MORPHOLOGICAL AND BlDCHEMlCAL ALTERATIONS UM NRRL B ~13‘68 AS PRODUCED BY AFLATDX‘IN 81 EGATERI- BACILLUS M IN Thesis for the Degree of Ph. D. ERSITY CHIGAN‘ STATE U‘NW 19:29:, D , A g . V L33... . . . . . V, . ,. . , , ,. . , A V ...M_f.un.”.9.... fl . . D“ A , . . ~ . D ., e. . , V . 7:. ... ._A. . 9 V , . . . 734......{3 : . . .z. .m»......,....,. 1.....“5/39... u. '9.9.. . .11.! _ . . . , . . . +m..¢...«.... . ‘BEUCHAT ..s r r . V4; In]: LARRY R M! ,5 LIBRAR 1-" I’HESIS ‘ Michigan S taco University This is to certify that the thesis entitled Morphological and Biochemical Alterations In Bacillus Megaterium NRRL 8-1368 As Produced by Aflatoxin Bl presented by Larry R. Beuchat has been accepted towards fulfillment of the requirements for Food Science IL degree in —_ i /’7 /’ , é’[// a [1311" 6 /Z Mainr nmfessor Date May 217 LQZCL ‘ 0-169 I ABSTRACT MORPHOLOGICAL AND BIOCHEMICAL ALTERATIONS IN BACILLUS MEGATERIUM NRRL B-l368 AS PRODUCED BY AFLATOXIN Bl Bv 9 Larry R. Beuchat Bacillus megaterium NRRL B-l368 cells and spores were produced on trypticase soy broth (TSB) and agar (TSA) Containing 3.8 pg/ml aflatoxin B analyzed for selected 1, chemical constituents and morphological characteristics, and compared to cells and spores of g. megaterium pro- duced on nontoxic trypticase soy medium. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3 1/2 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Morphologically abnormal cells were produced by g. megaterium in response to aflatoxin B Filamentous 1' forms were characterized by early granulation and unusually large and numerous deposits of poly-B—hydroxybutyric acid within the cells. bulb—like structures occasionally formed at the terminal portions of these filaments after Larry R. Beuchat 1“ hr of incubation, possibly indicating weakened cell wzill:: iri tine iiilannintCMls :forwns. Chemical analyses revealed an increase in protein, dvoxyribonucloic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when E. megatorium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid and N—acotylglucosaminc analyses of control and LPGL cell walls showed little, if any, difference in cell Wu] 1 Connulsitiiin. Transfer of aberrant forms to nontoxic TSA yielded macrocoicnies with daughter cells morphologically indistinguishable from untreated cells. Agar slide cultures of filamentous cells on nontoxic TSA also showed that normal size and shape daughter cells were formed, indicating that aflatoxin B did not produce a l permanent genetic alteration for the phenotypic expression of cell length in L}. megaterium. Pantoyl lactone was without effect as a reversing agent for the observed inhibition of cell septum formation and aflatoxin had no effect on apparent penicillinase formation. g. mcgatcrium completed the sporulation prOCess with about 97% efficiency after 3 days on nontoxic TSA while 6 dayr were required to obtain 65% sporulation on toxic TSA. Germination of spores was not inhibited by approximately Larry R. Beuchat ll.t) nypflnl :il'hittxxiii hilt (uuiqgrcnutii wens. N() si 3nii7iczint differences were observed in the hear resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA. Electron photo-micrographs showed a decreased number of mesosomen in filamentous cells as compared to control cells. Tiere were no observable morphological differences in JpnPPS formed on toxic or nontoxic TSA. MORPHOLOGJCAL AND BIOCHEMICAL ALTERATIONS iN UACILLUS MEGATERIUM NRRL B-l368 AS i‘R-DDUCRD RY AFLATOXIN Bl By Larry R . Beuchat A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1970 A C KI‘JO'xILEIDGi/IENTS My sincere thanks are extended to Dr. R. V. Lechowich for his guidance and assistance throughout the research program and during the preparation of this manuscript. Special thanks are eXpressed to Thayne R. Dutson for his advice and assistance in the electron microscopy [Mirtitnl of tfliis sunldy. Research was supported, in part, by the National institutes of Health Training Grant No. FD—OOOOl-O9. ii TABLE OF CONTENTS AC KNOWLEDGME. To LIST OF TABLES LIST OF FIGURES INTRCWMMYTION LITERATURE REVIEW Aflatoxin Chemi try and Formation . . . Occurrence of Fungi that Produce Aflatoxin Structure and Chemistry of the Aflatoxins Synthesis of Aflatoxin . . Factors Influencing Production of Aflatoxin Metabolism and Biochemical Effects of Aflatoxins in Living Cells . . 4 Effect of Aflatoxins on Plants Aflatoxicosis in Vertebrates Effects of Aflatoxins on Insects Toxicity of Aflatoxins to Microorganism Methods for Detection of Aflatoxins Problems in Methodology Screening Tests . . Presumptive Tests Confirmatory Tor ts Detoxification of Foods and Feeds Containing Aflatoxin . Physical Destruction Chemical Inactivation Implications of Aflatoxicosis in .Man METHODS AND MATERIALS Production of Vegetative Cells and Spores of Bacillus megaterium NRRL B-1368 Growth Media . Quantitation of Aflatoxin Bl Production and Harvesting of Vegetative Cells and Spores Observations on Vegetative Cell Division Phase Contrast Microscopy iii Page Macrocolony Formation . . . . . . . . Al Direct Counts, Viable Counts, and Absorbancy ()hzingje.3 . . . . . . . . Lil Effects of Cell Septum lnitiators on Cell Diviri ion . . . . . A2 Effect of Penicillin on Cell Division . . . . A3 Staining Studies . . . . . . . . . . A3 heat Resistance of Spores Formed in the Presence of Afl:1toxin P1 and the Effects of Aflatoxin Bl on Spore Germination . . . . . . . . . . . AA “(A (it llCSieJt/arlce StUdieS . o o e o o o e o Ll“ Germination Studies . . AA Chemical Analyses of Vegetative Cells and Spores‘ of Bacillus mggaterium Formed in the Presence and Absence of Aflatoxin Bl . . . . A5 Ireparation of Vegetative Cell Walls .for Amino Acid Analysis. . A5 Procedure for Amino Acid Analysis of Vegetative Cell Walls . . A6 Total Protein Analyses of Vegetative Cells and Spores . . A7 Total Ribonucleic Acid Analyses of Vegetative Cells and Spore. . A8 Total Deoxyrihonucleic Acid Analyses of .Vegeta— tive Cells and Spores . . . . . . A9 Dipicolinic Acid Analysi:3 of Spores . . . . 51 Ireparation of Vegetative Cells and Spores of Bacillus megaterium for Observation by Electron Microscopy . . . . . . . . . . . . . 51 RISUI.TS AND DIS ”(U MON . . . . . . . . . . 5A Growth Media . . . . . 5A Recovery of Aflatoxin Added to the Medium . . . 55 Iflifoct3 of Aflatoxin upon Growth of Bacillus megaterium . . . . . . . . . . . . . 59 Growth Curves . . . 59 Cellular Morphology as Affected by Aflatoxin . . 6O Pantoyl Lactone Studies . . . . . . . . 67 Penicillin Studiets . . 70 Effects of Aflatoxin upon Sporulation of Bacillus megaterium . . . . . . . . . . 72 Heat Resi13tance Studies . . . 73 Effects of Aflatoxin on Spore Germination and Outgrowth . . . . . . ' 76 Effect of Aflatoxin on Vegetative Cell Wall Composition . . . . . . . ‘. . . . . 77 Effect of Aflatoxin on Vegetative Cell and Spore Composition . . . . . . . . . . . . . 79 Electron Microscopy of Vegetative Cells and Spores 81 LlTHfliATTHiE (JITIH) . . . . . . . . . . . . _93 iv Table A. LIST OF TABLES Summary of physical, chemical, and spectral data on aflatoxins . . . . . . Absorbancy at 650 nm of spore suspensions in trypticase soy broth containing 3.8 ug/ml aflatoxin Bl (toxic) and trypticase soy broth (nontoxic) . . . . . . Concentration in11Moles per mg protein of lysine, glutamic acid, and alanine in cell walls of Bacillus megaterium formed in trypticase soy broth (control) and trypticase soy broth containing 3.8 ug/ml aflatoxin Bl Per cent protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) in vegetative cells and spores and per cent dipicolinic acid (DPA) in spores of Bacillus megaterium formed in trypticase soy medium (control) and trypticase soy medium containing 3.8 ug/ml aflatoxin B1 (treated) Page 77 78 80 Figure l. ’7 ‘) I 0 LIST OF FIGURES Structure of aflatoxins Growth curves for Bacillus megaterium cultured in trypticase soy broth (control) and trypticase soy broth containing 3. 8 ug/ml aflatoxin Bl (treated) . . . . Normal and aberrant forms of Bacillus megaterium produced during growth in tryptica13e soy broth and trypticase soy broth containing 3. 8 ug/ml aflatoxin 81’ respectively . . . . . . . . . . Micrographs showing bulb-like swellings at the terminal portions of filaments in a lA-hr culture of Bacillus megaterium grown in trypticase soy broth containing 3. 8 ug/ml aflatoxin Bl . . . . . Demonstration of capsular material in Bacillus megaterium cultured in (A) trypticase soy broth and (B) trypticase soy broth contain- ing 3.8 ug/ml aflatoxin B1 . . Time—lapse phase contrast micrographs of dividing filamentous Bacillus megaterium cells which were formed in trypticase soy broth containing 3.8 ug/ml aflatoxin Bl and then transferred to nontoxic trypticase soy agar . . . . . . . . . Survivor curves at 92 C (197 F) for Bacillus megaterium spores formed in trypticase soy agar (control) and trypticase soy agar con- taining 3.8 ug/ml aflatoxin B1 (treated) Ultrathin sections of Bacillus megaterium cells formed in trypticase soy broth . Ultrathin sections of Bacillus megaterium cells formed in trypticase soy broth con— taining 3.8 ug/ml aflatoxin B1 3 vi Page 57 61 63 65 68 7A 83 85 I"'i ytm'v Page IO. UlIrathin sections of Bacillus megaterium spores formed in trypticase soy agar . . . 88 Ll. Ultrathin sections of 53011118 megaterium case x spurns. I‘tjnr'mxi in ups/pt taininfl 3.6 ug/ml afla soy agar.con- in I51 . . . . . 90 vii INUTR(H)U(TPIIDN The word mycotoxin is not present in the third odition of Webster's unabridged dictionary (1966). The word myco is urfined as "a combining form from the Greek H mvkw , moaning fungus. Toxins are defined as "any of -J‘.“ ‘ various poisonous substancos that are specific products of the metabolic activities of living organisms . . . " A mycotoxin, then, is a poisonous material produced by a fungus. Mycotoxins are specific chemicals or mistures of chemicals. it is aflatoxin B a mycotoxin produced 13 along with at least seven other aflatoxins by the q. A.:;wrgiliu._z flavus group 01 fungi, that was under study airi wi 11 in} r<1x>rtcni or1 in tfliis rnaruuscriiat. Aflatoxins are the most sensational and probably tho most signifirant mycotoxins discovered to date. Aflatoxin—producing molds are ubiquitous and not particu- iurly fastidious in their requirements for growth and toxin production. The toxin 3 lethal to all animal spocies thus far studied and has inhibitory effects on Certain plants and microorganisms. Their extreme potentcy an carcinogens might be visualized in this way: an ounce of aflatoxin h properly administered, could kill a L) million ducklings. Certainly the threat to human health is roal and the problem of aflatoxins in foods must be eXplorod and solved from the standpoint of preventive contamination, decontamination, and detection. Of great concern 's the fact that aflatoxins are relatively stable compounds not significantly altered by cooking or ordinary decontamination measures used in food pruunflnlnfl. Although aflatoxins are rarely found in agricultural Commodities if proper harvesting, handling, and storage procedures are followed, it is desirable to have fast, accurate, and preferably inexpensive methods for detection of the toxin in suspect cases. Thin—layer chromatography in conjunction with confirmatory bioassays utilizing ducklings or embryonated chicken eggs are routinely used to quantitate aflatoxins. Problems asso- ciatod with duckling and embryonated chicken egg bioassays include their eXpense and demand for technical personnel. An alternative confirmatory bioassay providing simple and rapid techniques is based on the growth inhibition of bacillus megaterium. However, the microbial inhibition tost is not as sensitive as other bioassay methods. bosons of other mycotoxins may inhibit the growth of B. mogaterium, including toxic metabolites of the A. flavus group. The objective of this study was to chemically and morphologically analyze the aberrant cells and the spores formed by Q. megaterium in the presence of aflatoxin B1 in an attempt to derive additional information which could hl-llslul ll». u}dvlvnu Ht l~xi;d.lur:llatz1 av:1il:d>lc <>n ifl'OWlJI lnhitlllon. Such nwwly derived data might be used as additional confirmation for the presence of aflatoxin in Infrllnlltuivll conmswiititni. LITERATURE REVIEW More than lu0,000 young turkeys died in England within the course of a few months in 1960 from a disease termed "turkey X disease" (Blount, 1961). It was soon discchred that the disease was not restricted to turkeys as heavy mortality was also experienced with ducklirgs, pheasants, and partridges. Almost simultaneously an outbreak of trout hepatoma was reported in the United Btatos (Wolf and Jackson, 1963). Although there was no known relationship between the outbreaks, a toxic secondary metabolite of the mold Aspergillus flavus (christened aflatoxin for A. flavus toxin) was shown to H H be commonly present. The impact of this new mycotoxin gaVr stimulus to much research. No less than 1500 scientific publications concerning aflatoxins have appeared in the literature within less than a decade. No attempt is made here to thoroughly review the literature as several recent reviews dealing with many aspects of this mycotoxin are available (Adrian and Lunven, 1969; Uilai, 1969; boesenburg, 1969; Jones and Jones, 1969; Nesterin and Virsarionova, 1969; Scott, 1969b). In addition, an excellent book entitled "Aflatoxin," N describing its sciwntific background, control, and impli— cations, has recently been published (Goldblatt, 1969). Af"latmixiri Chcsnistrj/ arki Fadnnaticun ()c<'ui*rtuicr> of‘ EHaIsZi ttuit Produce Aflatoxin Aflatoxicosis is probably not a new phenomenon, although studied in depth only recently. The A. flavus group of molds (fl. flavus and g. parasiticus), which are now generally accepted as being solely reSponsible for the production of aflatoxins (Wilson _2 31., 1968), are ubiquitous and grow rapidly. It is their frequent role as contaminants in food of both animal and man that has attracted attention to their investigation. Interest has evolved to determine the suitability of nearly every type of food for the support of growth of A. flavus and subsequent production of aflatoxin. The Japanese, who consume a considerable amount of controlled and uncon~ trolled fermented foods in their diet, have carried out studies on the population of toxigenic fungi in foodstuffs (Kurata gt _l., 1965; 1968). Twenty-one strains of the Q. flavus group were isolated from several kinds of flours including wheat, red bean, and soybean. Five of the twenty-one strains were found to produce aflatoxin. The toxin has not been detected in takadiastase (Nose and Ishibashi, 1967). More than 60% of 1,626 isolates of A. flavus from groundnut kernels and soils in Israel produced aflatoxin in excess of 25 ug/g (Joffe, 1969). Surveys to establish the distribution and degree of occurrence of aflatoxin in groundnuts have also been made in South Africa (Sellschop 33 al., 1965) and the United States (Dickens, 1968; Schroeder and Ashworth, 1965). Aflatoxin-producing g. flavus have been isolated from red pepper (Christensen _3 _l., 1967; Schindler and Eisenberg, 1968) and from fish food_materials (Phelps, 1969). A. flavus has been isolated with regularity from molded soybeans and soy sauce but extracts of the molded products failed to reveal the presence of aflatoxins (Chung, 1966). Most reports have pointed to members of the g. flavus group for being responsible for production of aflatoxin (Uiener and Davis, 1966; Raper and Fennell, 1965; Senser, 1967), however a few researchers have indicated that certain members of the genus Penicillium (Hodges g2 gl., 196”; Kulik and Holaday, 1966; Wilson et al., 1967) as well as Aspergillus ostianus also produce aflatoxin. One Streptomyces species was reported to produce the toxin (Mishra and Murthy, 1968). Wilson 33 al. (1968) more fully examined the toxin-synthesizing capacity of several of the fungi previously reported to produce aflatoxins and noted that pitfalls in analytical technique have led to confusion on fluorescent materials with Rf values approximating those of the aflatoxins with the actual aflatoxins, thus producing falsely positive results. Stiwnrture znvi Chemttxtry of the Aflatoxins Figure 1 shows the structures of the eight reported aflatoxins. Table 1 lists various physical and Chemical characteristics of the toxins. Of Special interest are the melting and decomposition points which render the toxins stable under normal processing procedures used by the food industry. The coumarin-lactone type structure, common to all, is characteristic of many naturally occur- ring, physiologically active compounds but the bifuran structure, also common to all, is known to occur in only one other naturally occurring compound, sterigmatocystin ((iulilhliltt , 1968) . Methods for structural elucidation of the aflatoxins have involved the ultraviolet, infrared, nuclear magnetic resonance, and mass spectrograph (Asao gt a1., 1965). isolation and characterization of aflatoxins Bl, 89’ G1’ and 02’ four of the eight known aflatoxins, were the first to be established through separation on silica gel chromatoplates using chloroform:methanol (98:2) as a developing solvent (Hartley gt al., 1963). Ultraviolet light was used to determine the Rf values. Other researchers have worked independently to elucidate the chemical identity and biological activity of these 82¢ 520 Figure 1. Structures of aflatoxins. E: u Mgmn E: wmmo EC SGND d e: puma momoqsoomg * . some 3 3H an em osoocpmm: ecu cocsso .omfl . mam ao m o . :mmcm mooo.ma coco.mH u a QQAH N. 34. N4 MU msoocsmmm new Cessna .osm . 0mm o_.m ,o - mafia oos.om oooo.efi ,m mama .Hmeamnflox .mxm . 0mm wasmmpaa - mean mooo.fim poom.oa m: . a mama .Hteamaflo: .oflm - mmm paramefie - mass mooo.mH oom.HH H: mama .cmmos .osm emm 9mm eoHHmeHo so: cwuams osm.ma ooo.HH me .e J .4. mesa .zwmo: .osm saw mam ro_amp.o can cwuflms ooa.oa ooo.oa Ho toes .cemox .mmm mam sum mesa ado mm, mess ooe.sd oom.m mm x . a.” . \\;J 3 J J “H .Kn \IJ ) cosfi .cmio; ..co «or pHm 0o : Ho u.s mass oom.fim 004.0H Hm LUHOU EC mom EC mom ,, EC, 1».=., . pcflcm psmficz n«:E.On A c 1:; :1”me .. . r. .0 Ed c . L uufiuacx Lomzooaofi amazomacfi )L HmMH : on :HxOpmHm< noncoommhojam soapupono< poaofi>mcofiz .mcfixoomam co mome Hmcoooom new .HmoHEoco .Hmofimxcq go mnmeeswun.a monte lO aflatoxins (Chang gg gl., 1963; Van Dorp g: gl., 1963; Van Soest and Peerdeman, 1964; Wick g3 gl., 1965; Hrechbuehler :2 El}, 1967). Aflatoxins M1 and M2 were isolated from urine col— lected from sheep during two days after they had been do ed with aflatoxihs bl, 82, 01’ and G2, and resolved by paper chromatography (holzapfel g3 gl., 1966). Crystalline aflatoxin Ml was later isolated from urine and milk (Masri F‘ l aflatoxin M2 is U-hydroxyaflatoxin B2. The most recent ‘t gi,, 1967). Aflatoxin M is M—hydroxyaflatoxin B1 and aflatoxin to be isolated are aflatoxins 82a and aflatoxin GPa’ 2-hydroxy derivatives of aflatoxins 82 and G?’ respectively (Dutton and Heathcote, 1966). Formu— iation was made by means of ultraviolet, infrared, mass, and nuclear magnetic resonance spectra, and toxicity was confirmed by use of duckling assays (Button and Heathcote, 1968). The chemical identity of aflatoxin B2a (Poland g3 gi., 1968) and G2a (Poonoose, 1969) has been confirmed by their research reports. Synthesis of Aflatoxin Utilization of luC-labeled compounds has enabled researchers to hypothesize biochemical pathways leading to the formation of aflatoxins. The addition of methyl- labeled methionine to an 5, flavus culture medium resulted in the appearance of 1MC in the methoxy function (Adye and Matcles, 196U). Addition of acetate—l-luc as the sole 11 source of carbon yielded aflatoxin labeled in the ring carbons. Aflatoxin Bl produced by a phenylalanine- reduiring mutant, 5. flavus A77, grown in the presence of 1“! 1‘. a A ’ o ,‘ ' ‘ 0 1.,1,‘ .0 L-lULlefl (ring) hL-pnenylalanine had a relative isotopic conteht of less than 0.01, indicating that phenylalanine was not a precursor of B (Donkerslcot 1 ct al., 1968). similarly l“(J-labeled (ring) shikimic acid was shown not a precursor, however indications were that acetate was a precursor. Through the use of acetate- 1 _14 1M0 i C and acetate—Q- , a hypothetical scheme for biosynthesis of aflatoxin was recently proposed (Biollaz fl >t 21-: 1968a; 1968b). Labeled acetate, mevalonate, and leucine were demonstrated to be incorporated into afla— toxins by the mitochondrial fractions of reconstituted homogenates from g. flavus, indicating that enzymes inVoivcd in biosynthesis of aflatoxins were in the L mitochondria (hai 31 gl., 1969). This tends to confirm in part znn<)t’iiziri'i;:rn;: "— A survey to test the sensitivity of 329 micro- organisms to aflatoxin included 30 genera of bacteria, iU genera of fungi, A genera of algae, and l protozoan (hurmeister and Hes eltine, 1966). 3A streptomycete, a clo tridium, and l? Bacillus species were inhibited when crude aflatoxin (30 ug/ml; 36% pure) was incorporated into thH growth medium. Among the most sensitive bacteria was bacillus rw'gaterium and has thus been ro>osed as an A 3 as ay organism for aflatoxin in foods (Burmeister, 1967; Elements, 1968a, b; Jayaraman gt gl., 1968; Viitasalo and Gyllenterg, 1969). The technique is similar to anti- biotic assays and a zone of growth inhibition around the 23 test material is compared to standards to estimate the concentration of aflatoxin in the test material. In another study aflatoxins were found to be inactive against common Gram positive and Gram negative bacteria at concentrations 1 the growth of several Aspergillus and Penicillium species of 100 ug/ml (Arai et al., 1967). Aflatoxin B inhibited on modified Czapek Dox broth (Lillehoj gt al., 1967c). Aflatoxin uptake by Flavobacterium aurantiacum and the resulting toxic effects were studied (Lillehoj and Ciegler, 1967; Lillehoj gt 1., 1967a, b). The toxin inhibited DNA synthesis and, to a lesser extent, RNA and protein synthe- sis. Aflatoxin removed from the medium by E. aurantiacum cells could not be extracted but autoclaving of cells and cell wall preparations reversibly removed a fraction of the aflatoxin present in a test system. Aberrant forms of the bacterium were produced during growth in the presence of B and G . Binding capacity studies indicated that B1 1 1 has a greater affinity for DNA than B (Lillehoj and 2a Ciegler, 1968; Lillehoj and Ciegler, 1969). The incor- porating activity of Escherichia coli DNA polymerase was significantly lowered by 5 ug/ml B (Wragg, 1967; Wragg l t 1., 1967). The interaction of B f with polynucleotides Q) l coli was studied spectrophotometrically and a IE I O requirement for the amino group of adenine or possibly guanine for binding of aflatoxin was apparent (King and Nicholson, 1969). The same authors demonstrated that 2A aflatoxin B1 reduced tlw activity of DNA-dependent RNA polymerase in E. coli (King and Nicholson, 1967). Amino acid activation in E. coli preparations was inhibited by aflatoxin in vitro (Smith, 1965). Treatment of virulent bacteriophage with aflatoxin B1 rendered the phage incapable of lysing Streptococcus lactis cells (Jemmali, 1969), however the same toxin was reported to act as an inducing agent for lysogenic gtaphylococcus aureus (Legator, 1966). Dactylium dendroides, Absidia repens, and Mucor griseo—cyanus transformed about 50 to 60% of aflatoxin B1 in shake cultures to a new fluorescent-blue compound (Detroy and Hesseltine, 1968, 1969). Tetrahymena pyriformis was also observed to make a similar conversion (Teunisson and Robertson, 1967). Methods for Detection of Aflatoxins Problems in Methodology The main objective in aflatoxin methodology has been to develop a rapid and accurate procedure to analyze a large number of representative samples with adequate sensi- tivity to detect a few micrograms of toxin per gram of sample. Distribution of the toxin is not always homo- geneous (Ayres _t al., 1970) and methods satisfactory for one product may be completely inadequate for another. Consequently numerous screening, presumptive, and 25 confirmatory tests were developed for detection of aflatoxin in a wide variety of food materials. Methods for detection have been developed for milk (Purchase and Steyn, 1967; Roberts and Allcroft, 1968; Jacobson gt gl., 1969; Masri t 1., 1969), wine (Schuller g2 1., 1967; Frank and Eyrich, 1968; Wang _3 gl., 1968), fungal fer- mentation products (Denault and Underkofler, 1967), cocoa beans (Scott, 1969a), and green coffee (Levi and Borker, 1968; Scott, 1968). Procedures for aflatoxin analyses in oilseeds (Chen and Friedman, 1966; Gardner g2 g;., 1968; Velasco, 1969), peanuts and peanut‘products (Jayaraman, 1965; Holaday, 1968; Juszkiewicz and Stefaniakowa, 1969; Mayura and Sreenivasamurthy, 1969), stored corn (Johnson gg g;., 1969), cereal products (Lemieszek-Chodorwska g: al., 1969), and malt feeds (Kuehnert and Buchhein, 1968) have also been reported. ScreeningfiTests A rapid screening method requiring no special skills on the part of the examiner was described for aflatoxin detection in cottonseed (Whitten, 1969). Contaminated lots of cottonseed had stem ends that fluoresced a bluish color when eXposed to longwave ultraviolet light. A greenish— yellow fluorescence was previously associated with the growth of A. flavus on seedcoat and cottonseed fiber con— stituents (Ashworth g: gl., 1967; Ashworth g3 gl., 1968). 26 The color is believed to be formed under the influence of plant peroxidase on kojic acid produced by the fungus (Marsh gg g£., 1969). Several quick chromatographic techniques were pro- posed for aflatoxin analysis of food materials. Precise quantitation, however, is unlikely to be achieved. The "milli-column" technique for detecting aflatoxins in peanuts involves filling a A-mm i.d. glass tubing with silica gel, developing the column in a chloroform-methanol extract of the peanut sample, and then viewing the column under longwave ultraviolet light (Holaday and Passwater, 1969). Microslides were proposed for rapid thin layer chromatography (Eppley, 1969). Analytical methods for screening zearalenone, aflatoxin, and ochratoxin (Eppley, 1968) and aflatoxin, ochratoxin, and sterigmatocystin in cereals and groundnuts (Vorster, 1969) involved the use and manipulation of various solvents for extraction and development on TLC plates. Sampling mills which operate on the principle of size or color difference permitted effective separation of aflatoxin-contaminated peanuts prior to their incorpora- tion into food materials (Pattinson gg gl., 1968; Dickens and Satterwhite, 1969; Whitaker and Wiser, 1969). 2? Presumptive Tests Presumptive tests are more time consuming but provide better evidence than the previously described screening methods. Generally these physicochemical tests are not suitable for large samples. Preparation and extraction of the sample is usually performed in a Waring blender with solvents such as chloroform, methanol, benzene, acetonitrile (Yin, 1969), or aqueous acetone (Pons gg gl., 1966). Pigments are removed from solvent extracts with c0pper carbonate (Wiseman g3 gl., 1967). Cleanup pro- cedures involve liquid—liquid partitioning followed by partitioning on a Celite column and concentration by evaporation. Paper chromatography once was recommended for afla- toxin separation (Coomes and Sanders, 1963; Sreenivasa- murthy g2 g1., 196A) but was replaced by the more sensitive TLC method (Peterson and Ciegler, 1967; Nesheim, 1969; Stubblefield g3 g1., 1969). Silica Gel G-HR (Brinkman Instruments, Inc., Westbury, New York), Adsorbosil-l and Adsorbosil—S (Applied Science Laboratories, State College, Pennsylvania) are the most commonly used brands of adsorbents. Polyamide-impregnated film TLC was reported to give increased separation of aflatoxin B1 and G1 (Lee and Ling, 1967). Various commonly used solvents include chloroform, acetone, methanol, toluene, isopropanol, benzene, and I'ormate in various ratios and combinations ~Stoloff _g gl., 1968; Engstrom, 1969; Teng and Hanzas, 1969). The accuracy of the physicochemical procedures described above ultimately depends upon the reliability of the method used for evaluating the amount of toxin present. Visual estimates were reported to be accurate within 1 20 to 30% (Nesheim, 196“; Pons gg gl., 1966) whereas instrumental methods for fluorodensitometric quantitation are claimed to have precision of t 3 to A% (Beckwith and Stoloff, 1969; Pons gg gl., 1968). A method utilizing colored Polaroid recordings of TLC plates for precise, permanent records was presented (Chang and Lynd, 1968). Confirmatory Tests Chemical and biological confirmatory tests require fairly pure samples obtained from time—consuming cleanup procedures. EXposure of developed TLC plates to elemental iodine crystals was used to distinguish interfering materials from aflatoxins due to their continued fluorescence (Mislivec g3 gl., 1968). Depending upon the concentration and purity of the sample, aflatoxin B1 reacted with 2,A-dinitrophenylhydrazine to form a yellow to deep orange dinitrophenylhydrazone derivative (Crisan and Grefig, 1966; Crisan, 1968). Quantitation by both methods involved 29 visual comparison with standards. Modification (Stoloff, 1967) of a chemical confirmatory test (Andrellos and Reid, 196U) based upon derivative formation was adopted as "Official--First Action" by the Association of Official Analytical Chemists. Biological assays proposed as confirmatory tests for aflatoxins are generally more expensive and more time- consuming than chemical confirmatory tests. In addition to the use of g. megaterium as a bioassay organism (see Toxicity of Aflatoxins to Microorganisms), numerous other biological systems were tested. Chlorella pyrenoidosa (Ikawa gt gt., 1968; Ikawa gt gt., 1969), brine shrimp (Brown gt gt., 1968; Brown, 1969), mollusks (Townsley and Lee, 1967), chicken embryo liver cells (Kiyoshi and Miyaki, 1968), chicken embryos (Verrett gt gt., 196“), and corn seedlings (Mayer gt gl., 1969) were examined for their possible use as confirmatory bioassays for aflatoxins. Due to lack of specificity in most methods, proof of the presence of aflatoxin involves the use of physicochemi— cal as well as biological procedures. Only three of the tests suggested to date were collaboratively studied and given "Official--First Action" status by the A.O.A.C. (Ayres gt gt., 1970). These include two methods for peanuts and peanut products (A.O.A.C., 1966, 1968, 1969), one for cottonseed (A.O.A.C., 1969), and one for con— firmation of aflatoxin by derivative formation (A.O.A.C., 1967), 3O Detoxification of Foods and Feeds Containing Aflatoxin Physical Destruction Inactivation or removal of aflatoxins from contami- nated foods or feeds by methods presently investigated has generally left the food unfit to eat or, at best, with a decreased nutritional value. Early attempts to reduce toxicity of peanut meals by heating, cooking, or auto- claving were largely ineffective (Blount, 1961). Later reports, however, were more encouraging. Heating at atmospheric pressure at 60 to 80 C resulted in definite reductions in aflatoxin in cottonseed meals; heating cottonseed meals having 20% moisture at 100 C for 120 min resulted in an 80% reduction of aflatoxin B (Mann gt gl., 1 1967). Sterilization by autoclaving wet groundnuts at 15 lb per in.2 (121 C) produced a progressive reduction in toxicity with time (Coomes gt gt., 1966). However, heating dried fruits and various fruit juices in which A. flavus had grown and produced aflatoxin resulted in little toxin destruction (Frank, 1968). Buffered methanol containing aflatoxin received 30 Mrad of radiation which accelerated aflatoxin decomposition, but did not affect the biological toxicity of aflatoxin (Miyaka gt gt., 1966). Aflatoxins were reduced following roasting of contaminated peanuts (Sobolewski and Kmieciak, 1968; Lee et al., 1969). Natural or activated bleaching earth used 31 in peanut oil refining was responsible for detoxifying an oil containing 0.5 ug/g aflatoxin (Velan and Raymond, 1967). (V' 3"“ .7 r\ .":Vf‘-. chemical inactivation Ammonia is one of the most effective chemicals for inactivation of aflatoxins in contaminated food materials. Agricultural products contaminated with aflatoxin were detoxified by mixing with 0.3 g of ammonia per kg of product and heated (Dollear gt gt., 1968; Masri gt gt., 1969). Contaminated cottonseed and peanut meal were treated with ozone and aflatoxins B1 and G1 were readily destroyed, especially at high moistures, high temperatures (100 C), and at longer treatments (Dwarakanath gt gt., 1968). Sodium hypochlorite (5%) is recommended for decontamination procedures in the laboratory (Fischbach and Campbell, 1965; Stoloff and Trager, 1965). A micro— biological inactivation method using E. aurantiacum was proposed (Ciegler gt gl., 1966). Both growing and resting cells of this bacterium partially detoxified contaminated milk, peanuts, soy products, and corn without the develop— ment of new toxic products. Implications of Aflatoxicosis in Man Aflatoxins are probably the most potent hepatoma- inducing agents known. They are more toxic than benzopyrene and 1000 to 1500 times more effective than butter yellow, a butter additive that is universally prohibited (Hanssen and Hagedorn, 1969). The Food and Agriculture Organization of the United Nations in cooperation with the World Health Organization established in 1966 the maximum concentration of aflatoxin in foodstuffs as 30 ng/g (Anonymous, 1966). In the United States, Switzerland, and the German Federal Republic a zero tolerance was set; a 5 ug/g maximum tolerance was set by Great Britain, Canada, and the Netherlands. The ubiquitous nature of aflatoxin-producing molds, coupled with their nonspecificity on living cells of nearly every form of life, stimulated interest to determine the presence of the toxin in foods regularly consumed by man. in Uganda 15% of the samples of groundnuts sold for human consumption contained more than 1 ug/g aflatoxin B (Lopez, 1 1967). Toxic strains of g. flavus were isolated from Nigerian foods (Bassir, 196M); aflatoxin was detected in commercial milk samples in South Africa (Purchase and Vorster, 1968); from peanut oil, peanut cakes, peanut butter, and dried sweet potatoes in Taiwan (Ling gt gt., 1968; Tung and Ling, 1968); and from unrefined peanut oil in Malaya (Chong and Beng, 1965). Tortillas made by a method commonly used in Mexico for centuries contained aflatoxin (Ulloa-Sosa and Schroeder, 1969). On the other hand, exhaustive studies of fermented Japanese foods failed to reveal the presence of aflatoxin (Manabe et 1., 33 1968; Murakami gt gt., 1967; Murakami gt gl., 1968; Yokotsuka gt gt., 1968). Primary liver carcinoma is a relatively rare disease in Europe and North America but is much more common in certain parts of Asia and Africa (Foy gt g;., 1966; Purchase, 1967; Robinson, 1967). It is possible that there may be common links in the metabolic disorders leading to cirrhosis and to liver carcinoma in Africans, to hepatic fibrosis in aflatoxin treated rats, and to the hepatic lesions in acutely pyridoxine deprived baboons (Foy gt gt., 1966). High incidence of primary liver cirrhosis and carcinoma in the African may imply that their diets intermittently contain aflatoxin as a result of infection of porridges and brews by one or more molds. Other postulations were relative to the high incidence of carcinoma in areas where the consumption of fermented foods was high (Oettle, 1965). It is not suggested that aflatoxins are the only likely cause of hepatoma since many other factors may be implicated. The fact is that aflatoxins are highly suspect and should not be present in the human diet. METHODS AND MATERIALS Production of Vegetative Cells and Spores of Bacillus megaterium NRRL 8-1368 Growth Media Bacillus megaterium NRRL 8-1368, obtained from Dr. C. W. Hesseltine, Chief of Fermentation Laboratory, United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, was the organism used throughout this research. Any further reference to g. megaterium is reference to the above strain, unless otherwise indicated. The organism was cultured in Trypticase Soy Broth (TSB; Baltimore Biological Labora- tories, Baltimore) which contains the following constituents on a per liter of distilled water basis: 17.0 g trypticase (pancreatic digest of casein); 3.0 g phytone (soy peptone); 5.0 g sodium chloride; 2.5 g dipotassium phosphate; 2.5 g dextrose; and adjusted to pH 7.1. Five—hundred milli- liters of TSB were dispensed into 1000-m1 Erlenmeyer flasks, autoclaved at 121 C for 15 min, and used for the production of vegetative cells analyzed during the research project. Fifty milliliters of the T88 medium containing _2% agar were added to 1000-m1 pharmaceutical bottles and the agar was allowed to solidify along the flat side 3“ 35 surface in order to produce spore crops using a surface growth technique. Besides uring the "nontoxic" medium described above to culture B. megaterium, the same medium containing aflatoxin B1 (crystalline, dried in situ from chloroform, grade B, lot 9U0032, Calbiochem, Los Angeles) was also used to culture the organism for vegetative cell or spore analyses. The objective in the aflatoxin addition was to culture the organism under conditions of strain severe enough to inhibit the rate of normal cell division but not to decrease viability. An approximate aflatoxin con- centration of U.0 ug/ml produced these effects. Addition of aflatoxin was as follows. Crystalline aflatoxin B in l0-mg quantities, was dissolved in 10 m1 1’ of chloroform. Two milliliters or 0.2 ml of the solution was dispensed aseptically into 500 m1 of TSB or 50 ml, of tryticase soy agar (TSA), respectively. The medium was then steamed for 15 min, driving off the chloroform and leaving aflatoxin in the medium. Care was taken to shield the toxin and toxic media from light. Theoretically the medium should have contained A.0 ug aflatoxin per ml while the determined concentration was 3.8 ug/ml. Quantitation of Aflatoxin B1 The exact aflatoxin B1 concentration in the chloro— form solution added to the sterile T88 and TSA was deter- mined as was the concentration in the medium before steaming 36 and before culturing. These two quantitations allowed the per cent recovery to be calculated. The concentration immediately after steaming but before culturing was deter- mined to evaluate the stability of aflatoxin to heat. Concentration of aflatoxin in the spent broth upon termi— nation of culturing was also established. To obtain this information, the following procedure was carried out. First the sample to be quantitated was treated ultra- sonically with a Branson Sonifier Model S—75 sonic oscillator (Heat Systems—Ultrasonic Inc., Plainview, New York) operated at 5 amperes for 10 min. Fifty milliliters of the sample were poured into a 250-ml separatory funnel along with 50 ml of chloroform. The mixture was shaken vigorously for l min and allowed to separate into two phases before drawing the bottom (chloroform) layer off through a 2—cm layer of anhydrous sodium sulfate in a Butt tube. The partitioning was repeated with two additional 50-ml aliquots of chloroform after which the pooled chloroform extracts were evaporated to near dryness on a steam bath and the toxin was dissolved in sufficient chloroform to permit quantitative transfer to a vial. After evaporation to dryness, the vials were flushed with nitrogen and refrigerated until quantitated. The dry extract was dissolved in chloroform and spotted on TLC plates, along with an aflatoxin standard containing 1.0 pg B1, 0.3 pg 82’ 1.0 pg G1, and 0.3 ug G2 per ml. The 37 aflatoxin standards were obtained from Dr. Leo A. Goldblatt, Chief of Oilseed Crops Laboratory, United States Department of Agriculture, Southern Utilization Research and Develop- ment Division, New Orleans, Louisiana. The TLC plates used were coated with Adscrbosil-S (20 X 20 cm, 250—pm layer) obtained from Applied Science Laboratories, State College, Pennsylvania. The plates were developed with chloroformzacetone:2-propanol (26:6:1, v/v/v) in unlined, unequilibrated tanks (Desaga Multiplate Glass Tank 25-10- 95, Brinkman Instruments, Inc., Westbury, New York). After drying the plates for 15 min in the dark, measure- ment of the emitted fluorescence of aflatoxin Bl upon eXpo ure to longwave ultraviolet was made. The basic fluorometric densitometry system consisted of a Photovolt Model 530 densitometer equipped with a longwave ultra— violet lamp, No. 265 primary filter and 6 x 19 mm primary slit, a stage for TLC plates equipped with automatic 0 drive, a No. U65 secondary filter and an ultraviolet sensitive phototube, and a No. 520—A multiplier photometer. A Varicord Model “3 recorder equipped with an automatic recording integrator (Integraph Model A9) was used to record the intensity of the fluorescent radiation. Integrator area counts were used to calculate aflatoxin R concentrations. The content of a sample was 1 Calculated according to the following equation: 38 (Ax) (vs) (Cs) (sd) Aflatoxin B (pg/ml) , (AS) (Vx) (V) l where: (AX) = average area count of duplicate 131 sample spots (Vs) = pl of standard spotted (CS) = pg of B1 per ml of standard (8d) = volume of final extract in pl .’\ :> ,- J V II average area count of the 8 standard spot l < ll pl of sample extract spotted (V) ml of original sample represented by the final sample extract (50 ml in this case) Production and Harvesting of Vegetative Cells and Spores A stock suspension of E. megaterium spores was heat shocked for 8 min at 80 C and inoculated into TSB. Trans- fers were made at 8, 6, and A hr into 1000-ml Erlenmeyer flasks containing 500 ml of medium and incubation was at 32 C in a New Brunswick Model 025 incubator gyratory shaker at 150 RPM. Vegetative cells were harvested by centrifugation at 9000 x g at a time late in the loga- rithmic growth phase. Cells were then washed seven times with 0.1 M phosphate buffer pH 7.1 and stored at -20 C until they were analyzed for chemical constituents. Cells used for electron microsCOpy studies were harvested in 39 early stationary phase and were not frozen, but stored overnight at A C. Since it was impossible to get good sporulation in toxic liquid medium, TSA in the 1000—ml pharmaceutical bottles was employed for spore production. Two milli- liters of a 2—hr-old culture of §° megaterium were inoculated on the surface of toxic or nontoxic agar slabs in pharmaceutical bottles and incubated at 30 0. Culture samples were observed at various times with an American Cptical mircoscope with phase contrast optics ’American Optical Co., Buffalo, New York) at 1000 x magnification. All phase dark and phase bright spore forms were counted as positive indices of sporulation. The per cent sporulation was computed by dividing the number of sporangia by the total number of cells counted and multiplying by 100. Spores were harvested from the nontoxic medium after 3 days and the toxic medium after 6 days with cold 0.1 M phosphate buffer (pH 7.1) and transferred to centrifuge bottles. Spores were washed twice with phosphate buffer, treated 1 hr at 37 C with 0.075% lysozyme, and again washed 5 times with phosphate buffer. Spore crops were stored at A C until chemical, germination, heat resistance, and electron microscopy studies were performed. “0 Observations on Vegetative Cell Division Phase Contrast Microscopy Cell division and morphological development of g. megaterium either grown in toxic or nontoxic TSB were followed. Cells were observed with either an American Optical microscope or a Leitz Ortholux microscope, both of which were fitted with photomicrographic equipment. Photographs were taken at several magnifications and times during the growth cycle. The ability of elongated vegetative cells formed in toxic TSh to revert to normal cellular division and morphology when placed on nontoxic TSA was studied with phase contrast microscopy. Observations of cell division in one or more stationary E. megaterium cells were made on agar slides. A layer of liquified control TSA (u% agar) was placed near the center of a standard 3 x l in. glass slide and allowed to harden and dry for about 2 hr. The edges of the agar layer were trimmed to about 22 mm square and a loop of the vegetative cells was placed on the medium; a caver slip was placed on top of this and sealed with warm paraffin to prevent further drying. Agar temperature was maintained at 30 i l C as measured potentiometrically with a 250-Watt infrared brooder lamp. Photographs were made throughout the progression of cell division. Ml MacrOColonygFormation Aberrant filamentous cells produced by g. megaterium in toxic TSB were surface plated on nontoxic TSA and incubated at 32 C. Colony formation was visually examined and morphology of daughter cells from different areas in the colony were examined by phase contrast microscopy to determine whether permanent genetic changes affecting phenotypic characteristics had occurred in the filament. Direct Counts, Viable Counts, and Absorbancy Changes The obvious differences in cell size of g. megaterium grown in toxic TSB versus cells grown in nontoxic TSB . required the determination of viability and absorbancy changes during growth. Direct microscopic counts were made on the nontoxic cultures at different times in the growth cycle using a Fetroff—Hausser counting chamber. A l to A dilution of the suspension was added to 3 ml of 30% glycerol, the counting chamber filled with one lOOpful of the suspension, and the organisms in 80 small squares were counted. Total counts were calculated according to the following equation: 6 Total Count = (Number 08110 Counted)(Dilution)(2 x 10 ) (Number Small Squares Counted) U2 Viable counts were made at selected times during the growth cycle by making appropriate dilutions in sterile 0.2% peptene and plating in triplicate on TSA. Incubation was at 30 C and counts were made U8 to 72 hr after pouring. Cellular growth as measured turbidimetrically was also correlated with viable counts of @. megaterium cultured on toxic and nontoxic TSA. Absorbancy of the culture at bot nm was plotted versus time using a Bausch and Lomb Spectronic 20 spectrophotometer. Effects of Cell Septum lnitiators on Cell Division Pantoyl lactone, a septum-inducing agent, was added to E. megaterium in toxic TSB to determine its effects on cell septum formation. The chemical was added at a con- centration of 0.075 M initially or after 1 1/2 hr of growth to toxic and nontoxic TSB. Fifty milliliters of growth medium were added to 250-ml Erlenmeyer flasks and incubated at 30 C in a shaker-incubator at 150 RPM (New Brunswick Model G25). Absorbance at 660 nm and viable counts using TSA were made throughout the growth cycles. The effects on cell division of 5 ug/ml Co++, Fe+++, ha , Zn , Cu++, and Cr++ in toxic TSB were investigated using the same culturing techniques as described for pantoyl lactone. “3 Effect of Penicillin on cell Division Both 15 and 30 units/ml of penicillin G were added to 80 ml of nontoxic and toxic TSB in 250—ml Erlenmeyer flasks. Agitated incubation was performed as previously described. Cultures were examined visually for turbidity and microscopically to determine the development of pos- sible morphological changes due to the interaction of penicillin and aflatoxin or to penicillin alone. Staining Studies Six-hour cultures of E. megaterium grown in toxic and nontoxic TSB were stained for cell wall development. A smear was prepared and fixed by heat. Three drops of 0.3M% aqueous cetyl pyridinium chloride were placed on the smear followed by the addition of 1 drop of saturated aqueous Congo red. The slide was washed with distilled water after 1 min and counterstained with methylene blue for 10 sec. The stain was then rinsed once more with distilled water, dried, and examined microscopically. Sudan black B (0.3% in 70% ethanol) was flooded over a wet smear of @. megaterium cells to demonstrate the existence and relative amounts of poly-B-hydroxybutyric acid at different stages of growth. The smears were washed with distilled water, dried, and examined micros00pically. AU Heat Resistance of Spores Formed in the Presence of Aflatoxin B1 and the Effects of Aflatoxin B1 on‘Spore Germinationri ' .- -' .e...” - n ,4: k. heat Resistance stud es Cne tenth milliliter of stock 3- megaterium spore suspension formed in toxic or nontoxic TSA was dispensed into 16 x 150 mm screwcap test tubes containing 9.9 ml of water tempered at 92 C 1197 F). Aliquots were removed after 10, 20, 30, 50, and 70 min of heating, appropriately diluted, and plated in TSA. Incubation was at 30 C and counts were made after “8 hr. Decimal reduction times (D values) were calculated by plotting the viable count versus time of heating in min at 92 C on semi log paper. Germination Studies fl. megaterium spores produced in TSA as described previously were heat shocked at 60 C for 10 min and diluted 7 to yield approximately 10 viable spores per ml. One milliliter of the suspension was inoculated into 6 ml of T88 and to 6 m1 of TSB containing 3.8 ug/ml aflatoxin Bl. The TSB media was tempered at 32 C. Absorbance at 650 nm was recorded each 15 min for the first 2 hr and at 3, 1A, and 2A hr of incubation. lit ) Chemical Analyses of Vegetative Cells and Spores of Bacillus megaterium Formed in the Presence and Absence of Aflatoxin Bl Chemical analyses described below were performed on vegetative cells produced in toxic and nontoxic TSB which were stored at —20 C and on spores formed in toxic and nontoxic TSA which were stored at A C. Except for a few minor changes in procedure, the method for a particular analysis of vegetative cells or spores was the same. Analys>s were performed in triplicate on vegatative cells and spores formed both on toxic and nontoxic media. Dry weights were determined in quadruplicate on cell and spore suspensions. Preparaticnitd'\Hfi;etative Cell Walls for Amino Acid Analysis Cells were resuspended in 200 ml of cold distilled water. one-hundred milliliters were then sonicated for 7 min (filamentous cells formed in toxic TSB) or 12 min (control cells formed in nontoxic TSB) at a setting of 5 amperes. Sonication was about 98% complete as evidenced by phase contrast microscopy. The sonicated cell sus- pension was centrifuged at 10,000 x g for 10 min and the supernate was discarded. The pellet, containing cell walls, intact cells, and coarse debris, was resuspended in 50 ml cold water and centrifuged at 3,000 x g for 5 min. The pellet was discarded and the supernate, containing A6 cell walls and fine particles, was centrifuged at 10,000 x g for 20 min. The supernate was discarded and the pellet or crude cell wall fraction was washed four times in l M NaCl; after each washing the cells were centri- fuged at 10,000 x g for 20 min and the supernatant fluid was discarded. The pellet was then suspended in cold water and centrifuged at 3,000 x g for 5 min. The supernate, containing cell walls, was centrifuged at 10,000 x g for 20 min and the pellet was dried in pre- paration for amino acid analysis. All centrifuging was done at ll C. Procedure for Amino Acid Analysis of Vegetative Cell Walls Sufficient dried cell wall material, as determined by semimicro Kjeldahl analysis (Kabat and Mayer, 1948), was dissolved in 1 ml of 6 N hydrochloric acid in lO-ml ampoules to give a 0.4% protein solution. The tube was drawn to a small neck, cooled, and placed in a dry ice bath to freeze the contents. After freezing, the tube was evacuated, the sample was allowed to melt, and the tube was sealed at the constriction with an air-propane flame. The sealed tube was then placed in an oil bath in an oven at 110 C for 20 hr or 70 hr. Following hydrolysis and cooling, the hydrochloric acid was quickly removed on a vacuum evaporator and the sample was dissolved in 5 ml of 0.067 M citrate-hydrochloric acid buffer, pH 2.2. “7 An aliquot of 0.2 ml was removed from each sample for analysis on a Beckman Amino Acid Analyzer Model 120 C (Moore gt gi., 1958). The amino acid composition of the protein samples was eXpressed as uMoles amino acid per mg of protein. Total Protein Analyses of Eggetatlve Cells and Sporgg Breakage of vegetative cells was by sonication for 7 or 12 min as described earlier. Spores were mechanically ruptured by means of a Bronwill Disintegrator (Type 2876, Bronwill Scientific Inc., Rochester, New York). The supernate and three washings from the glass beads used for disrupting the spores were pooled for the analysis. The Biuret reaction (Gornall g: gl., 19u9) and the method of Lowry g3 gl. (1951) were used to estimate the protein concentration in the supernatant fluids collected from the cell sonicates and in the spore disintegrates. Standard protein solution was made using crystalline bovine albumin (Nutritional Biochemicals Corporation, Cleveland, Ohio). The total protein analysis described in this section and the RNA analysis described in the following section were performed using supernatant fluid collected by centrifugation following sonication of cells and on spore disintegrates. However, calculations were made to express A8 the resultant quantities of each component as a percentage of dry weight of the whole cell or spore. Total Ribonucleic Acid Analyses of Vegetative Cells 'nd Spores The RNA isolation procedure described by Clark (196“) was modified to determine the RNA content in the supernatant fluid collected from cell sonication and in the spore disintegrates. An equal volume of 88% phenol was added to the chilled supernate and the mixture was stirred at room temperature for 30 min. The emulsion was then cooled in an ice bath for 5 min before separating by centrifugation at 3,300 x g for 15 min. The aqueous upper supernatant layer and most of any intermediate layer containing denatured protein was decanted from the brown phenol phase. Removal of denatured protein from the aqueous phase was accomplished by centrifugation at 8,000 x g for 5 min. A volume of 20% (w/v) potassium acetate (pH 5.0), 1/10 the volume of the aqueous fraction, was added to the aqueous fraction followed by precipita- tion of RNA by the addition of two volumes of cold 95% ethanol. The suspension was chilled in an ice bath 5 min before collecting the precipitate by centrifugation at 1,000 x g for 20 min. The precipitate was washed once each with ethanolzwater (3:1, v/v), absolute ethanol, and anhydrous ether and then air dried. All centrifuga- tion was performed at 0 to 5 C. “9 The dried precipitate was dissolved in 10 ml of 0.5 N potassium hydroxide and hydrolysis proceeded at room temperature for 24 hr. Sufficient 20% (v/v) hydrogen perchlorate was added to the chilled hydrolyzed solution to reduce the pH to about 2 followed by removal of pre— cipitated potassium perchlorate, DNA, and/or protein by centrifugation at 1,000 x g for 10 min. The pH of the supernate was adjusted to 3.5 with 1.0 N potassium hydroxide. A modification of the Ashwell (1957) procedure for colorimetric analysis of sugars was used to determine the RNA content of vegetative cells and spores. The acidified hydrolyzed sample of RNA was appropriately diluted in deionized water and suitable aliquots were mixed with 6 ml of orcinol acid reagent (0.1% FeCl3 in concentrated RC1) and 0.“ ml of 6% (w/v) ethanolic orcinol. Total volume was brought to 10 ml with deionized water. The solutions were heated 20 min in a boiling waterbath, cooled, and absorbancies were measured at 660nm. A standard of 10-“ M nucleic acid (Ribonucleic acid, Nutritional Biochemicals Corporation, Cleveland, Ohio) was used. Total Deoxyribonucleic Acid Analysis of Vegetative Cells and Spores The DNA isolation procedure of Clark (196“) was used. Twenty milliliters of sonicated cell suspension or 50 disintegrated spore suspension was mixed with 80 m1 of 15% (w/w) sodium lauryl sulfate in 0.1“ M sodium chloride: 0.01 M sodium citrate. The mixture was homogenized and stirred 30 min. Two-hundred milliliters of absolute ethanol were added slowly and the precipitate was Col- lected by centrifugation at 3,000 x g for 5 min. The precipitate was then washed three times with 5 m1 of 1.0 M sodium chloride, collecting the pellet formed each time at 10,000 x g for 20 min and pooling the supernates. The pooled supernates were mixed with an equal volume of absolute ethanol; DNA was collected on a glass rod and air dried. The diphenylamine reaction was used to quantitate the DNA obtained. Two milliliters of diphenylamine reagent (1.0 g diphenylamine dissolved in 100 ml of glacial acetic acid plus 2.75 ml of concentrated sulfuric acid) were added to 1.0 ml of sample dissolved and diluted in sufficient water to contain approximately 200 ug of DNA and the mixture was heated for 10 min in a boiling waterbath. The absorbancy was read at 600 nm upon cooling. Sperm deoxyribonucleic acid (Nutritional Biochemicals Corporation, Cleveland, Ohio) was used as a standard. 51 Dipicolinic Acid Analysis of_§pores Dipicolinic acid analysis was performed according to a modified Janssen g_ gl. (1957) procedure. Spore suspensions containing 25 to 35 mg (dry weight) of spores were diluted to 2.5 ml with deionized water in a 10-ml volumetric flask. Hydrochloric acid (1 N) was added to the suspension to bring the pH to 2.0 and the hydrolysate was autoclaved for 1 hr at 121 C. The flask was then cooled and 3 ml of u% trichloracetic acid was added to precipitate interferring materials. Flasks were placed in an ice bath for 1 hr to complete precipitation followed by removal of the precipitate by centrifugation at 10,000 x g for 20 min. Aliquots of the supernate made to A ml with deionized water were mixed with 1 m1 of color reagent (1% ascorbic acid and 1% Fe(NHu)2(SOu)2 in 0.5 M sodium acetate) and the absorbancies were determined at ““0 nm. Standard dipicolinic acid solution (100 ug/ml) was also monitored. Preparation of Vegetative Cells and Spores of Bacillus megaterium for Observation by Electron Microscopy Production of vegetative cells and spores was pre- viously described. Cell types were fixed by adding suffi- cient cell or spore suspension to 13 x 100 mm screwcap test tubes to yield pellets about 0.5 mm thick upon centr- fugation. Both cell and spore pellets were treated identically. The pellets were suspended in a 50% aqueous solution of glutaraldehyde diluted to 6.25% with Sorensen's phosphate buffer, pH 7.2, and kept at room temperature for 2 hr. The samples were then washed three times in 1 hr with Sorensen's buffer and suspended in a 2% aqueous solution of osmium tetroxide diluted to 1% with Sorensen's buffer. The samples were dehydrated after 1 1/2 hr at room temperature for 10 min each in 25, 50, 75, and 95% ethanol and twice for 15 min each in 100% ethanol. The samples were treated for 30 min twice in propylene oxide and kept overnight in propylene oxide plus EPON complete mixture (1:1, v/v). The EPON complete mixture was prepared according to the following formula: Solution A: 80 g EPON 812 (W.P.E. = 160) plus 94 g dodecenyl succinic anhydride Solution B: 100 g EPON 812 plus 78 g nadic methyl anhydride Solutions A and B were mixed in ratio A:B::6:A and A drops of hardener, dimethyl amino methyl phenol, were added for every 10 ml of the mixture. Samples were collected on the following day by centrifu- gation and dried with a stream of nitrogen. The samples were then suspended in the complete EPON mixture and kept (Mlernight in a desiccator. Approximately 2 drops of the Sample were placed on top of fresh EPON complete mixture in gelatin capsules, the desiccator was evacuated, and the SaUKDles were permitted to settle to the bottom of the 53 capsule overnight. Hardening was accomplished by heating the samples for 12 hr at 35 C, 12 hr at “5 C, and 12 hr at 65 C. The gelatin was removed from the hardened EPON upon cooling and ultrathin sections were made on an LKB ultratome (LKH, Type A801 A, Rockville, Maryland) and examined with a Philips electron microscope (Model EM-100 B, 1TH? Netln‘rlaiuis). RESULTS AND DISCUSSION Growth Media A sufficiently aflatoxin-sensitive bioassay micro- organism was sought to investigate the mechanism of toxicity of aflatoxin. Since most available data describe metabolic responses of higher organisms to the toxin, additional data are needed on simpler organisms. Bacillus megaterium NRRL B—l368, previously found to be inhibited by 15 ug/ml aflatoxin (Burmeister and Hessel- tine, 1966), was chosen for this purpose. Various morphological and biochemical responses of the vegetative cell to aflatoxin were evaluated and sporulation, germi- nation, and outgrowth processes as affected by aflatoxin were studied. To date no reports are available involving the effects of aflatoxin on sporulation and germination. The organism was initially cultured on several media to determine a suitable medium for both vegetative cell and sporulation studies. Media evaluated were nutrient broth (Difco), peptone broth (Difco) plus 10 ug/ml Mn, trypticase soy broth (Baltimore Biological Laboratories), and a special modified sporulating medium containing metal salts, glucose, and casamino acids (Webster and Lechowich, 1970). Trypticase soy broth 5A (TSB) was superior with respect to rate of cell growth for culturing vegetative cells and for sporulation (TSB plus 2.0% agar, TSA) and was therefore chosen as the growth medium . Hocovvfij of Aflatoxin Added to the Medium The calculated aflatoxin concentration in the toxic “SB quantitatively obtained by fluorodensitometry against aflatoxin standards should have been “.0 ug/ml prior to inoculation. The medium was steamed for 15 min to drive off the chloroform in which the aflatoxin was dissolved, and a 5% loss in aflatoxin B1 concentration was observed. This loss could have been due to thermal inactivation or degradation to some other compound, although no additional fluorescent spots were detected by thin layer chromatography. This loss could also be due to the failure in quantitative removal of the toxin from the TSB by the partitioning procedures used for quantitation. The per cent recovery of aflatoxin from TSB before steaming was about 97%, indicating that the 5% loss was probably due to a combina- tion of both phenomena. Only 1.7 ug aflatoxin B1 per ml or about 45% of the original B added to the TSB were detected after 10 1/2 1 hr of culturing B. megaterium at 30 C on a gyratory shaker. The decrease may have been caused by oxidation during the culturing period, utilization of the toxin by the organism, 56 and/or inefficient extraction of the toxin from the ruptured cellular components liberated by sonication. Decrease due to utilization of the toxin might be inferred from the observed initial 30% kill of B. megaterium during the first 3 1/2 hr after inoculation (Figure 2). The lethal effect was followed by an extended lag and possible adaptation period before logarithmic growth commenced. During such an adaptation period, mutation could have occurred to yield cells potentially capable of utilizing the toxin. The apparent loss of aflatoxin B is more likely l principally due to chemical oxidation since a 2.A ug/ml concentration (60% of that initially added) of B1 was detected in noninoculated TSB after 10 1/2 hr at 30 C on a gyratory shaker or to inadequate extraction of the ruptured cellular components resulting from sonication. Lillehoj and Ciegler (1968) reported that repeated chloroform washing of macerated B. megaterium cells was required to quantitatively remove aflatoxin from cell walls, protoplasts, and nucleic acid. Aflatoxin concentration in the sporulation medium was 3.2 ug/ml after 6 days of incubation. Figure 2.—-Growth curves for Bacillus megaterium cultured in trypticase soy broth (control) and trypticase soy broth containing 3.8 ug/ml aflatoxin B1 (treated). 58 um 099 u: Kaunqlosqv t 5 «E: NdL v.0... 0.0.1 0.0.... o._l «.74 ¢._1 4 I70/ .W.\ ..V.. \. \ \o“\ o o\«\4 \ \ I \ 4 . \ . v\.\ uoaomtfiocofoma<£ IIIIII \ III .. ”\ .223 $055336: _. “.33: J53 030$. o .223 .230 23;; “>3. (Juno: alqngA) [Lu .Iacl '°N 601 59 Effects of Aflatoxin upon Growth of Bacillus megaterium Growth Curves It is interesting to examine and study the growth curves of B. megaterium cultured in toxic and nontoxic TSB which are shown in Figure 2. There was a decrease in the viable number of cells during the first 3 1/2 hr of incubation in toxic TSB followed by logarithmic cell division at a rate considerably less than the growh rate of the control culture. Absorbance of treated cells at 660 nm measures increase in total cellular mass, and was observed to be more like the control culture, although the slepe of the control culture was greater. The apparent differences when comparing the rates of growth and rates of cell division of control and treated cells are probably due to the difference in size of the Viable units capable of forming colonies on nontoxic TSA. The aberrant single cell forms have increased length and mass after exposure to aflatoxin B1 in comparison to cells grown in nontoxic TSB. This results in a concommitant decrease in the number of viable cell units and thus the observed slope differences. Generation times were calculated to be 75 min for the treated cells and 20 min for the control cells. The shape of the viable units also had an effect on cellular chemical composition which will be discussed lateru 60 Ce 1 lulu!" Morlihology as Affected by Aflatoxin Figure 3 shows the filamentous cells formed in the presence of aflatoxin B1 and normal B. megaterium cells. Filaments often reached 50 u and intertwined to such an extent that made direct microscopic counts impossible. Massive accumulations of poly-B-hydroxybutyric acid and swelling, usually terminal, can be seen in mature aberrant cell forms (Figure u). The bulb-like formations might indicate a weakening of cell wall structure with age or a greater sensitivity to osmotic change. Lending some support to this reasoning were the observed times required to rupture normal and treated cells. Control cells required 12 min and treated cells required only 7 min for satisfactory rupture by sonication. However, long filamentous cells would naturally be more susceptible to breakage and thus should require a shorter sonication CIWQ1LUKMIt. The affinity of cell wall or capsular material of the two cell types for the stains cetyl pyridinium chloride and Congo red was evaluated. Photographs of stained 6-hr cultures of control and treated cells are shown in Figure 5. The striated character of the distribution of poly— saccharide and the coincidence of major striations with septa of control cells is of interest. Although striations can be seen perpendicular and irregularly along the long 61 Figure 3.--Normal and aberrant forms of Bacillus megaterium produced during growth in trypticase soy broth and trypticase soy broth containing 3.8 ug/ml aflatoxin 8%, respectively. (A) control at 3 1/2 hr incubation, ( ) 3.8 ug/ml 81 at 3 l/2 hr, (C) control at 5 1/2 hr., (D) 3.8 ug/ml B1 at 5 1/2 hr. Micrographs taken through a phase microscope X500. 62 63 Figure U.--Micrographs showing bulb—like swellings at the terminal portions of filaments in a lA-hr culture of Bacillus megaterium grown in trypticase soy broth containing 3.8 ug/ml aflatoxin B1. Stained with 1% tannic acid and 0.“% basic fuchsin in 0.5% NaCl. Micrographs were taken at X500. 64 65 Figure 5.——beminstraiion of capsular material in ngillui megaterium cultured in (A) trypticase soy broth and (H) trypticase soy broth containing 3.8 pg ml aflatoxin B-. Note the striated Character of the distribution of polysaccharide capsular material and the coincidence of striations with septa between dividing cells of the control culture. Few SEpta were observed in filaments of the treated culture. Micrographs were tzikt>n :ii, X‘SUL). 66 67 filaments, septa were rarely detected in these filaments by phase contrast microscopy or by electron microscopy. Thus the accumulation of polysaccharide near regions of septa formation must be independent of the inhibitory effect of aflatoxin on septum formation. Aflatoxin-induced mutation is unlikely, due to the reversion of filamentous cells formed in toxic TSB to normal morphology when transferred on nontoxic medium (Figure 6). Visual examination of colonies of daughter cells formed from filamentous cells streaked on TSA indicated no morphological differences from cells never exposed to aflatoxin. Transfer of filamentous cells to nontoxic TSB resulted in growth patterns identical to the control cells. Thus there does not seem to be permanent genetic damage in the filamentous cells formed in toxic TSB. Pantoyl Lactone Studies Mutant cells of Escherichia coli formed after eXposure to ionizing radiation is thought to be genetically controlled (Adler and Hardigree, 1964). Such filaments do not give rise to macrocolonies. Pantoyl lactone, an agent that initiates cross plate formation, allows these filaments to divide normally and produce macrocolonies. Reversal of inhibition of division by D-serine, D-histidine, D-phenylalanine, and penicillin in an Erwinia sp. was observed to occur after the addition of pantoyl lactone 68 Figure 6.-—Time-lapse phase contrast micrographs of dividing filamentous Bacillus megaterium cells which were formed in trypticase soy broth containing 3.8 ug/ml aflatoxin Bl and then transferred to nontoxic trypticase soy agar. Note the formation of normal sized daughter cells. (A) 0 hr incubation at 30 C, (B) 1 hr, (C) 2 hr, and (D) 8 hr. Micrographs taken through a phase micro- SCope X1950. 69 70 to the growth medium (Grula and Grula, 1962). Pantoyl lactone has two distinct roles in metabolism, one as a precursor to pantothenate and another undefined role in cell ivision. To determine the reversibility of aflatoxin inhibition of division in B. megaterium by pantoyl lactone, 0.075 M was added initially and after 1 l/2 hr incubation to control and aflatoxin-treated cultures. Data derived from turbidimetric measurements and viable cell counts of the growing cultures indicated that pantoyl lactone had neither an inhibitory nor stimulatory effect on the rates of growth or division in Control and treated cultures. The apparent lack of effect may be due to the inability to distinguish formation of septa by turbidity and viable counts. Observations by phase contrast microscopy, however, did not reveal these morphological structures. Penicillin Studies Penicillin inhibits the formation of cell walls in Gram positive bacteria by interferring with the attachment of the tetrapeptide to the N-acetylglucosamine-N- acetylmuramic acid backgone. Cells formed in media con- taining penicillin may divide at a reduced rate, sometimes producing penicillinase which destroys the penicillin and permits reversion to normal cell growth and division. Data from eXperiments involving the addition of 15 or 30 units/ml of penicillin G to aflatoxin-containing and 71 nontoxic media indicated that the metabolic actions of penicillin and aflatoxin were apparently independent of each other. Media containing high or low concentrations of penicillin or penicillin plus aflatoxin resulted in similar growth lags, the lags being greater than those observed for aflatoxin Bl alone. Once growth commenced, cells formed in TSB plus penicillin were morphologically normal with only occasional bulging. Filamentous cells formed in TSB plus penicillin and aflatoxin were morpho- logically indistinguishable from filaments formed in TSB with aflatoxin alone, with the exception of occasional bulging. Aflatoxin 81 had no apparent effect on the inhibition or enhancement of apparent penicillinase formation in this particular strain of a. megaterium. Penicillinase was not assayed, however cultures contain- ing penicillin alone and penicillin plus aflatoxin behaved similarly after similar extended lag periods. It is inferred that the formation of penicillinase was inducible during the lag periods. Lillehoj and Ciegler (1969a) also observed that simultaneous addition of 50 pg of B per ml and penicillin to an inducible strain of l g. cereus did not interfere with penicillinase synthesis. Exposure of the cells to toxin for 2 hr before incubation, however, reduced penicillinase production US to 60% compared to the control. 72 Effects of Aflatoxin upon Sporulation of Bacillus megaterium Sporogenesis in g. megaterium begins when a portion of an axial thread of chromatinic material becomes posi- tioned at one end of the sporangium and a centripetally growing thin septum encloses the chromatin and a small amount of cytoplasm. The sporulation process is believed to be irreversible or committed at this stage. Since one of the morphological alterations in Q. megaterium grown in toxic TSB was the apparent inhibition of septum formation, a study was made to determine the effect of aflatoxin on the initial septum formation phase of sporulation. Completion of septum formation is compulsory for the development of the mature spore. Sporulation was followed on toxic and nontoxic TSA. After 3 days, 97% sporulation occurred in the control culture as measured by direct micrOSCOpic count. Only 68% sporulation was obtained in the test culture after 6 days in the toxic medium. The 65% figure is only approxi- mate due to the difficulties in counting caused by abnormal length of the filaments and the frequency of multiple spores being formed within these filaments. Old filaments were observed to lay down septa at close inter- vals to form a chain-like arrangement of cells, the length and width of which were nearly the same. The cells failed to separate, perhaps because of incomplete septum formation. Spores were often formed within these 73 irregular partitionings of the filaments. Although the medium contained 3.2 ug/ml aflatoxin (16% decreased from initial) on a total volume basis when spores were harvested on the sixth day of incubation, it is probable that the content may haVe been decreased in the immediate area of colony formation due to oxidation and dilution brought about by increased cell mass. Aflatoxin concentration may have been decreased to a level which was not detri- mental to either septum formation in dividing cells or septum formation as an initiator of sporogenesis. lieat lieciz‘tarnu‘ Stiniies Once the spores were formed in a medium initially Containing 3.8 ug/ml aflatoxin, were they physically, chemically, or morphologically different from sporls formed on nontoxic medium? An examination of the wet heat resistance of spores formed on control and toxic TSA indicated no significant difference. Survivor curves for spores formed on toxic and nontoxic TSA are shown in Figure 7. The (time in minutes to destroy 90% of D 197 the population at 197 F) for the control was 17.0 min; the qu7 for the test culture spores was 16.9 min. The D values are extremely low, indicating little heat resistance. The chemistry and morphology of control and treated spores will be discussed later. 7“ Figure 7.--Survivor curves at 92 C (197 F) for Bacillus megaterium spores formed in trypticase soy agar (control) and trypticase soy agar containing 3.8 ug/ml aflatoxin Bl (treated). log No. per ml (viable count) 75 '0 key: 0, Viable count, control A.Vioblo count, treated 5 l o I I I I I I l IO 20 3O 4O 50 60 70 Time (min at 197 r) 76 Effects of Aflatoxin on Spore Germination and Outgrowth Germination of spores is characterized by loss in heat resistance, permeability to dilute stains, loss in turbidity or cry matter, and darxening under phase con- trast (Murrell, l961). The loss in turbidity and phase darkening were followed to study the effect of aflatoxin of germination of spores which were formed on nontoxic TSA. initiation of germination is usually brought about by chemical substances and can occur even if environmental conditions are unfavorable to germinal development. Germination initiation involves among other processes, the uptake of water with a coincident swelling and decreased refractility of the spore. Germinal development following initiation involves either rupture or adsorption of spore coats followed by the elongation of a germ cell and is generally defined as outgrowht. Results from germination studies using normal spores exposed to a suitable germination environment and the same environment plus aflatoxin indicated that the toxin had no effect on germination initiation but suppressed outgrowth for an extended period. Table 2 lists absorbancy readings at 650 nm of spore suspensions in toxic TSB and nontoxic TSB at various times during incubation at 30 C. The degree of outgrowth inhibition, that is the per cent of spores which initiated germination but failed to develop through the outgrowth stage, cannot be estimated 77 TABLE 2.——Ahsorhancy at 050 nm of spore suspensions in 1rypticase soy broth containing 3.8 ug/ml aflatoxin B1 (toxic) and trypticase soy broth (nontoxic). w.--‘ w. —— ._—_~—.-.— -.—._—___——___ Aigxirhnnicy 765C) hm) \Niti.'Tinn? (hr) i , ,1 -a . - .—-———-— _--_-~———. Toxic Tar .690 .569 .538 .523 ,u95 .U69 .u32 .553 Noni (3X10. TSB .638 .569 .538 .523 .U95 -“69 .85“ -957 due to experimental design. More than 90% of the spores turned phase dark in both control and treated spore sus— pensions. A lag of more than 1“ hr was required for outgrowth of treated spores compared to about 5 hr for control spores. it would only require a small percentage of spores to progress through the outgrowth stage and begin dividing to increase the turbidity as an indication of growth. Results from the germination studies indicate, then, tha1 the catabolic processes occurring during germination initiation are not inhibited by aflatoxin but the outgrowth phase, in which anabolic mechanisms are at their maximum rates, is inhibited. Effeet of Aflatoxin on Vegetative Cell Wall Com osition it is obvious Upon examination of the aberrant cell forms resulting from growth of E. megaterium in toxic TSB that there is more cell wall material per living unit of 8 and probably less cell wall material on a dry weight basis than in shorter control cells. This can be concluded by virtue of the vhape of the cells, assuming there is no difference in cell wall thickness. But there have been no reports on the ”emposition of cell walls of these filamentous forms. For this reason it was decided that the amino acid composition of cell walls of control and treated cultures would be analyzed. Although the cell wall preparaiions were not particularly clean, ratios of alanine, glutamic acid, and lysine, the three amino acids making up the tetrapeptide bridge in most Gram positive bacteria, were not significantly different when chromato- grams of the extracts were compared. Table 3 lists the “Moles amino acid (lysine, glutamic acid, and alanine) per mg protein in the vegetative cell walls of control and test cells. Altheugh amounts of the constituents were greater in cell wall preparations of the control cells, TAHLE 3.-—Concentration in uMoles per mg protein of lysine, glutamic acid, and alanine in cell walls of Bacillus megaterium formed in trypticase soy broth (control) and trypticase soy broth containing 3.8 ug/ml aflatoxin Bl (treated). Control Treated (uMoles per (uMoles per mg protein) mg protein) Lysine 3.31 2.73 diutamic acid 6.97 5.25 ‘J'i R) CI) Alanine 6.37 —.—--- 79 approximate ratios of lysinezglutamic acidzalanine in test _l:?.l:l.9\ and control (l:l.9:l.9) cell walls were similar. Apparent higher concentration: of the cell wall constituents in control cells was most likely due to inqnwwper' prfqulratlf arnirio alzitis . The constituents N—acetylglucosamine, N—acetylmuramic acid, (nullliaudrnnuimelic eu:hi were rmn;1flln as sfixnriards, howeVer, no significantly different peaks were observed. it was concluded that aflatoxin B does not affect the 1 chemical rompwsltlon of cell walls formed in its presence. Effect of Aflatoxin on Vegetative Cell and Spore Composition Chemical analyses performed on whole cells and spores formed in toxic and nontoxic TSB revealed different results. Table A list: the total protein, DNA, and RNA of control and treated cells and spores along with the total dipicolinic acid (DPA) content of spores. Analyses of whole cells revealed that the percentages of protein, DNA, and RNA an a dry weight basis were greater when the cells were grown in toxic TSB. At the same time-it should be noted that total synthesis of all three components was decreased on a total culture basis. The increase in per cent protein, DNA, and RNA can be eXplained in part by considering cell shape. Assuming the concentration of the 80 1 TAbe 4.—-fer cent protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) in vegetative cells and spores and per cent dipicolinic acid (DPA) in spores of Bacillus mernterium fO‘med in trypticase soy medium (control) and Tripticace cry medium containing 3.8 ug/ml aflatoxin B1 ftrwvated). - - . —_ —_. .____.-~—._-_v_ -__. _. _.. .- —. .. m”..— Veggetad;iv<> Celils Sprnoes tiontrw>l 'freatewi Ckuitrol Treailni Protein bl.0£* 73.0% 57.1% WQ.L% um 1:.i 5.3 2.8%:- 2.73 mm ll.” 114.6 10.93 10.3 iii-ii - — 6.8 6.3 fer cent «f each component is on a dry weight basis. three components on a cytoplasmic volume basis to be equal in bvth filamentous and short cells, then the per cent of each compwnent on a dry weight basis would be greater in the filamentous cells due to the decrease in per cent of cell wall material. Cell wall material, of course, would contribute no DNA nor RNA, and little protein to cell mass. A similar imbalance in macromolecular biosynthesis was reported in E. aurantiacum grown in the presence of MU uyvfinl ai‘lclioxiniii (Lillffiluj arui£3ieglin7, 1967). ’fhe imbalance, particularly in DNA production, might have been responsible for the decrease in viability of multiplying Q. megaterium (Lillehoj and Ciegler, 1968). The decreased total synthesis of DNA, RNA, and protein as reported by Lillehoj aini Ciiwjler"is lll agrwnemerdj witli th6>