THE NUTRITION OF BRUCELLAE Chemically …mmbr.asm.org/content/22/2/81.full.pdf · the usual concentrations of peptone and glucose either blood or agar but mayrequire considera-maybecomelimiting

THE NUTRITION OF BRUCELLAE

PHILIPP GERHARDTDepartment of Bacteriology, University of Michigan Medical School, Ann Arbor, Michigan

CONTENTSI. Introduction........................................................................... 81

II. Composition of Media..................................................................... 81A. Undefined Media ...................................................................... 81B. Chemically Defined Media ............................................................. 82

III. Basic Requirements....................................................................... 84A. Inorganic Materials.................................................................... 84B. Nitrogen Sources ...................................................................... 85C. Carbon and Energy Sources............................................................ 86D. Accessory Growth Factors ............................................................. 87E. Oxygen........................................................................... 88F. Carbon Dioxide ....................................................................... 89G. Other Physicochemical Factors ........................................................ 89

IV. Selective Factors in Population Changes................................................... 90V. Practical Applications..................................................................... 92

A. Primary Isolation ..................................................................... 92B. Mass Propagation ..................................................................... 93C. Preservation of Viability .............................................................. 94D. Identification and Classification........................................................ 94

VI. References................................................................................ 94

I. INTRODUCI1ON by Bruce (18), have been used in the cultiva-

Sir David Bruce in 1887 isolated on peptone- tion of brucellae. The beef liver infusion mediumbeef extract agar the causative "micrococci" of Stafseth (94) is typical and earlier found con-from fatal cases of human brucellosis (18). Dur- siderable use. Potato infusion agar (93) wasing the next forty-five years of investigation of recommended by the Bureau of Animal Indus-the brucellae, the major basic finding in their try, U. S. Department of Agriculture, and isnutrition was the observation that freshly iso- still in use for the preparation of standardizedlated Brucella abortus requires an added amount antigen and of vaccine for cattle.of carbon dioxide for growth (6, 41, 100). About The variability in composition and nuisance of1932, the comprehensive work of ZoBell and preparation of such infusion media were obviatedMeyer (103-106) established a solid foundation about 1938 by the commercial development of afor the study of growth requirements in defined peptone in dehydrated form, which was preparedenvironments and since then significant advances from a pancreatic digest of casein and whichin knowledge of the nutrition of brucellae have supported superior growth of brucellae (42). Inbeen accomplished. The limited objectives of its present form, this medium contains 2 perearly investigation, better methods of isolation cent peptone, 0.1 per cent glucose, 0.5 per cent

and classification, have since been enlarged to sodium chloride, and 0.5 mg per cent thiamin.include other practical applications, a directed A number of similar casein-digest media areapproach to chemotherapy in brucellosis, and available commercially; the choice betweenperhaps most importantly, basic information on them is largely a matter of individual preference.the physiology of these pathogens and of bacteria Media prepared with acid-hydrolyzed casein,in general. preferably if the hydrolyzate is subsequently

deionized, also support excellent growth ofII. COMPOSITION OF MEDIA brucellae (82, 87).

Modifications of these complex media haveA. Und~efined Media appeared from time to time in the literature and

A variety of undefined media, usually meat in the manufacturers' formulas. Yeast (67, 74)infusion comparable to that originally employed and liver (40, 88) extracts and hemin (59) have

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82 PHILIPP GERHARDT [VOL. 22

been reported to enhance growth in some cases, isolated from autoclaved cystine solutions, andand the growth-stimulating effect of thiamin the previously observed toxicity was duplicatedwhen this vitamin is added to some undefined by the addition of colloidal dispersions of sulfurmedia suggests that it may sometimes be a from this or other sources in concentrations aslimiting factor (53, 67, 85). The addition of iron low as 0.06 ,ug per ml of medium (92). Sulfurand higher concentrations of glucose increased toxicity, which develops with aging of the pep-the growth of Brucella 8uis in one medium (67). tone studied and possibly of other peptones asWhen the supply of oxygen is greatly increased, well, is neutralized in culture media containingthe usual concentrations of peptone and glucose either blood or agar but may require considera-may become limiting (85). Yeast extract, thiamin, tion if such broth media are not supplemented.sodium bisulfite, and phosphate have been in- The growth of brucellae on undefined mediacluded in various commercial preparations along may be inhibited by certain fatty acids. Boyd andwith the usual peptones, salt, and glucose. Casman (7) found that such components prob-Some of the highest yields reported for growth ably account for the toxicity of media filtered

of brucellae, up to 1 X 1012 viable cells per ml, through cotton or for the original toxicity of ahave been attained in a medium consisting of 3 commercial peptone. These toxic materials, toper cent peptone, 3 per cent glucose, 1 per cent which strains of the organism apparently varyyeast extract, and 0.15 per cent Na2HPO4 at in sensitivity, may be extracted by fat solventpH 6.4, sterilized by filtration (94a, 98a). In at- or neutralized by starch. Huddleson similarlytempts to confirm these results. Tyrrell and reported (46, 48) the enhancement of growth,Gerhardt (unpublished) found that nearly a especially of Wilson strains of B. abortus, afterdecimal increase in the yield could be attributed treatment of peptones with charcoal, serum, orto the use of filtration rather than heat for Tween; the original toxicity was duplicated insterilization of the medium. nontoxic peptones by the addition of oleic acid.A comprehensive investigation of the toxicity Living animals, cell suspensions, and body

of some lots of a peptone for brucellae was re- fluids constitute exceedingly important "media"ported in a series of papers by Schuhardt and for growth of brucellae. Embryonated chickens'his associates at the University of Texas (89-92); eggs (93), leucocytes (80a), and tissue culturestheir observation has been confirmed, and the (39a) provide especially useful in vivo systems.peptone also has been reported to affect agglu-tinability of brucellae (19). The Texas group B. Chemicaly Defined Mediainitially detected a brucellacidal factor which Critical study of the nutritional requirementscould be extracted and concentrated 60-fold. of brucellae inevitably led to development ofIt was neutralized by agar, blood, and other media in which all components were known. Suchsubstances, and seemed to consist of oxidized an approach not only resulted in a rational im-amino acids (89). Subsequently it was also provement of undefined media (67) but has pro-found that the toxicity developed upon aging vided controlled environments for allied studiesand was not the result of gross exposure to labora- of intermediary metabolism, regulatory mech-tory environments (91). When amino acids were anisms, genetics, and virulence.systematically added to a synthetic medium Koser and Rettger in 1919 (55) were amongoriginally lacking amino acids, cystine was the first of several workers who unsuccessfullyfound to be toxic (90). However, the apparent attempted to grow brucellae in a syntheticcystine toxicity in synthetic medium could be medium. The comprehensive investigation ofneutralized by adding nontoxic peptone, and ZoBell and Meyer (103-106) and ZoBell andcystine reversed the inhibition of growth caused ZoBell (107), beginning in 1930, resulted in suc-by adding toxic peptone. When cystine was cessful cultivation of brucellae in chemically de-heat-sterilized at pH 7 and was then added to fined media; however, relatively large inoculanontoxic peptone, the resulting medium became were required, the growth period was prolonged,toxic for brucellae (91). These findings impli- the actual cell yields were low, and serial transfercated a degradation product of cystine as the was not possible. In this connection ZoBell andantibrucella factor and this interpretation sub- Meyer correctly noted that the failure of smallsequently was confirmed. Elemental sulfur was inocula to grow "may be interpreted as a mani-


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19581 NUTRITION OF BRUCELLAE 83

TABLE 1Composition of some chemically defined media formulated for the growth of brucellae*

.mposn . lKoser et l. McCullough g Rode d.al. Sanders McCullough and Gerhardt andcomposition (54) al. (67) (84) d al. (87) Dick (66) Wilson (31)

mg/mlSodium chloride 8.0 8.0 7.5 7.5 7.5Dipotassium phosphate 1.0 1.0 1.0 1.74 1.0 10.0Sodium thiosulfate 0.1 0.1 0.1Glucose 3.0 10.0 4.0 25.0 1.0Glycerol 1.0 30.0Lactic acid 5.0

Mg++ 10.0 10.0 10.0 62.0 10.0 10.0Fe++ 4.00 0.10 2.8 0.10Mn++ 0.10 0.10 5.5 0.10Thiamine*HCI 0.20 0.10 0.20 0.15 0.20 0.20Nicotinic acid 0.20 0.80 0.20 2.0 0.20 0.20Ca pantothenate 0.20 0.30 0.04 0.04 0.04Biotin 0.0001 0.0001 0.0001 0.0001 0.0001Ammonium sulfate 500 500DL-Asparagine 3000Glycine 200 337 100DL-ca-Alanine 500 100 1200DL-Valine 100 100DL-Leucine 100 50DL-Isoleucine 59 50DL-Aspartic acid 665 200L-Glutamic acid 500 411 (DL) 3000 3900 (1500)tDL-Serine 15 16 25DL-Threonine 15 47 25L-Proline 100 100L-Hydroxyproline 100L-Cystine 150 192 5 100DL-Methionine 100 104 50 940DL-Phenylalanine 100 99 25L-Tyrosine 50 49 25L-Tryptophan 200 20 25L-Arginine 100 82 100 770L-Lysine 200 146 (DL) 100 770L-Histidine 200 4464 100Initial pH 6.8-7 7 6.8-7 7.5 6.8-7.2 6.8-7

* Modified from a table by Rode et al. (84).t Recommended now in place of asparagine.

festation of the requirement of a vitamin, hor- reported partial success with synthetic media.mone, or accessory growth factor." They also Although limited by the absence of knowledge ofobserved the enhancement of growth from small vitamins, these early studies provided a wealthinocula when preparations of killed brucella of valid information on nitrogen and carboncells or products of the growth of other bacteria sources, inorganic salts, minerals, and physical-were added to the medium. These results now chemical balances required for the growth ofmay be attributed to a lack of accessory growth brucellae.factors in the media and a probable carry-over of Probably the first fully defined and nutri-traces of such substances in the inoculum. Con- tionally adequate medium was published bycurrently, McNutt and Purwin (68), Olitzki Koser et al. in 1941 (54), at which time theand Bromberg (78), and Huddleson (see 106) also specific preformed vitamin requirements of the


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brucellae were discovered. The constituents of be obtained by dispensing with the glycerol andthis and several subsequently described media lactate and using 0.5 per cent L-glutamate as theare summarized in table 1. sole source carbon, energy, and nitrogen (30).The medium of Koser et at. supported mod- In retrospect, the development and applica-

erately high cell yields (0.5 to 1 X 109 viable tion of synthetic media have tended toward eithercells per ml) in 3 days from small inocula (1 X of two types: media that contain multiple sources104) of 7 of the 8 strains tested and permitted of nitrogen and give maximum growth (54, 67,serial transfers, which are reasonable standards 84, 87, 102a) or media that contain a singleof nutritional adequacy. Their results have been source of nitrogen and have maximum sim-confirmed by others (64, 84) and recently their plicity compatible with moderate growth (31,medium has been modified to contain fewer 66). Very frequently these media have been de-amino acids (56a, 102a). veloped for a selected strain and then tested withThe Fort Detrick group published reports on a representative group (31, 67, 87). Even if the

two intensive studies of the nutrition of single goal has been general (84), differences in species,strains (67, 87). In both cases the cell yields ob- strains, level of inoculum, and aeration havetained (ca. 1 X 1010 viable cells per ml) were as tended to make comparisons of the value of thehigh as or higher than those attainable at that several formulations relative. All of the moretime with undefined media and the results were recent media that contain multiple amino acidsapplicable to most other strains. McCullough support growth of most strains from a smallet al. (67) and Sanders et al. (87) used strains of inoculum at rates and to levels at least com-B. &uis and Brucella melitensis, respectively. parable to those obtainable with undefined media.These investigations demonstrate what may be Even the simpler synthetic media often produceaccomplished in nutritional studies involving yields within a logarithmic unit of those ob-specific objectives and specific organisms. tainable with undefined media and support

In contrast, the medium of Rode et al. (84) growth from small inocula, particularly whenwas designed to promote growth from small thickening agents are added. The choice of ainocula of a large number of strains of the 3 medium, then, depends on the purpose of thespecies of Brucella. Of the 61 strains tested, in- study in which it is to be used.cluding 47 that required additional CO2, all grewin this medium, although wide differences were III. BASIC REQUIREMENTSobserved in the nutritional requirements of the A. Inogani Matalsvarious strains.The early demonstration of the adequacy of The need for a high buffering capacity near

the ammonium ion as a nitrogen source (68, 105) neutrality, for inorganic phosphate, and proba-and the subsequent definition of the added vita- bly for potassium ions has made the incor-mm requirements for growth of brucellae (54, 64) poration of K2HPO4 common to all definedled to the formulation by McCullough and Dick media for brucellae. Sodium ions usually have(66) of a different type of chemically defined been added at the same concentration as in themedium, which contains a minimum number of traditional "physiological saline" (0.85 per centconstituents and an ammonium salt as the nitro- NaCl), although the addition of NaCl probablygen source. This medium and others like it offer has little effect (87). Sodium and potassium ionsthe considerable merit of simplicity, although appear interchangeable (54, 105).this usually is obtained at the expense of high The requirement for sulfur may be met by anyyields of cells. of a number of inorganic or organic sources;The disadvantages in using ammonium salts thiosulfate and cystine have been used most

were overcome in the medium of Gerhardt and commonly. ZoBell and Meyer (105) recognizedWilson (31), who employed instead a single the relatively low levels of sulfur required by theamino acid. This medium was further studied and organisms and also present in many chemicals.modified (27, 30, 35, 72), and L-glutamate rather They found that cystine or cysteine, thiogyl-than the DL-asparagine used originally is now colate, sulfate, sulfite, and thiosulfate wererecommended as the nitrogen source. For growth utilized less well in the order given; sulfide andof at least one strain, maximum simplicity may elemental sulfur did not support growth. In a


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1958] NUTRITION OF BRUCELLAE 85

more recent and detailed investigation of the B. Nitrogen Sourcessulfur nutrition of B. suio in sulfur-free media, The success of early workers (68, 78, 103, 105)the Texas group (57, 58, 83) found that none of in obtaining growth of brucellae by using an10 strains assimilated inorganic sulfur at an sat, or a readily deaminated organicoxidation level above that of hydrosulfite. All compound, strongly indicated the sufficiency ofcultures utilized thiosulfate, sulfide, and hydro- the ammonium ion as a nitrogen source. Thissulfite, but not elemental sulfur. L-Cystine or since has been amply confirmed by experimentsL-cysteine seemed to be the best organic source using adequately supplemented media, smallof sulfur. The toxic effect of elemental sulfur ing su emented meia smallinocula, and serial transfers (31, 66). Any of theand the production of it on autoclaving cystine ammonium salts serve equally well, althoughhave been discussed previously in this review. they are unstable on autoclaving, and growth

Relatively large amounts of magnesium are from small inocula often is obtained oniy if theobligatory for growth of brucellae. Since the medium is semisolid (31, 106). Nitrates andcomponents of synthetic media usually are con- nitrites usually are reduced to aminoma by thetaminated with traces of various minerals, the brucellae (79), yet alone they fail to supportessentiality of such an element can best be buele(9,ytaoete alt uprdessentialitydo she anbeleeneofgrowthcnbes

a

b growth, possibly because the necessarily large

extracted with chelatingcenoro in media initial concentration of these oxidized nitrogenexracted wicursythhelatin gen ofsubcultur, es tsources poises the medium at an unfavorablygrowth occurs, by the failure of subcultures to high Eh. Readily deaminated compounds such

grow in such a depleted medium. An absolute as ea .Ramin ad amidescircumnt uheas urea, amilno acids, or amides circumvent therequirement of brucellae for magnesium was disadvantages of MUM salts and singlydemonstrated by these methods (27) and was replace them (30, 31, 105). Of these substances,strongly implied by less rigorous methods (67 appears to be the most satisfactory.105). Magnesium apparently cannot be replaced Consideration of the amino acid composition ofby manganese (27). casein, hydrolyzates of which provide the baseTreatment of the medium with chelating of most undefined media, has led to several veryagents revealed an absolute requirement for iron satisfactory combinations of amino acids, de-by strain 19of B. abortus (27), althoughinsimilar pending on the cultural conditions, strains, andexperiments iron appeared only stimulatory for criteria employed. The simplest combinationstrain PS-1 of B. suis (99), as well as for other consistent with maximum growth of B. melitendsstrains in studies in which special precautions indicated a requirement for glutamate, alanine,were not taken to remove traces of iron (67, 105). lysine, histidine, methionine, and cystine in oneSuch apparent differences in the need for iron instance (87) and for aspartate, cystine, arginine,and other trace elements and the rather wide histidine, phenylalanine, and seine in anothervariation in the amounts reported to support (56a, 102a). In an investigation of a B. iuts strainmaximum growth may reflect the degree of de- ( c h t ppletion in the basal medium, subsequent preven- and typtonwe reptetosbe esentialandtion of cont on, carry-over in the inoculum, 9an tr amptopaanwere reported to be essential and

presence of sequestering compounds in theo

medium, strains used, population levels reached, larger survey of 61 strains, the full componentX of 18 amino acids appeared necessary (84). Upon

oTher varefiiabl effects, of theadditionofotherassessing the amino acids apparently essentialThe beneficial effects of the addition of oer or even common to such formulations, one gainsthe impression that a balance rather than anypronounced. Although manganese often is in- specific amino acids are essential for

cluded in defined media for brucellae and appears growth of brucellae. However, differences inmoderately stimulatory, the one attempt to synthesizing capacity among strains also maydemonstrate a requirement for the element, gave account for the apparent inconsistencies in suchequivocal results (27). Calcium has been re- an analysis.ported to be stimulatory (67, 105) and traces The exact function of exogenous amino acidsoften are added in the form of calcium panto- in the nutrition of brucellae is debatable. Pre-thenate. Low levels of cadmium, cobalt, zinc, or formed amino acids may be assimilated per se,nickel are either ineffectual or toxic (67). relieving the necessity for their synthesis and


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thus enhancing the rate or maximum extent of metabolized by and support growth of smoothgrowth. However, it seems more probable that strains of brucellae, but that better utilizationmixtures of amino acids may simply supply a of, or lesser sensitivity to, the D-forms may bebalanced reservoir of ammonium or amino one of the distinguishing properties of non-nitrogen, which serves as the primary source for smooth mutants. These apparent differencesgrowth. This concept is supported by observa- between mutant types emphasize the importancetions that the optimum mixtures of amino acids of recognizing and controlling populationvary considerably (table 1), that single amino changes in nutritional studies on brucellae (12,acids are adequate for growth (30), that there is 33).a positive correlation between the nutritional Using a casein-digest medium to which wereand metabolic specificity for single amino acids added the D- or DL-amino acids essential in human(29a, 30), that amino acids are aerobically nutrition, Yaw and Kakavas (101) found thedeaminated (29), and that the ammonium ion racemic mixtures to be toxic for three strains ofsupports growth of the brucellae (31, 66, 103, B. abortus. A slowly bactericidal as well as a bac-105). teriostatic effect was indicated for the separate

Utilization by brucellae of organic nitrogen D-isomers of five of the amino acids. Continuedsources other than amino acids has received little subculture in the presence of any of several suchattention. It is not known whether simple pep- amino acids selected the less virulent mucoidtides will suffice or offer advantages for growth, mutants (25, 51, 102).although alanyl dipeptides recently have been The metabolism of sulfur-containing aminoinvestigated for their effect on population changes acids and the utilization of the stereoisomers by(3). Catalytic amounts of ribose nucleic acid or brucellae have been studied in detail by Lank-any of its component purines or pyrimidines ford et al. (57, 58) and Rode et al. (83).stimulate the rate of growth of B. suis in a mul- Glycine added in high concentrations to atiple amino acid medium (67). growth medium uniquely causes the formation ofOne of the very intriguing aspects of the nu- protoplast-like bodies from brucella cells. Un-

trition of this group of bacteria concerns their published results by the author have shown thatutilization of D-amino acids. The extensive work several strains incubated for 16 to 24 hr inon the apparent relationship between D-alanine Albimi broth containing 1.25 per cent glycineand the selective establishment of nonsmooth are converted almost entirely to these bodies.mutants will be considered subsequently in this Gentle agitation during the incubation, a heavyreview. Population changes are observed when inoculum of actively growing cells, the strain ofmost smooth strains of Brucella are grown under the organism, and the concentration of glycinenonaerated conditions in Gerhardt-Wilson me- were found critical, whereas moderate variationdium where DL-asparagine is employed as the in pH near neutrality or the addition of sucrosesole nitrogen source. D-Asparagine, alone or as made little difference. The brucella "protoplasts"the racemate, supports growth of several smooth appeared in the phase microscope as round ob-strains to varying degrees, but it greatly affects jects having a delicate membrane, which re-the accumulation of intermediates and the selec- mained after plasmoptysis; unfortunately, thetion of the nonsmooth mutants (35), and even is cytoplasm often appeared to be retracted to aassociated with formation of a red pigment (30). small portion of the "protoplast" and theThe utilization of D-alanine in a synthetic medium original cell (or cell wall) appeared to adhere(33) or of other D-amino acids in undefined media, to it, thus limiting their potential application tosimilarly is associated with the selection of non- physiological and immunological problems.smooth mutants (86, 101). Some of these variantsdevelop a requirement for alanine, presumably in C. Carbon and Energj Sourcesthe D-form (33). B. melitensis can be grown on Glucose traditionally is included in mostD-glutamate, D-methionine, D-cystine, and D-ala- bacteriological media as a source of carbon andnine, although the last two were tested as the energy. The concentration generally employedracemate (87). D-Cystine and D-methionine were for brucellae has been 0.1 to 0.4 per cent; how-reported to support growth of B. suis (83). It is ever, higher levels (1 to 3 per cent) were foundapparent that at least several D-amino acids are optimum and were completely utilized in several


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instances characterized by unusually high aera- that physical and not chemical factors aretion and yields (67, 85, 87, 94a, 98a, 98b). Other responsible for the growth-promoting propertiessugars may be employed less satisfactorily: 2.5 of glycerol" (105). Non-nutritive properties mayper cent glucose can be replaced completely by also apply in part to the effect of glucose, which2.5 per cent galactose or fructose (but not in a has reducing properties and, when heat-sterilizedcasein-digest medium), partially by 1 per cent in the medium, was shown to neutralize thearabinose or xylose, but not at all by ribose, toxicity associated with some peptones (89).lyxose, or various disaccharides or polysaccharidesfor growth of a strain of B. melitensis in a syn- D. Accessory Growth Fatrethetic medium (87). Using 12 strains of the three The beneficial effect of supplements such asspecies in McCullough-Dick medium, McCul- liver extract in natural media upon growth oflough and Beal (63) found that the lag period was brucellae was recognized early (40). Kerby (53)shortest and all strains could be carried through noted that growth of B. abortus on a peptoneserial transfers when erythritol was used as the medium was enhanced by the addition of nicotinicsole carbon and energy source. Glucose appeared acid and thiamin. In 1941, Koser et at. (54) firstnext best of 9 carbohydrates tested. Since strains demonstrated that seven representative strainsvary in their carbohydrate requirements, classi- of the three species require thiamin, calciumfication within the genus on the basis of glucose pantothenate, nicotinic acid (or nicotinamide),utilization during growth was advocated (60, 61) and probably biotin for growth in a chemicallybut later refuted (104). Fermentation of carbo- defined medium. The need for biotin was subse-hydrates by resting cells similarly has been sug- quently confirmed and the pyrimidine, but notgested (80) and questioned (1) as a convenient thiazole, component of thiamin was found to becriterion of speciation. required (56). McCullough and Dick (65) were

Several compounds support a greater rate of unable to correlate a vitamin requirement withrespiration than does glucose and consequently their earlier failure to obtain growth of C02-have been used as a carbon and energy source in sensitized strains of B. abortus in a defineddefined media. i-Glutamate appears the most medium (54); after acclimatization to normallikely replacement and its value when used atmospheric conditions, however, 30 of 41 suchsingly or in combination (table 1) as a nitrogen strains grew in the medium, 23 of these with onlysource possibly may be attributed in part to the thiamin and biotin as added vitamins. Quantita-concomitant oxidation of its carbon skeleton. tive requirements for the four vitamins subse-The same may be said for asparagine. Used alone, quently were determined for various strains andeither was found to support growth as the sole media (64, 67, 87). Recent investigations havenitrogen, carbon, and energy source (30). Of the centered on the role of pantothenate in popula-non-nitrogenous organic acids, L-malate is tion changes (3, 73) and they will be discussedrapidly metabolized and was used by Marr and later in this review. Mika et al. (73) concludedWilson (72) to replace lactate and glycerol in that strains vary in their utilization of preformedGerhardt-Wilson medium. In this medium pantothenate and that it is a stimulatory ratherlactate and the ammonium ion support the than an absolute nutritional requirement forgrowth of B. abortus strain 19 (31) but in a pep- brucellae. In reviewing these data on vitamins, ittone medium lactate offers no advantages as a appears that thiamin is indispensable for growthreplacement for glucose (85). A unique effect of of all strains, niacin is usually so for B. guis andlactate, permitting the utilization of methionine B. melitensis, and biotin is essential for B.and cystathionine, has been reported (83). abortus; niacin, biotin, and pantothenate often

Glycerol was recommended as a medium are stimulatory, if not absolutely required.supplement in several early investigations (78, Vitamin B12 has been studied in regard to its105). Although the effect of glycerol in these ex- effect on utilization of sulfur-containing aminoperiments may be partially attributed to a acids (58). Vitamin A has been reported to be anprobable contamination with vitamins, the high essential growth factor for some strains ofconcentration optima observed both in these Brucela (95).vitamin-deficient and in a subsequent vitamin- Several other supplements have been reportedsufficient medium (31) "are very suggestive to accelerate the initiation and rate of multi-


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plication of brucellae. As noted before, ribose atmosphere is humidified. Sanders and Huddle-nucleic acid, a mixture of its constituents, or son (85) reported that a flow of pure oxygen overadenine, guanine, or cytosine alone stimulated shaken cultures dramatically increased the ratesthe rate of growth but not the yield of cells from of growth and the total yields of three strains.small inocula of B. suis in a multiple amino acid This effect was especially pronounced when themedium (67). Two attempts have been made to concentrations of peptone and glucose were in-demonstrate a new growth factor in autolyzed creased to 3 and 2 per cent, respectively. Repre-yeast, which frequently is observed to stimulate sentative strains of the three species differed ingrowth of brucellae even in relatively complete their response to various 02 and CO2 atmospheresmedia. In both cases (23, 87), the factors were (85). However, a correlation between growth ofsoluble in water and ethanol, insoluble in ethyl brucellae and direct determinations of dissolvedether, adsorbed by charcoal, and stable to acid oxygen in the medium has not been made andand alkaline hydrolysis. In neither case was the there is reason to believe that the maximummaterial singly replaced by a mineral, known oxygen demand of brucellae has not yet beenvitamin, amino acid, purine, or pyrimidine. realized.

Aerobiosis also is a major selective factor inE. Oxygen population changes of brucella cultures, anoxia

The popular emphasis on chemical factors in generally favoring the growth of nonsmoothbacterial nutrition is evident also in studies with mutants (5, 8, 13, 86). This important factor willbrucellae, although the obvious effects of aeration be considered in detail subsequently in thisand the requirements of some strains of B. abortus review.for an increased pCO2 have focused attention on Oxygen is only slightly soluble and diffusesgaseous requirements, in particular where small slowly into static media. However, tradition andinocula are employed or large cell crops are convenience continue to favor the use of testdesired. tubes for cultivating aerobes such as brucellae,The brucellae are aerobic and do not grow even in nutritional work where the limitation of

under anaerobic conditions (106). In exception growth by a vital requirement casts doubt on con-to this statement, Zottner (108) reported the clusions. This limitation seems more readily ac-isolation of B. abortus in "pure" C02; however, it cepted with regard to vitamin deficiencies thanseems likely that the commercial CO2 he used with oxygen starvation. Shaken flasks or bottlescontained some oxygen or that he inadequately have been employed with considerable success toremoved air from the containers before replacing insure adequate aeration. Cuvette tubes that arewith CO2. Earlier, Wilson (100) observed growth attached as a sidearm to shaken flasks (23), di-in CO2 if as little as 0.5 per cent oxygen was rectly shaken longitudinally (27), swirled rapidlypresent. on a rotary shaker, or agitated internally withAs with most aerobes, growth initiation of magnetic impellers provide reasonable aeration,

brucellae is favored by a reduced air supply can be read directly in a colorimeter, and stillbut considerable increases of both the rate and retain the other advantages of test tubes.extent of growth result when cultures are ade- A modification in this recommendation forquately aerated by either shaking or sparging aeration is warranted when the sole criterion of(28,29, 31,67, 82, 85, 87, 98a). Agitation alone is "growth" is its initiation, especially from a smallineffectual (85) but turbulence is necessary for a inoculum. Then, an increase in oxygen supplyhigh rate of oxygen transfer. The type of sparger becomes deleterious, possibly because of a higherand the antifoam agent used are important Eh, and measures to prevent or neutralize thefactors in sparger aeration systems for mass pro- absorption of air actually become desirable. Addi-duction of brucellae; air economy and better tion of a thickening agent such as 0.1 per centfoam control result from the use of a gradually agar to provide a semisolid medium appears toincreasing rate of aeration (28). It is important to be the most effective measure, although strati-recognize that high aeration rates from shaking fication of growth results. It was demonstratedor sparging of cultures often result in a con- that the action of the agar is physical and thatsiderable and variable amount of evaporation this effect of agar may be duplicated with gelatinduring the usual incubation period unless the or synthetic methyl cellulose (31, 106) but not


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with various concentrations of reducing agents closed containers with cultures of Bacillus ubtilisuch as thiosulfate or cysteine. An attractive (77) and in sealed tubes (81). Later, followingalternative to the above idea is supplied by the others' clarification of the similar requirement byexperimental observations and literature review gonococci and meningococci, Huddleson (41,by Schuhardt et al. (89) concerning the possible 49) clearly showed that the increased pCO2neutralization of toxic materials by small amounts rather than reduced P02 was responsible for stim-of agar. In any case, the use of semisolid media is ulating growth of B. abortus, but he concludedrecommended for growth from a very small that this C02 in part "was necessary to produceinoculum of brucellae, especially in isolation pro- the pH change in the atmosphere." In 1931,cedures from clinical specimens (26). Wilson (100) demonstrated unequivocally that

Chemical reducing agents presumably favor CO2 is required per se.initiation of growth from small inocula, although On continued subculture, isolates of B. abortusit is difficult to separate this Eh effect from the frequency lose the requirement for added C02primary purpose for which such components are and may then be grown in air alone. Marr andusually added, and no adequately controlled Wilson (71) found that this "acclimatization"experiments have been reported. Thiosulfate as a consists in the selective establishment of spon-sulfur source and glucose as a carbon and energy taneously occurring mutants rather than thesource probably are also effective in lowering the gradual adaptation of the culture as a whole.Eh, and the high concentration optimum for The mutation rate for nine strains was shown toglycerol suggests that its growth-promoting be fairly constant and relatively low, varyingproperties are due to physical factors, possibly from 1.5 to 6.4 X 10-1° mutations per cell di-including Eh. vision, which indicated a high degree of stability

Several workers have described the negative for the C02 requirement in these cultures.drift of Eh that occurs with growth of brucellae The biochemical basis of the C02 requirement(29, 96, 106), although the observation in itself is unknown. Unlike comparable requirements inhas dubious significance. some other bacteria, the added C02 needed by

B. abortus apparently cannot be replaced by cellF.Carbon Dsoxtde extracts, complex nutrients, Krebs cycle inter-

Many freshly isolated strains of B. abortua mediates, or purines or pyrimidines (32, 72). Adiffer from others of the group in requiring a basic similarity in their other growth require-quantity of C02 in excess of that needed by most ments and in their oxidative metabolism appar-heterotrophs. Five per cent C02 in an air at- ently exists for both parent and mutant C02mosphere seems adequate for such dependent types (70). With isotopic techniques, the majorstrains (44, 45); 10 per cent frequently is used but products of C02 fixation in the protein of a de-may result in unfavorable pH changes (44, 45), pendent strain were identified as glycine andalthough either these increased levels or com- alanine (70), and in nucleic acids as pyrimidinesplete exclusion inhibits the growth of nonde- (75, 76); however, in neither case did a C02-pendent strains (62). Zottner reported that B. independent mutant differ from its parent in theabortus could be isolated equally well in "pure" distribution of the fixed C02 (70, 75, 76).C02 (108); if confirmed, the observation wouldpermit a simpler apparatus for isolation, as he G. Other Physicochemiial Factor8suggested, but it seems probable that such "These organisms thrive in rather wide rangesstrains are obligately aerobic, as discussed of hydrogen ion concentration. The maximumpreviously. multiplication was observed between pH 6.6 andThe C02 requirement of B. abortus apparently 7.4 with some growth at from pH 6.1 to 8.4.

was first studied by Bang in 1897 (6); observing Hydrogen ion concentration has a more specifica subsurface layer of growth in semisolid medium effect on the viability of the organisms suspendedincubated in air and using different atmospheres in salt solutions." These statements of Zobell andof 02 and C02, he thought that growth of the Meyer (106) still prevail, although confirmatoryorganism was influenced by lowered PO2. This data of comparable detail are wanting. A signifi-idea of a reduced oxygen tension also was used cant qualification, especially in primary isolationto explain why primary isolates grow well in of B. abortus, was emphasized by the experi-


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ments of Huddleson (44). These indicated a an observation of Goodlow et al. (34) that smoothgreater pH sensitivity of some C02-dependent B. abortus cells growing in unaerated Gerhardt-strains and the necessity for increased buffering Wilson medium (31) containing DL-asparagine ascapacity and/or a higher initial pH to counter the sole source of nitrogen are supplanted by athe acidity resulting from added C02 and blood. predominantly rough population late in theThe several defined media (table 1) are adjusted stationary phase of growth, at about which timeinitially to pH 6.8 to 7.5 and contain 0.1 to 1.0 alanine accumulates extracellularly. Furtherper cent phosphate buffer; in these media, the pH investigation revealed that the addition of olddoes not appreciably change during growth of culture filtrate or of DL- or D-alanine to smoothbrucellae. cultures early in growth accelerated the out-

Tradition and convenience often have dictated growth of alanine-resistant, nonsmooth types inthe use of an incubation temperature of 37.5 C the population; L-alanine had no effect except infor brucellae. A scant literature (23) and unpub- very high concentrations (33). When smooth B.lished information indicate that both the rate and abortus cells were inoculated into Gerhardt-extent of growth are greater at 34 C. Moreover, Wilson medium containing the L-isomer of as-a precipitous drop beyond the optimum usually is paragine, the change to a predominantly roughseen in a temperature curve for bacterial growth, population did not occur, nor did alanine accumu-suggesting the practicality of underestimating the late; with D-asparagine, the result was similarincubation temperature. Fortunately, adiabatic to that in the racemic mixture (35). However,cooling probably occurs in aerated cultures, so these effects associated with alanine are usuallythat actual temperatures are somewhat below delayed until late in the growth cycle, suggestingincubation temperatures. the possibility of an indirect action of alanine on

In apparently the only pertinent investigation the synthesis or utilization of a substance vital(106), the optimum osmotic pressure for growth for the multiplication of alanine-susceptible cells.of brucellae, as determined by cryoscopic The known association of alanine with panto-methods, seemed to be between 2 and 6 atm, thenic acid synthesis led to the investigation ofwhich is somewhat lower than that of usual cul- this relationship with respect to populationture media (about 7 to 8 atm). Solutions as hy- changes of brucellae. Mika et al. (73) observedpertonic as 10 atm are inimical both to multipli- that increased levels of pantothenate depressedcation and to viability. The organisms apparently the effect of alanine, that pantothenate-starvedare more sensitive to hypertonic than to hypo- cells displayed more pronounced populationtonic salt solutions. changes than pantothenate-rich cells (although

Surface tension influences the growth and, even the latter comparison was unfortunately mademore so, the morphology of brucellae, although with two different basal media), and that someonly qualitative data have been obtained (106). inhibitors of pantothenate synthesis duplicated

the effect of alanine. It is tempting to concludeIV. SELECTIVE FACTORS IN POPULATION CHANGES that D-alanine selects nonsmooth cells by inter-Mutation in brucellae is readily observed as fering with pantothenate synthesis, possibly

smooth-rough colony changes on solid media. competing with f3-alanine as a substrate analogueSince such "dissociation" also is correlated with (12, 73). This interpretation was furthered by thevirulence, considerable research has been di- concurrent observation of Altenbern and Ginozarected to this aspect of genetics and, pertinent to (2) that smooth cells can synthesize panto-this review, to the nutritional basis for population thenate more rapidly than rough cells. However,changes involving such genetically altered types. a rigorous analysis of the competition betweenThere is ample evidence that such changes repre- D-alanine and ,B-alanine has not been published.sent spontaneous mutations and that the emer- Interference of D-alanine with pantothenate syn-gence of a particular type in a population results thesis could not be demonstrated in resting cellsfrom environmental selection (9). Braun has (2); D-asparagine, however, was shown to inter-periodically reviewed the subject (9, 11, 12) and fere with pantothenate synthesis in resting cellspapers by Henry (38) and Huddleson (43) are at pH 5.4 but not at pH 7.4 (2). Recently, Alten-basic to the study of dissociation in brucellae. bern et al. (3) have found that the selective actionAn important segment of this work stems from of D-alanine in promoting population changes was


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antagonized competitively by L-alanine and ap- sociated types within a population was obtainedparently noncompetitively by L-alanyl-L-valine when broth with a lowered oxidation-reductionand L-alanyl-L-leucine, and that there was no potential was used." However, considerable sub-effect of L-alanine in suppressing population sequent work has indicated the opposite: rapidchanges in cultures not supplemented with growth specifically associated with aerobic con-D-alanine. Resting smooth cells synthesized and ditions brings about markedly reduced outgrowthexcreted panthothenate, whereas rough cells of nonsmooth types. Although interpreted inutilized the pantothenate present in the medium; terms of "population pressure," Braun in thethis difference was reported to be reflected in a same paper (8) observed that little dissociationgreater rate of pantothenate uptake by rough occurred when growth took place on the surfacecells, although small cell masses were used and of a solid medium. Goodlow et al. (12, 35) notedthe rate differences were not great. briefly that less dissociation in Gerhardt-WilsonThe evidence to date on alanine and panto- medium occurred when cultures were shaken or

thenate may be summarized as follows: (1) held statically with a thin layer of medium in thealanine, presumably the D-isomer, accumulates flask. The critical role of aeration in dissociationin nonaerated cultures of originally smooth B. was then clearly recognized by Braun et al. (13),abortus growing in a D-asparagine medium, and who found that the enhancement of dissociationthere occurs at about the same time a selective by homologous antiserum did not occur in aeratedestablishment of nonsmooth, alanine-resistant cultures grown in either complex or defined media.mutants in the population; (2) it cannot be con- Sanders and Huddleson (86) definitely estab-cluded unequivocally that pantothenate syn- lished that the increased population changes ofthesis is the primary site nor even that D-alanine originally smooth brucellae, brought about by theis the primary cause of the selective establish- addition of sugars or DL-alanine to a peptonement of nonsmooth mutants. medium, could be reversed by an adequate supply

This degree of skepticism is also supported by of air or oxygen. In a mixed inoculum of smooththe diversity of other environmental changes and nonsmooth cells in a glucose medium, thethat effect population changes of smooth to growth of nonsmooth types was suppressed byrough types, suggesting some underlying com- aeration and enhanced by the absence of aeration.mon mechanism. Thus, population changes in These findings were substantiated by the ob-originally smooth brucella cultures were found to servations of Altenbern et al. (5) that the popu-be suppressed by normal serum and globulin frac- lation change to nonsmooth types was greatly ac-tions of susceptible animals (10), pyrophosphate celerated by incubation under low air pressure(22), or chelating agents (21), and to be enhanced and that a rough variant grew more rapidly thanby homologous antiserum (8, 9), pH, Eh, and its smooth parent at reduced pressure. They madetemperature (8), penicillin (16), metallic ions the further observation that other hydrogen ac-(22, 99), valine (35), sugars (86), and oxygen de- ceptors (nitrate, methylene blue, or resazurin) inficiency (5, 8, 13, 35, 86). Kinetin (14) and break- Gerhardt-Wilson synthetic medium or in severaldown products of deoxyribonucleic acid (14, 17) undefined media could replace oxygen in sup-uniquely favor change from rough to smooth pressing population changes to nonsmooth types.colonial types and may have to be considered It thus appears clear that the supply of oxygen,separately. Of the factors listed, oxygen de- acting either as a hydrogen acceptor or as anficiency offers the most likely alternative to the oxidizing agent, is a critical factor in populationD-alanine-pantothenate hypothesis. changes of brucellae.

Recognition of the effects of aeration on popu- How may this fact be reconciled with thelation changes has developed over a period of effects of alanine and of the other factors observedtime. In his initial work on dissociation of bru- to be influential? At present, only partial ties arecellae, Braun (8) observed that lowered growth apparent. The brucellae are known to excreterates generally were associated with reduced pyruvate and alanine and to possess the en-population changes; this effect occurred with zymes necessary for interconversion and racemi-"daily disturbance" of the culture tubes, and "the zation of these products (4, 69); greater amountsmost striking demonstration of the effect of of such products commonly occur in bacterialgrowth rates upon the establishment of dis- metabolism as a consequence of anaerobiosis or


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of excess carbohydrate supply. Thus the natural essentially is one of obtaining growth of a strain-accumulation of D-alanine, or even valine, in variable heterotroph from an often small inocu-brucellae may be the result of anoxia; this possi- lum. under an environment different from thebility is borne out by the fact that alanine usually host. The essential prerequisite is a clinicalappears in detectable concentrations only after sample that actually contains the organism; inat-conditions selectively favoring nonsmooth types tention to this requirement undoubtedly ac-have been established, at least for strain 19 (3). counts for many failures. However, even idealAlthough added D-alanine is demonstrably sampling of blood at the crest of pyrexia, orselective, the amount required is considerably in biopsy of bone marrow or lymph nodes, oftenexcess of that produced naturally by cultures (3). will yield specimens containing relatively fewMoreover, L-alanine at still higher levels (33), or brucellae. Consequently, preliminary concen-for that matter several sugars (86), will produce tration of the few cells by centrifugation or filtra-the same effect in unaerated media. A possible tion (15) makes good sense.relationship between aeration and pantothenate Frequently described as fastidious in theiris not immediately apparent. In the case of en- growth requirements, the brucellae actually arehancement of dissociation by metals, and of the fairly typical gram-negative organisms withopposing action of pyrophosphate or specific basically simple nutritional requirements andchelating agents or possibly other factors that relatively complete synthetic capacities. Althoughmay act by sequestering metals, the effects may strains differ in specific requirements, the inclu-be related to electron transport systems con- sion of complex nutrients with the clinical sampletaining metals, such as the cytochromes. That is, makes the selection of a basal medium relativelyrough and smooth types conceivably may differ unimportant. The sulfur toxicity occurring inin their dependence on these pathways to oxygen. some lots of peptone undoubtedly is neutralizedPossible relationships between aeration and other under the conditions of primary isolation.selective factors are obscure and generally have A semisolid medium with 0.1 to 0.3 per centnot been examined, although anoxia was found agar (26, 31, 105) has much to recommend it overto explain the seemingly selective effect of liquid or solid media alone or even Castaneda'shomologous antiserum (13) and aeration was widely used combination (20). Heating and cool-reported (Braun, personal communication) to ing the tube of semisolid medium just before use,antagonize rough-to-smooth changes in the a routine procedure with pneumococci, would bepresence of breakdown products of deoxyribo- a simple precaution against excessive oxidation.nucleic acid. It may be that there is no simple A solid sample (i.e., one collected on a membranerelationship between oxygen availability and the filter) should be arranged longitudinally in thesurvival ability of a mutant. However, it seems tube as growth stratification may be expected todoubtful that an understanding of the physio- occur.logical basis of population changes in brucellae The crude but rational procedure of enrichingcan be furthered by enumeration of separately a culture by adding killed cells or extracts ofeffective factors, which must be endless, or by brucellae to the medium (45, 47, 48, 103) alsothe detailed study of any one of them without seems worthy of a wider test for primary isolation.adequate consideration of the others. This procedure has been found especially useful in

inducing growth of the so-called Wilson strains ofV. PRACTICAL APPLICATIONS B. abortus, which frequently are missed in the

A. Primary Isolation usual procedures (45, 47, 48).Huddleson (44) recently has re-emphasized

Laboratory procedures for the primary iso- the necessity of pH control in the isolation oflation of brucellae from clinical samples have C02-dependent strains from blood. Any of severalfrequently and adequately been reviewed else- procedural faults may allow the pH to fallwhere (93). Although a complete appraisal of critically below the optimum (pH 7.2 to 7.6) forsuch methods is beyond the scope of this review, initiation of growth from small numbers of suchbasic nutritional investigations have disclosed strains: inadequate levels of buffer, or inhibitoryseveral principles that would seem applicable. types, e.g., KHPO4; an atmospheric concentra-The problem of primary isolation of brucellae tion of CO2 above the adequate level of 5 per


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cent; or failure to compensate for the acidifying offers considerably increased efficiency as well aseffects of erythrocytes. A practical means of ac- a physiologically uniform product (28a); how-complishing adequate control and of dispensing ever, these advantages are largely lost unlesswith the usual CO2 jar is to add to a diaphragm- subsequent operations also are done continuously.sealed container sufficient carbonate to bring the Since concentration of the cells imposes thepH of the medium initially to 7.5 (44). greatest safety problem, the possibility of grow-Methods for isolating brucellae from sources ing brucellae in concentrated culture has ob-

other than the usual clinical specimens have been vious merit. One approach to this objective wassummarized by Spink (93). A highly selective to cultivate the cells in a charcoal-cellophanemedium was described recently by Morris (74a). system contained in a shaken flask (36). A thin-

walled cellophane sac containing the nutrientmedium was immersed in saline containing char-

The need for large numbers of cells requires coal and the growing organisms. As an exampleefficient and safe laboratory methods for growth of the results, populations of strain 19 of B. abortusand concentration of brucellae. The usual pro- rose to 1.4 X 1011 viable cells per ml, compared tocedure, and possibly the best for good yield and an equal-volume control of 5.4 X 1010, a concen-minimum dissociation, is simply to inoculate tration ratio of 2.6. Unfortunately, this compari-evenly a large, previously dried surface of agar son did not fully take into account aeration andmedium in a Roux, Povitsky, or penicillin-culture toxicity differences, and the presence of charcoalbottle; the resultant growth is harvested by dis- with the cells may be impractical for some appli-lodging the growth with a small amount of cations. Recently Sterne (94a) successfully useddiluent. The suspension then is removed by a cellophane tube apparatus for production ofaspiration and the cells are washed by centrifuga- strain 19 vaccine, with yields up to 1 X 1012 viabletion in plugged cups. cells per ml reported.When larger numbers of cells are needed, it be- This principle of physically confining growth so

comes necessary to turn to aerated liquid cul- as to obtain concentrated cell populations wastures in large containers. Although limited by further exploited by using a biphasic growthvolume requirements, large shaker flasks have system, which consists simply of a layer of solidthe obvious advantages of simplicity and relative nutrient medium overlaid with a small volumesafety. In using carboys or similar vessels, the of nutrient broth in a flask aerated by shakingmain consideration is to provide adequate agita- (97). Such cultures of three BruceUa species in ation and aeration; in all probability, it will be heat-sterilized casein-digest medium yielded 0.3physically impossible to over-aerate, and one may to 2.6 X 1011 viable cells per ml as compared toin practice be guided by the limit of foam control. populations in control flasks of 0.3 to 1.6 X 1010,Vortex aeration may offer some advantages with concentration ratios of 10 to 20. When a(98b). Several procedures are worth special at- filter-sterilized medium was used, yields of straintention: use of a gradually increasing rate of 19, for example, rose to 1.2 X 1011 viable cellsaeration (28); oxygen-enriched air, while main- per ml in the biphasic system as compared totaining adequate C02 levels, together with a more 2.2 X 1010 in the control flask, with a concentra-concentrated medium (85); a means for obtaining tion ratio of 5.5 (Tyrrell and Gerhardt, unpub-turbulence as well as air passage; and an effective lished). Adequate aeration of the broth and thebut nontoxic antifoam. agent. A stable silicone depth rather than quantity of the nutrient agaremulsion such as General Electric antifoam no. 60 dept rathe th quantit ofte ientag... . . * ~~~were found to be important in determining theis excellent; it is effective and nontoxic in con- . . . . tcentrations of 50 parts per million. Considerable otimu ratio of lqist s ase. th vreincreases in yields can be effected by sterilizing tive efficiency of the system was found to varythe medium by filtration rather than by heat with different media and organisms(94a, 98a). Precaution should be taken against A remarkable concentration of B. abortus inthe rapid decline of viability and the selective the placental cotyledon of the bovine foetus hasestablishment of nonsmooth mutants after been reported by H. Smith, J. Keppie, and R.maximum populations are obtained. Fuller, in a personal communication; these

Continuous culture of brucellae is possible and workers have separated masses of in vivo grown


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cells from this tissue for the purpose of chemical It is now commonly recognized that distilledand antigenic studies. water, saline, buffered saline, or other similar

In all the above cases, the eventual problem of diluents are toxic to dilute suspensions of bru-separating the cells from the menstruum, a cellae, and some quantitative data on toxicitynormally simple procedure, is here complicated have been published (24, 107). Dilute peptoneby the infectious hazard associated with the most (24) or peptone-saline are generally preferred aseffective methods, e.g., continuous centrifuga- diluents for counting. Among seven diluentstion. Enclosure of centrifugal equipment in an tested in unpublished work by the writer,autoclave, use of settling columns with a hydro- phosphate-saline (pH 6.8, M/15 phosphate, 0.85philic flocculating agent such as methyl cellulose per cent NaCl) supported a relatively constantor gelatin (39), or various types of filtration (52) endogenous and exogenous (glucose, glutamate,may prove practical in special situations. Repli- or asparagine) respiration of heavy suspensionscated batch sedimentation in some kind of a of strain 19 of B. abortus for the period of at leastbucket centrifuge usually proves to be the most 1 week.practical method in a laboratory and high speed, D. Idifan and Clsificationlarge-capacity centrifuges are available.

C.Pre.oofViabilityNutritional studies have found considerableC. Preservation of Viability application in the identification and classification

Maintaining the viability of brucellae over of brucellae. These methods have been appraisedperiods of time becomes of concern in the use of in Spink's recent monograph (93).living vaccines, stock cultures, dilute suspensions VI. REFERENCESfor counting, and concentrated suspensions formetabolic work. Several observations concerning 1. AIKEN, M. A., AND WEAVER, R. H. 1956these problems may be mentioned. Fermentation reactions with Brucella.

Freeze-drying has obvious merit and is widely Bacteriol. Proc., 53.used for preserving permanent stock cultures 2. ALTENBERN, R. A., AND GINOZA, H. S. 1954andvaccines.The latterpurposerquirPantothenic acid synthesis by smoothand vaccines. Tee latter purpose requires a hgher Brucella abortu&. J. Bacteriol., 68, 570-576.

viability retention during the freeze-drying 3. ALTENBERN, R. A., GINOZA, H. S., AND WIL-process, when most of the killing occurs. A pre- LIAMs, D. R. 1957 Metabolism and popu-liminary study of some physical factors that in- lation changes in Brucella abortus. I. Rolesfluence the survival of B. abortuo during freeze- of alanine and pantothenate in populationdrying was reported by Hutton et al. (50). Van changes. J. Bacteriol., 73, 691-696.der Scheer (98) recently reviewed the pertinent 4. ALTENBERN, R. A., AND HOUSEWRIGHT, R. D.literature. He also reported the experimental de- 1953 Transaminases in smooth Brucellavelopment of a suspension medium containing 2 abortus, strain 19. J. Biol. Chem., 204,per cent peptone, 0.75 per cent thiourea, and 0.5 159167.percentfreshlyprepar andneutralizedas- 5. ALTENBERN, R. A., WILLIAMS, D. R., KELSH,

per centbcchic prepared and neutralized as- J. M., AND MAUZY, W. L. 1957 Metabo-corbic acid, which gave over 82 per cent survival lism and population changes in Brucellaof B. abortue strain 19. It has been pointed out abortu8. II. Terminal oxidation and oxygen(11) that lyophilization does not necessarily pre- tension in population changes. J. Bac-clude the appearance of mutant types. teriol., 73, 697-702.

Brucellae also may be stored effectively and 6. BANG, B. 1897 Die Aetiologie des seuchen-conveniently on slants under paraffin oil (37) or haften (infectiosen) Verwerfens. Z. Thier-as suspensions in an equal mixture of horse serum med., 1, 241-278.and peptone broth (93), in either case frozen 7. BomD, D. M., AND CASMAN, E. P. 1951 In-hard to minimize mutation. This serious and hibition of a strain of Brucella abortue byfrequentlyinueddifiulyamedium filtered through cotton. U. S.frequently incurred difficulty also may be les- Public Health Repts., 66, 44-49.

sened by growing the cultures before storage as 8. BRAUN, W. 1946 Dissociation in Brucellabriefly as feasible, certainly not beyond the abortus: a demonstration of the role of in-logarithmic phase, and by transferring the cul- herent and environmental factors in bac-tures from selected and tested small single terial variation. J. Bacteriol., 51, 327-349.colonies (12). 9. BRAUN, W. 1947 Bacterial dissociation. A


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critical review of a phenomenon of bacterial 24. DEMELLO, G. C., DANIELSON, I. S., ANDvariation. Bacteriol. Revs., 11, 75-114. KIsER, J. S. 1951 The toxic effect of

10. BRAUN, W. 1949 Studies on bacterial varia- buffered saline solutions on the viability oftion and selective environments. II. The Brucella abortus. J. Lab. Clin. Med., 37,effects of sera from Brucella-infected ani- 579-583.mals and from normal animals of different 25. DEVERELL, M. W., AND KAKAVAS, J. C. 1955species upon the variation of Brucella abor- Virulence determination of D-valine adaptedtu8. J. Bacteriol., 58, 299-305. strains of Brucella abortus using chick em-

11. BRAUN, W. 1950 Variation in the genus bryo. Bacteriol. Proc., 105.Brucella. In Brucellosis, pp. 26-36. Am. 26. Diagnostic procedures and reagents 1950 3rdAssoc. Advance Sci., Washington, D.C. ed., pp. 17, 246. Am. Public Health Assoc.,

12. BRAUN, W. 1953 Bacterial genetics. W. B. Inc., New York, N. Y.Saunders Co., Philadelphia. 27. EVENSON, M. A., AND GERHARDT, P. 1955

13. BRAUN, W., ALTENBERN, R., KELSH, J., AND Nutrition of brucellae: utilization of iron,SANDOVAL, H. 1956 Anoxia as a major magnesium, and manganese for growth.factor causing population changes in anti- Prodl Soc. Exptl. Biol. Med., 89, 678-680.serum-containing cultures. J. Bacteriol., 28. GEE, L. L., AND GERHARDT, P. 1946 Bru-71, 417-420. cella suis in aerated broth culture. II.

14. BRAUN, W., FIRSHEIN, W., AND WHALLON, J. Aeration studies. J. Bacteriol., 52, 271-281.1957 Effects of desoxyribonucleic acid 28a. GERHARDT, P. 1946 Brucella suis in aer-breakdown products on bacterial popula- ated broth culture. III. Continuous culturetion changes and virulence. Science, 125, studies. J. Bacteriol., 52, 283-292.445-447. 29. GERHARDT, P., AND GEE, L. L. 1946 Bru-

15. BRAUN, W., AND KELSH, J. 1954 Improved celia 8uis in aerated broth culture. I. Pre-method for cultivation of Brucella from the liminary studies on growth assays, inocu-blood. Proc. Soc. Exptl. Biol. Med., 85, lum and growth characteristics in an im-154-155. proved medium. J. Bacteriol., 52, 261-269.

16. BRAUN, W., KRAFT, M., MEAD, D. D., AND 29a. GERHARDT, P., LEVINE, H. B., AND WILSON,GOODLOW, R. J. 1952 Effect of penicillin J. B. 1950 The oxidative dissimilation ofon genetic changes and temporary modifi- amino acids and related compounds bycation in populations of brucellae. J. Bac- Brucella abortus. J. Bacteriol., 60, 459-467.teriol., 64, 41-47. 30. GERHARDT, P., TUCKER, L. A., AND WILSON,

17. BRAUN, W., AND WHALLON, J. 1954 The J. B. 1950 The nutrition of brucellae:effects of DNA and enzyme-treated DNA utilization of single amino acids for growth.on bacterial population changes. Proc. J. Bacteriol., 59, 777-782.Natl. Acad. Sci., 40, 162-165. 31. GERHARDT, P., AND WILSON, J. B. 1948 The

18. BRUCE, D. 1887 Note on the discovery of a nutrition of brucellae: growth in simplemicroorganism in Malta fever. Practi- chemically defined media. J. Bacteriol.,tioner, 39, 161-170. 56, 17-24.

19. CARRhRE, L., RENOUX, G., AND QUATREFAGES, 32. GERHARDT, P., AND WILSON, J. B. 1950G. 1951 A propos de Faction de certaines Attempts to replace the added carbon diox-peptones (tryptose) sur les Brucella. Ann. ide required by some strains of Brucellainst. Pasteur, 80, 321-322. abortus. J. Bacteriol., 59, 311-312.

20. CASTANEDA, M. R. 1947 A practical method 33. GOODLOW, R. J., BRAUN, W., AND MIKA, L. A.for routine blood cultures in brucellosis. 1951 The role of D-alanine in the growthProc. Soc. Exptl. Biol. Med., 64, 114-115. and variation of Brucella abortus. Arch.

21. COLE, L. J. 1952 Suppression of colonial Biochem., 30, 402-4.variation in Brucella abortus by metal-com- 34. GOODLOW, R. J., MIKA, L. A., AND BRAUN,plexing compounds. J. Bacteriol., 64, W. 1950 The effect of metabolites upon847-853. growth and variation of Brucella abortus.

22. COLE, L. J., AND BRAUN, W. 1950 The J. Bacteriol., 60, 291-300.effect of ionic manganese and magnesium 35. GOoDLOW, R. J., TUCKER, L., BRAUN, W., ANDon the variation of Brucella abortus. J. Bac- MIKA, L. A. 1952 Effect of the isomericteriol., 60, 283-289. configuration of the source of nitrogen on

23. COPELAND, J. M. 1955 Characterization of changes in population and metabolism ina stimulatory factor for Brucella abortus cultures of brucella. J. Bacteriol., 63,from yeast autolysate. M.S. Thesis, Uni- 681-685.versity of Wisconsin. 36. GORELICK, A. N., MEAD, D. D., AND KELLY,


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E. H. 1951 The growth of bacteria in a tus (Bang). J. Infectious Diseases, 40,charcoal-cellophane system. J. Bacteriol., 352-368.61, 507-513. 50. HUTTON, R. S., HILMOE, R. J., AND ROBERTS,

37. HARTBELL, S. E. 1953 The preservation of J. L. 1951 Some physical factors that in-bacterial cultures under paraffin oil. Appl. fluence the survival of Brucella abortus dur-Microbiol., 1, 36-41. ing freeze-drying. J. Bacteriol., 61, 309-

38. HENRY, B. S. 1933 Dissociation in the 319.genus Brucella. J. Infectious Diseases, 52, 51. KAKAVAS, J. C., AND YAW, C. 1953 Anti-374-402. genicity and virulence of Brucella abortus

39. HODGE, H. M., AND METCALFE, S. N. 1958 (strain 19) modified to grow in D-aminoFlocculation of bacteria by hydrophilic acids. Bacteriol. Proc., 48.colloids. J. Bacteriol., 75, 258-264. 52. KAPLAN, A. M., AND ELBERG, S. 1946 Con-

39a. HOLLAND, J. J., AND PICKETT, M. J. 1956 centration of Brucella suis from broth cul-Intracellular behavior of brucella variants tures. J. Bacteriol., 52, 513-517.in chick embryo cells in tissue culture. 53. KERBY, G. P. 1939 Nicotinic acid and thia-Proc. Soc. Exptl. Biol. Med., 93, 476-479. min hydrochloride as growth-promoting

40. HOLTH, H. 1911 Untersuchungen uber die factors for Brucella. J. Bacteriol., 37,Biologie des Abortusbazillus und die Im- 495-499.munitititsverhaltnisse des infektiosen Abor- 54. KoSER, S. A., BRESLOVE, B. B., AND DORF-tus der Rinder. Z. Infekt. Haustiere, 10, MAN, A. 1941 Accessory growth factor207-273. requirements of some representatives of the

41. HUDDLESON, I. F. 1921 The importance of Brucella group. J. Infectious Diseases, 69,an increased carbon dioxide tension in 114-124.growing Bact. abortus (Bang). Cornell 55. KOsER, S. A., AND RETTGER, L. F. 1919Vet., 11, 210-215. Studies on bacterial nutrition. The utiliza-

42. HUDDLESON, I. F. 1939 Brucellosis in man tion of nitrogenous compounds of definiteand animals. The Commonwealth Fund, chemical composition. J. Infectious Dis-New York. eases, 24, 301-321.

43. HUDDLEBON, I. F. 1952 The dissociation 56. KoBER, S. A., AND WRIGHT, M. H. 1942pattern in the species of genus Brucella. Further experiments on accessory growthMich. State Coll. Agr. Expt. Sta., Memoir factor requirements of the Brucella group.6, 7-63. J. Infectious Diseases, 71, 86-88.

44. HUDDLEBON, I. F. 1954 Important factors 56a. KtTWAHARA, S., YOKOYAMA, K., INOUE, Z.,influencing the growth of a C02-dependent AND TANAKA, J. 1957 Studies on thestrain of Brucella abortus in a liquid cul- amino acid requirements of brucellae. I.ture medium in the presence of blood. Simple amino acid media for each speciesQuart. Bull., Mich. Agr. Expt. Sta., 37, 3-13. of Brucella. Japanese J. Microbiol., 1,

45. HUDDLESON, I. F. 1954 Effect of killed bru- 117-123.cella cells and an extract from sonically dis- 57. LANKFORD, C. E., RODE, L. J., AND SCHU-integrated brucella cells in culture mediums HARDT, V. T. 1952 Effect of methionineon the growth of Brucella abortus. Quart. on utilization of DL-homocystine by Bru-Bull., Mich. Agr. Expt. Sta., 37, 14-22. cella suis. Proc. Soc. Exptl. Biol. Med.,

46. HUDDLESON, I. F. 1955 Effect of esters of 80, 727-731.long chain fatty acids in agar mediums on 58. LANKFORD, C. E., RODE, L. J., AND SCHU-the growth of Brucella abortus in the pres- HARDT, V. T. 1956 Factors influencingence of blood. Bacteriol. Proc., 122. utilization of the stereoisomers of homo-

47. HUDDLESON, I. F. 1955 The effect of bac- cystine by brucella. J. Bacteriol., 71,terial cells and sonic extracts from bacterial 582-587.cells in culture mediums on the colonial 59. LIBERMAN, J. D., AND PICKETT, M. J. 195Zgrowth of Brucella abortus. Am. J. Vet. Hemin requirements of brucellae. Bacte-Research, 16, 264-268. riol. Proc., 54.

48. HUDDLESON, I. F. 1956 Improvement of 60. MCALPINE, J. G., PLASTRIDGE, W. N., ANI>culture mediums for the growth and species BRIGHAM, G. D. 1929 Studies on thecharacterizations of Brucella abortus type metabolism of the abortus-melitensis group.II (Wilson). Bacteriol. Proc., 53. 5. Factors influencing sugar utilization. J.

49. HUDDLESON, I. F., HASLEY, D. E., AND TOR- Infectious Diseases, 45, 485-489.RZY, J. P. 1927 Further studies on the 61. McALPINE, J. G., AND SLANETZ, C. A. 1928isolation and cultivation of Bacterium abor- Studies on the metabolism of the abortus-


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melitensis group. 3. Glucose utilization. J. 74a. MORRIS, E. J. 1956 A selective mediumInfectious Diseases, 42, 73-78. for Brucella spp. J. Gen. Microbiol., 15.

62. MCALPINE, J. G., AND SLANETZ, C. A. 1928 629-631.Studies on the metabolism of the abortus- 75. NEWTON, J. W., MARR, A. G., AND WILSON,melitensis group. 4. Effect of various con- J. B. 1954 Fixation of carbon14 dioxidecentrations of carbon dioxide. J. Infectious into nucleic acid constituents by BrucellaDiseases, 43, 232-240. abortus. J. Bacteriol., 67, 233-236.

63. MCCULLOUGH, N. B., AND BEAL, G. A. 1951 76. NEWTON, J. W., AND WILSON, J. B. 1954Growth and manometric studies on carbo- Carbon dioxide requirements and nucleichydrate utilization of Brucella. J. Infec- acid synthesis by Brucella abortus. J. Bac-tious Diseases, 89, 266-271. teriol., 68, 74-76.

64. MCCULLOUGH, N. B., AND DICK, L. A. 1942 77. NOWAK, J. 1908 Le bacille de Bang et saPhysiological studies of Brucella. I. Quan- biologie. Ann. inst. Pasteur, 22, 541-546.titative accessory growth factor require- 78. OLITZKI, L., AND BROMBERG, J. 1931 Ver-ment of certain strains of Brucella. J. In- wendungsstoffwechsel und Differential-diag-fectious Diseases, 71, 193-197. nose in der Bruzellagruppe. Zentr. Bakte-

65. MCCULLOUGH, N. B., AND DICK, L. A. 1942 riol. Parasitenk., 120, 347-364.Physiological studies of Brucella. II. Ac- 79. PICKETT, M. J., AND NELSON, E. L. 1954cessory growth factor requirement of re- Speciation within the genus Brucella. III.cently isolated strains of Brucella abortus. Nitrate reduction and nitrite toxicity tests.J. Infectious Diseases, 71, 198-200. J. Bacteriol., 68, 63-6.

66. MCCULLOUGH, N. B., AND DICK, L. A. 1943 80. PICKETT, M. J., AND NELSON, E. L. 1955Growth of Brucella in a simple chemically Speciation within the genus Brucella. IV.defined medium. Proc. Soc. Exptl. Biol. Fermentation of carbohydrates. J. Bac-Med., 52, 310-311. teriol., 69, 333-336.

67. MCCULLOUGH, W. G., MILLS, R. C., HERBST, 80a. POMALES-LEBRON, A., AND STINEBRING,E. J., ROESSLER, W. G., AND BREWER, C. R. W. R. 1957 Intracellular multiplication1947 Studies on the nutritional require- of Brucella abortus in normal and immunements of Brucella suis. J. Bacteriol., 53, mononuclear phagocytes. Proc. Soc. Ex-5-15. ptl. Biol. Med., 94, 78-82.

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70. MARR, A. G., AND WILSON, J. B. 1950 The HARDT, V. T. 1951 Studies of sulfurcarbon dioxide requirements of Brucella metabolism of Brucella suis. J. Bacteriol.,abortus. In Third Inter-American Congress 62, 571-582.on Brucellosis. World Health Organiza- 84. RODE, L. J., OGLESBY, G., AND SCHUHARDT,tion, Washington, D. C. V. T. 1950 The cultivation of brucellae

71. MARR, A. G., AND WILSON, J. B. 1950 Ge- on chemically defined media. J. Bacteriol.,netic aspects of the added carbon dioxide 60, 661-68.requirements of Brucella abortus. Proc. M. SANDERS, E., AND HUDDLESON, I. F. 1950Soc. Exptl. Biol. Med., 75, 438-440. The influence of atmospheric gases on the

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74. MooRs, T., AND MITCHELL, C. A. 1945 A C. R. 1953 The nutrition of Brucellameli-method of cultivating Brucella abortus for tensis. J. Bacteriol., 66, 294-299.antigen without the use of agar. J. Am. 88. SCHUBERT, J. H. 1951 A catalase liver ex-Vet. Med. Assoc., 107, 226-227. tract as a growth stimulant for fastidious


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Brucella cultures. Am. J. Clin. Pathol., 21, 98b. VAN DRIMMELEN, G. C. 1958 The Bru-894-896. cella vortex aeration culture apparatus.

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90. SCHUHARDT, V. T., RODE, L. J., AND OGLESBY, iron in the biology of Brucella suis. I.G. 1949 Toxicity of certain amino acids Growth and virulence. J. Bacteriol., 66,for brucellae. J. Bacteriol., 58, 665-674. 82-89.

91. SCHUHARDT, V. T., RODE, L. J., OGLESBY, 100. WILSON, G. S. 1931 The gaseous require-G., AND LANKFORD, C. E. 1950 The de- ments of Br. abortus (bovine type). Brit.velopment of peptone toxicity for bru- J. Exptl. Pathol., 12, 88-92.cellae with aging and the correlation of 101. YAW, K. E., AND KAKAVAS, J. C. 1952this toxicity with the probable oxidation Studies on the effects of D-amino acids onof cystine. J. Bacteriol., 60, 655-660. Brucella abortus. J. Bacteriol., 63, 263-268.

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94. STAFszTH, H. J. 1920 Studies in infectious Japanese J. Bacteriol., 11, 535-538.abortion. Technical Bull. No. 49, Mich. 103. ZoBELL, C. E., AND MEYER, K. F. 1930Agr. Expt. Sta. The metabolism of the Brucella group in

94a. STFERNE, M. 1958 The growth of Brucella synthetic media. Science, 72, 176.abortus strain 19 in aerated dialysed media. 104. ZoBELL, C. E., AND MEYER, K. F. 1932 Me-J. Gen. Microbiol., 18, in press. tabolism studies on the Brucella group.

95. STERZL, J. Z. 1948 Oil soluble growth fac- VII. Dextrose utilization. J. Infectioustors and Brucella abortus. 'Casopis ldkiru Diseases, 51, 109-116.6eskych, 87, 1215-1219. 105. ZoBELL, C. E., AND MEYER, K. F. 1932

96. TUTrLE, C. D., AND HUDDLESON, I. F. 1934 Metabolism studies on the Brucella group.Oxidation-reduction studies of growth and VIII. Nutrient requirements in syntheticdifferentiation of species of Brucella. J. mediums. J. Infectious Diseases, 51, 344-Infectious Diseases, 54, 259272. 360.

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98a. VAN DRIMMELEN, G. C. 1955 Brucella 108. ZOTTNER, G. 1942 Contribution & 1'Ftudeabortus strain 19 cultivated in aerated de d'isolement de Brucella abortus in COtliquid medium. Nature, 175, 999-1000. pur. Compt. rend. soc. biol., 136, 369-370.


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THE NUTRITION OF BRUCELLAE Chemically …mmbr.asm.org/content/22/2/81.full.pdf · the usual concentrations of peptone and glucose either blood or agar but mayrequire considera-maybecomelimiting