The Effects of an Extract of. Media. I. W. Gibby, P. S. Nicholes, J. T. Tamura and Lee Foshay

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1 CONTENT ALERTS The Effects of an Extract of Blood Cells upon the Cultivation of Bacterium tularense in Liquid Media I. W. Gibby, P. S. Nicholes, J. T. Tamura and Lee Foshay J. Bacteriol. 1948, 55(6):855. Updated information and services can be found at: These include: Receive: RSS Feeds, etocs, free alerts (when new articles cite this article), more» Downloaded from on April 7, 014 by PENN STATE UNIV Information about commercial reprint orders: To subscribe to to another ASM Journal go to:

2 THE EFFECTS OF AN EXTRACT OF BLOOD CELLS UPON THE CULTIVATION OF BACTERIUM TULARENSE IN LIQUID MEDIA I. W. GIBBY, P. S. NICHOLES, J. T. TAMURA, AND LEE FOSHAY Department of Bacteriology, College of Medicine, University of Cincinnati, and the Cincinnati General Hospital, Cincinnati, Ohio Received for publication March 1, 1948 Until satisfactory synthetic media become available, a clear liquid medium of defined composition that will support sustained growth from small inocula and provide large yields of cells would fill many needs. Further study and exploitation of the properties of an aqueous extract of blood cells have resulted in the incorporation of these characteristics into improved liquid media of the type devised by Tamura and Gibby (1943). The constituents of unknown composition are the acid hydrolyzates of the selected proteins and the blood cell extract (BCE). For this study we are not concerned with the composition of BCE but with certain of its properties and practical uses. Preparation of blood cell extract. Although satisfactory extracts were prepared from rabbit and horse bloods, most were made from human blood, using cells obtained from the hospital's blood bank. Nine volumes of distilled water were added to each volume of packed, unwashed cells. The laked suspension was heated with constant stirring and boiled for 3 minutes. While a temperature between 95 and 0 C was maintained, concentrated HCl was added to bring the ph to 6.3. After acidification this temperature was maintained for 5 minutes, whereupon, while still hot, the coagulum was removed by filtration through fluted paper. The fluid was refiltered through the same paper until the filtrate was clear. The volume was recorded as filtrate A, of which each ml represented 1 ml of original cells. This filtrate was concentrated in a vacuum still to one-fiftieth or less of its volume. The concentrate was restored to ph, heated to the boiling point, and filtered through paper until clear. The filter was well drained, and the volume of the clear filtrate was recorded as filtrate B. This liquid concentrate was used for current cultural needs. The ratio of the volumes of filtrates B to A was used to calculate the volume of filtrate B that was required per liter of medium to give a concentration of BCE of any desired percentage in terms of the original packed blood cells. For example, if filtrate A was concentrated to one-fiftieth of its volume, each 1 ml of filtrate B was equivalent to 5 ml of blood cells. Hence for a medium to contain 5 per cent BCE in terms of original cells, it required ml of concentrate per liter. Storage of unsterilized BCE concentrate at 5 C was unsatisfactory except for short periods. Preservation without loss of activity was accomplished either by rapid freezing and storage in a dry ice chest or by dehydration from the rapidly frozen state. The lyophilized material offered the advantages of safe storage of the ground powder in stoppered bottles and of gravimetric additions to media. 855

3 856 I. W. GIBBY, P. S. NICHOLES, J. T. TAMURA, AND LEE FOSHAY [VOL. 55 Basal media. Two basal media were used to study the metabolic properties of BCE. Their formulas were: Basal medium A per L 3.0 g Gelatin hydrolyzate to make N content... Cystine Glycerol NaCl (total) NaHP041 H KHPO4.0.5 MgSO Basal medium B pff L 3.0 g Gelatin hydrolyzate to make N content... Cystine Glycerol NaCl, (total) Substitutions were made in basal medium B as follows: hydrolyzates of crude casein, vitamin test casein, or of a partly defatted soybean protein for gelatin hydrolyzate; glucose for glycerol. The hydrolyzates were prepared, with slight modifications, according to the method of Williams et al. (1941). Since neutralized acid hydrolyzates varied in chloride content, it was necessary to determine chlorides (as NaCl) for each lot. The difference between each determination and the totals recommended in the media above must be made up by additional NaCl. Most strains tested here grew better and faster with glycerol than with glucose, but some grew just as well with glucose, and an occasional strain seemed to prefer it. All media were adjusted to an initial ph of unless otherwise stated. Culture tubes. Provision for a high ratio of surface to volume of media, and for frequent determination of growth by means of the turbidity of cultures, was made by blowing bulbs of mm diameter from the closed ends of 18-mm pyrex tubes, and by sealing 16-cm side arm tubes, made from selected pyrex tubing of mm outside diameter, into the large tubes just above the level of the bulbs. The distal ends of the side arms were sealed. These side arm tubes fitted vertically into the holder of a photoelectric turbidity comparator. Cultures could be tipped into the side arms at will for turbidity measurements without risk of contamination. Inocula for cultures. Unless otherwise stated all inocula contained about 40 millions of thrice-washed cells in 0.1-ml volumes. They were prepared from 4-hour cultures in gelatin hydrolyzate medium. All cultures were incubated at 37 C. Most work was performed with strains Memp and Schu; occasionally others were used. Methd for quantitative determination of growth. A fairly accurate approximation of the number of bacteria per unit volume was obtained with the photoelectric comparator described by Krebs and associates (194). Measurements of turbidity were obtained in terms of microamperes (M. A.). Many virulence titrations in mice indicated that suspensions in.5-mm tubes, adjusted to 4.0 M. A., contained.5 to 3 billions of bacteria per ml. Calibration of the instru-

4 1948] EFFECT OF BLOOD CELL EXTRACT ON BACTERIUM TULARENSE ment with dilutions of bacterial suspensions, and with dilutions of standard silicate suspensions, showed that M. A. readings were almost directly proportional to turbidity throughout its range. Table 1 indicates the approximate numbers of bacteria in suspensions with various M. A. readings. Some physical and chemical properties of BCE. The color of dried extract varies from light yellow-brown to light reddish-brown. It is readily soluble in water. Although its composition may vary from lot to lot, a large representative sample gave the following test results: The biuret and xanthoproteic tests were faintly positive. The Molisch and Bial tests were strongly positive. Benedict's test was negative. Sulfur and phosphorus were present, and nitrogen formed 5.4 per cent of the dry weight. It was irreversibly reduced, with bleaching of TABLE 1 Approximate equivalents of turbidity in microamperes measured in.6-mm tubes and numbers of Bacterium tularense Microamperes Bact./ml in millions , 1 1, ,670 0, 4, , , , ,550 color, by sodium hydrosulfite. In 8 per cent aqueous solution there was no light absorption in the visible spectrum. Physiological properties of BCE; effect upon growth. The addition of BCE to a basal medium that will not support growth of Bacterium tularense with large washed inocula gives it a capacity for sustained multiplication from small washed inocula. The growth-promoting effects of ascending concentrations of a typical sample in basal medium A are shown in table. The inoculum for each culture contained about 50 million organisms. Media made from hydrochloric acid hydrolyzates of gelatin supported sustained multiplication from washed inocula that contained from 5 to organisms, to 5 ml of medium, on repeated testing. Identical media made with sulfuric acid hydrolyzates or from hydrolyzates of other proteins were not capable of initiating growth from such small inocula. Stabilizing effect ofbce on the ph of culture media. One of the difficulties early encountered in the cultivation of this organism in liquid media was the extraordinary and unpredictable fluctuations of the ph of cultures. In some cultures 857

5 858 I. W. GIBBY, P. S. NICHOLES, J. T. TAMURA, AND LEE FOSHAY [VOL. 55 end points of ph were reached after 4 hours of incubation, invariably with very scanty growth. In others, usually those with good growth, end points of ph 8.3 occurred, which caused cessation of multiplication. These difficulties with still cultures were magnified in degree and accelerated in appearance by either shaking or aeration. The stabilizing effect of BCE upon the ph after 7 hours of incubation is also shown in table, in which turbidity and ph determinations are tabulated against increasing increments of BCE in otherwise identical cultures. Since these ph determinations were made with bromthymol blue, it is probable that the two upper readings were higher than the values actually reached. TABLE The effect of increasing concentrations of blood cell extract on growth and on ph values in basal medium A after incubation for 7 hours BCZ PER CET M. A. PH or less or less Many attempts were made to find suitable buffers to stabilize the ph of growing cultures. Phosphate buffers that maintained reactions at or near neutrality in concentration of 0.03 M, but not in lesser concentrations, inhibited growth in concentrations above 0.0 m. Caxbonate and acetate buffers gave similar results; growth was approximately inversely proportional to the concentrations of the buffers. Sodium glycinate did not inhibit growth, nor did it stabilize the ph. Variations in concentrations of hydrolyzates of gelatin or of casein were also ineffective. No stabilizing buffer system was found that did not adversely affect growth. It was even shown that the amounts of phosphates present in basal medium A were inhibitory, and that increased yields of cells were obtained after they and the magnesium salt were omitted. Since BCE had revealed a stabilizing effect upon the ph levels of growing cultures, its capacity was tested further by adding it in per cent concentration to basal medium B and making a duplicate series of tubes with initial ph values ranging from 5.0 to 7.8 in steps of 0.4. Turbidity measurements and ph deter-

6 1948] EFFECT OF BLOOD CELL EXTRACT ON BACTERIUM TULARENSE 859 minations were made daily for 3 days. These measurements are shown in table 3 and figure 1. All cultures within the initial ph range of 5.8 to 7.8 developed TABLE 3 Growth and ph changes of culture8 with different initial ph values during $ days of incubation in basal medium B containing per cent of BCE INITIL PH PH AT 4 x M. A. AT 4 PH AT 48 X. A. AT 48Ht PH AT 7 M. A. AT LA I5 ' ph Hr Hr FIG. 1. THE GRAPH AT THE LEFT ILLUSTRATES THE RATE OF GROWTH AND THE TERMINAL DENSITIES IN TERMS OF MICROAMPERES OF THE DUPLICATE CULTURES WHOSE INITIAL PH VALUES, AND DAILY PH VALUES FOR 3 DAYS OF INCUBATION, ARE SHOWN AT THE RIGHT ' values that steadily approached neutrality. Those within the 5.8 to ph range multiplied rapidly and steadily. Even those with initial ph values of 7.4 gave good yields after 7 hours although they grew more slowly. The 7.8 cul-

7 860 I. W. GIBBY, P. S. NICHOLES, J. T. TAMURA, AND LEE FOSHAY [VOL. 55 tures grew still more slowly and failed to give satisfactory yields. Cultures with initial ph values of 5.4 or less showed little or no growth. Further studies confirmed that BCE alone supplied substances that not only markedly stimulated growth but also stabilized the ph of unbuffered gelatin or casein hydrolyzate cultures. The mechanism of ph 8tabilization of cultures by BCE. The addition of BCE to uninoculated media does not alter the ph. The remote likelihood that it could serve as a buffer was shown to be untenable. Serial tubes of basal medium B containing ascending quantities of BCE from 1 to 5 per cent were adjusted to a ph of, were inoculated with washed cells and incubated for 7 hours, whereupon turbidity, ph values, and titratable acidity values were determined. Cultures containing 1 per cent of BCE showed the least growth, the lowest ph values, and the highest titratable acidities. With ascending quantities of BCE the determinations, recorded in table 4, showed progesively increased growth and ph values, and decreased titratable acidity values. Cultures that contained 5 TABLE 4 The effect of ascending concentrations of BCE upon growth, ph, and titratable acidity after incubation for 8 days in basal medium B (The initial ph of the medium was ) BCE PER CENT M. A. PH TITRATABWIX ACDITY * MI of 0.01 N NaOH required to titrate to ph. per cent BCE showed a fall in ph from to 6.3 in 7 hours, and 0.5 ml of 0.01 N NaOH were required to restore the ph to. Cultures that contained 1 per cent of BCE showed a fall in ph from to 5.1 during the same period, and required.0 ml of 0.01 N NaOH to restore the ph to. If BCE had exerted a direct buffering action the cultures containing 5 per cent should have produced as much, if not more, titratable acid as did the cultures conta ning 1 per cent BCE, whereas the latter actually produced eight times as much. Hence the bufferlike action of BCE on growing cultures is a result of its effects on bacterial metabolism, apparently dependent upon some component of the extract that accelerates amino acid utilization with resultant formation of base, this process keeping pace with and offsetting the effect of acid production from carbohydrate oxidation, including glycerol, if an appropriate concentration of BCE is present. Repeated tests failed to detect a trace of volatile base during the growth of still or aerated cultures that became progressively more alkaline. Dialyzability and heat stability of the metabolically active components of BCE. Dialysis of BCE in a cellophane sack against distilled water in a small vessel

8 1948] EFFECT OF BLOOD CELL EXTRACT ON BACTERIUM TULARENSE disclosed that both the growth-promoting and the ph-stabilizing components disappeared from the sack and appeared in the dialyzate within 4 hours. A sample of concentrated liquid BCE was adjusted to ph, brought quickly to boiling, and then rapidly cooled in running water. The precipitate was sedimented by centrifugation. A portion of the supernatant was removed for assay, and the remainder was autoclaved for minutes at pounds pressure. The precipitate was sedimented from the autoclaved portion, and the supernatant was removed for assay. Each supernatant was added in increasing amounts to separate series of tubes containing basal medium A; all tubes were adjusted to ph, and inoculated together with a third series containing identical quantities of unheated BCE. The 7-hour growth yields and the ph values at 4, 48, and 7 hours for each comparable BCE concentration were identical for the boiled and the unheated extracts. The growth yields of the autoclaved series, for each level of BCE used, were about one-third less than those of the corresponding tubes of the other series, and the ph values fell in all tubes to 6.0 or below by 48 hours. Momentary heating to the boiling point did not diminish metabolic activity of the extract, but autoclaving near neutrality destroyed almost all of the ph-stabilizing component. Other natural sources of substances with properties similar to those of BCE. A search for substances with growth-promoting and ph-stabilizing properties similar to those of BCE revealed that these functions were present, though to lesser degrees, in extracts prepared from powdered egg yolk, liver cake, yeast, leaf mold, and compost heaps, and marsh and river muds. The extracts were tested in basal media by substitution for BCE. Cultural conditions for successful cultivation in liquid media. The demand for a free supply of oxygen must be satisfied. Vessels for still cultures must provide a high ratio of surface area to volume of medium. Suboptimal growth is inevitable from all except large inocula if the medium is dispensed in 5-ml amounts in the usual 18-mm tubes. Growth is still poorer if larger volumes are used. Tubes of 5-mm diameter are satisfactory for 5-ml cultures. Cultures in larger volumes should be grown in Erlenmeyer flasks, using a size that for the desired volume of medium will give a layer of fluid not more than 5 to 8 mm deep. Aqueous solutions of BCE deteriorated at refrigerator temperatures. Poor or irregular growth resulted from solutions held at 5 C for 6 days. They are more stable in complete media and will hold in that form for weeks at 5 C. The preferred dehydrated powder maintained its activity for many months at room temperatures in tightly stoppered bottles, DISCUSSION Cultures of Bacterium tularense on solid, or in liquid, media produce acids from fermentable carbohydrates, including glycerol, and nonvolatile bases, presumably amines, from amino acids. The reaction of the inoculated medium at any time represents the relative differences in magnitudes and velocities of these processes. Determinations of the ph of cultures may or may not betray evidence of carbohydrate utilization; they are treacherous and unsuitable methods for that pur- 861

9 86 I. W. GIBBY, P. S. NICHOLES, J. T. TAMURA, AND LEE FOSHAY [VOL. 55 pose. The ability of the organism to produce either acidic or alkaline end points in similar media, and indeed first one and then the other during cultivation on a single medium, was also noted by Downs and Bond (1935) and by Francis (194), who studied the utilization of carbohydrates by means of indicators. Growth on highly buffered media is seldom restricted by fluctuations in ph, but in nutritionally adequate but unbuffered liquid media most failures to obtain good yields result from high concentrations of hydrogen or hydroxyl ions. In the absence of a buffer system that does not of itself restrict growth it is impossible to say that limitation of growth is not due to exhaustion of an essential substrate or enzyme activator, or possibly to an accumulation of inhibitory end products. Since the addition of BCE to liquid cultures that have ceased to grow in the presence of a low ph permits rapid and progressive multiplication for an additional 5 days at 37 C, the importance of waste products is minimized, but, owing to our incomplete knowledge of the composition of the extract, it does not permit discrimination between the effect of an enzyme activator and the exhaustion of an essential substrate that may be present in the extract. However, there is convincing evidence that regulation of the reaction of liquid media within the noninhibiting ph range of 6.4 to 7.6 can be effected by appropriate quantities of BCE, and that this regulation depends upon unidentified components that permit the organisms to produce constantly a sufficient quantity of base from amino acids to offset acid production from concurrent oxidation of glucose or glycerol. Exclusive of substances present in basal media, including those made from hydrolyzates of vitamin test casein, BCE apparently supplies all other substrates and growth factors in quantities and proportions that make the complete medium a close approximation to the nutritional needs, for no other simple medium has produced comparable cell yields per unit of time from such small inocula in still cultures. The presence in leaf molds, and in marsh and river muds, of substances with properties similar to those of BCE offers support to the hypothesis suggested by Parker, Steinhaus, and Kohls (1943) that even such an organism as Bacterium tularense, generally considered to be fastidious in its nutritional requirements, might, under suitable conditions, persist and multiply in nature outside of animal, insect, and arachnid hosts. Subsequent to the epizootic of tularemia among beaver that was reported by Jellison, Kohls, Butler, and Weaver (194), Parker and his associates demonstrated a prolonged and heavy contamination of river waters in the Northwest that could not reasonably be attributed to successive or continuous wild life sources. They showed that marsh mud contained cystine, and that the organism persisted in naturally contaminated, refrigerated mud for 1 weeks. The extracts that we prepared from samples of marsh and river muds sent by Dr. Parker contained moderate amounts of substances with cultural properties similar to those of BCE. Hence, from a nutritional viewpoint there is little reason to doubt that Bacterium tularense could malntain itself in muds, and possibly even in waters, in the absence of any living host. Since per cent NaCl is not inhibitory to growth, maintenance of the organism is possible in muds covered or bordered by waters of considerable salinity.

10 19481 EFFECT OF BLOOD CELL EXTRACT ON BACTERIUM TULARENSE The media described here, made from acid hydrolyzates of gelatin, soybean protein, casein, or vitamin test casein, and containing 00 to 300 mg per cent of dehydrated BCE, do not neutralize the actions of sulfonamides, PABA, penicillin, or streptomycin. When used with suitable conditions of cultivation they are capable of large bacterial yields. Since all constituents of these media are dialyzable, quantities of cells sufficient for chemical or other analyses are obtainable with relative ease and are entirely free from foreign materials. The simplest complete medium permits continuous subcultivation at room temperatures with weekly transfer of 0.1 ml of culture to 5 ml of fresh medium with little loss of virulence. Strain Memp was maintained continuously in this medium for more than years. SUMARY An aqueous extract of blood cells provided substances that effected a metabolic stabilization of the ph, and supported excellent growth of Bacterium tulkrene from small inocula in simplified, unbuffered, liquid media. The simplest medium contained four ingredients otherthan the extract and water. This medium is suitable for many strains and many purposes, including the production of large quantities of bacterial cells free from all foreign material. REFERENCES DOWNS, C. M., AND BOND, G. C Studies on the cultural characteristics of Pasteurella tularensi8. J. Bact., 30, FRANCIS, E. 194 Fermentation of sugars by Bacterium tularense. J. Bact., 43, JELLISON, W. L., KOHLs, G. M., BUTLER, W. J., AND WEAVER, J. A. 194 Epizootic tularemia in the beaver, Castor canadensis, and the contamination of stream water with Pasteurella tularensis. Am. J. Hyg., 36, KREBS, R. P., PERKINS, P., TYTELL, A. A., AND KERSTEN, H. 194 A turbidity comparator. Rev. Sci. Instruments, 13, 9-3. PARKER, R. R., STEINHAUS, E. A., AND KOHLS, G. M Tularemia in beavers and muskrats and the contamination of natural water and mud with Pasteurella tularensis in the northwestern United States. J. Bact., 45, TAxuRA, J. T., AND GIBBY, I. W Cultivation of Bacterium tularense in simplified liquid media. J. Bact., 45, WILLIAMS, R., et al Studies on the vitamin content of tissues. Univ. Texas Pub. No Refer to p