Observations of growth were made at several different intervals over a period of 10 days or occasionally longer. The rapidity of development of the organisms as well as the dilution in which growth occurred served to give a measure of the potency of the extracts. Altogether over 30 preparations were tested in this manner. The results may be summarized as follows:† Veal infusion +++, skim milk +++, calf liver ++++, ash from calf liver -, calf thymus +++, calf heart ++, calf spleen ++++, calf kidney +++, calf lung +++, pig embryo ++, chicken liver +++, shad roe +++, human liver with carcinoma +++, human placenta +++, human urine +, oat sprouts ++, wheat germ ++, rice bran +++, tea -, lettuce ++, sprouted soy beans +++, canned tomato juice +++, turnips +++, whole white potatoes +++, ash of potatoes -, potato sprouts +, potato tuber left after removal of sprouts ++, carrots ++, cocoanut milk +++, bakers yeast ++++, several cultures of Aspergillus and Penicillium + or ++, Pseudomonas fluorescens +.

Adsorption of Physiologically Active Substances by Activated Charcoal.

During the progress of some studies on adsorption, it became necessary to have some information regarding the action of activated charcoal on physiological substances. A search through the literature did not reveal any previous work except a paper by Guerrant and Salmon 1 on the adsorption of quinine. We were not concerned in this case with the mechanism of adsorption or activation. We merely wanted to know whether or not certain drugs would be adsorbed from aqueous solution by activated charcoal. To insure uniformity of results, we decided to use an activated charcoal easily obtainable on the open market.∗ The following drugs were studied: strychnine sulphate, brucine sulphate, adrenalin hydrochloride, histamine hydrochloride, acetylcholine hydrobromide, ephedrine hydrochloride, tyramine hydrochloride and diamino butane hydrochloride. The activity of the drug was studied by intravenous injection. The solutions were prepared as follows: The drug was dissolved in water or physiological salt solution. 25 cc. of the solution were put into a 100 cc. flask as a control solution. Another 25 cc. portion was added to a 100 cc. flask containing 1 gm. of the active charcoal. Both solutions were then shaken for 20 minutes and filtered through a folded filter. The activity of these filtered solutions was determined by intravenous injection. Nine dogs under ether anesthesia were used as the test animals. All injections were made into the left femoral vein. When strychnine, brucine, adrenalin, histamine and tyramine are treated with activated charcoal they are quantitatively inactivated either through adsorption or modification. In the case of acetylcholine and ephedrine the inactivation is not quite complete.

Studies on Bacterial Nutrition. II. The Distribution of a Growth Stimulating Factor in Animal and Plant Tissues.

In a previous study of the requirements for growth of the more exacting bacteria it was found that a growth stimulating factor1 could be removed from veal infusion. This factor, when added to a simple culture medium, rendered the medium suitable for growth of some of the more fastidious organisms. In certain instances small amounts of added substance amounting to only 0.0006 to 0.00006 milligram of total solids per cubic centimeter of medium were sufficient to permit development in a synthetic medium in which the organisms ordinarily refused to grow.2 Additional study of the factor responsible for this effect seemed desirable. However, before attempting chemical purification of the growth substance obtained from veal infusion, it was considered best to take up first the question of its occurrence in various natural sources. There is considerable evidence in bacteriological literature that growth stimulating substances can be found in a number of animal and plant tissues. In many of these reports, however, it is not clear whether the stimulating effect is due simply to a more abundant supply of readily available food or to the presence of accessory factors which may be required by certain types in addition to the ordinary nutritive material. While no attempt will be made to give a comprehensive survey of the literature, since this has been summarized by Peskett,3 certain references which bear upon the present study should be mentioned. The important role of plant or animal tissue extracts in cultivation of Hemophilus influenzae has been emphasized especially by Davis,4 Thjotta and Avery,5 Fildes6 and others. Substances of a supposedly vitamin-like nature which exerted a strong growth-stimulating activity on hemolytic streptococci and yeast were reported by Freedman and Funk7 in

Growth factors in relation to development of certain "fastidious" bacteria.

It is well known that many of the more fastidious bacteria require special media such as fresh meat infusions for their development and refuse to grow, or at best grow poorly, on the ordinary standard agar and broth made with peptone and meat extract. These organisms, almost without exception, also refuse to grow in a chemically definite or synthetic medium. The nature of the necessary substances in the more complex media is largely a matter of conjecture at present, although it constitutes one of the very important problems of bacterial nutrition. In the present investigation we have attempted to separate from ordinary veal infusion the factors responsible for growth of some of the more exacting bacteria. It seems reasonable to suppose that a more exact knowledge of these substances would not only reveal many practical applications in bacteriological work but would also have a rather general biological significance. The removal of growth factors from protein digests or infusions by the use of charcoal has been reported by Robinson and Rettger, 1 Thjotta and Avery, 2 Mueller, 3 and Freedman and Funk. 4 In our work it was found that treatment of an ordinary veal infusion with charcoal resulted in the removal of unknown substances necessary for the growth of certain microorganisms. Other commonly used adsorbents, such as Fullers earth, kaolin, talc, Lloyds reagent, filter paper, calcium phosphate, Supercel, or silica gel were relatively ineffective. The unknown substances or growth factors, after removal by charcoal from an infusion, could be recovered by extraction from the charcoal with hot ethyl alcohol or hot acetone. These extracts, after evaporation of the alcohol or acetone and subsequent solution of the residue in water, were effective in “activating”an infusion from which growth factors had been removed, or in rendering a standard beef extract-peptone broth more suitable for growth of certain exacting organisms.

A Comparative Study of the Proteins of Citrus Seeds

The seeds of citrus plants are particularly adapted to comparative studies of the proteins of different plant species. They can be obtained relatively easily and yield a crystalline globulin which has been given the name “pomelin”. To isolate the protein the seeds are ground to a fine meal and the oil, which constitutes about 35% of the total weight, is removed with benzene. The defatted meal is covered with 10 times its weight of a salt solution which has been heated to 55°. (The concentration of the salt solution may vary from normal to saturation.) The salt solution is recovered by filtration or centrifuging and saturated with ammonium sulfate. The precipitated protein is collected by filtration and redissolved in a minimum of water. If the solution is not clear any sediment is removed in the centrifuge. The solution is dialyzed in viscose bags for 48 hours against cold (6-10°) running distilled water which causes the globulin to separate in a crystalline form. No preservative is used. The protein is collected and washed with several changes of alcohol starting with 30% and running up to absolute. After a final washing with absolute ether and air-drying, the protein is ready for analysis. The results of the analysis for various nitrogen fractions are shown in Table I. The analytical figures show that the pomelin from grapefruit, lemon and orange is the same in each case. The analysis of pomelin from tangerine seeds also agrees with these figures although there was not enough material available for complete analysis. The crystal form as far as can be seen under the microscope is the same in each case. The authors conclude that the seeds from the citrus plants: grapefruit, lemon, and orange, and probably tangerine, all yield the same crystalline globulin, pomelin.