— 1 %—+ ™ AEROBIC MESOPHILIC SPOREFORMING BACTERIA \> ««• Miscellaneous Publication No. 559 \l U. S. DEPARTMENT OF AGRICULTURE . UNITED STATES DEPARTMENT OF AGRICULTURE MISCELLANEOUS PUBLICATION No. 559 Washington, D. C. May 1946 AEROBIC MESOPHILIC SPOREFORMING BACTERIA By Nathan R. Smith, senior bacteriologist, Ruth E. Gordon, formerly assistant soil bacteriologist, and Francis E. Clark, i bacteriologist, Division of Soils, Fertilizers, and Irrigation, Bureau of Plant Industry, Soils, and Agricultural Engineering, Agricultural Research Administration 2 CONTENTS Page Page Summary 2 Descriptions of species studied—Con. Need for classification 3 Group 1—Continued. Studies on dissociation 6 Bacillus megatherium-Bacillus Dissociation of Bacillus mycoides. 6 cere us intermediates Historical background 6 Bacillus cereus Dissociants obtained 7 Bacillus cereus var. mycoides. Vitamin requirements 7 Bacillus cereus var. anthracis Examination of dissociants . 8 Bacillus subtilis Lysis of dissociants by bac- Bacillus subtilis vox. aterrimus teriophage 9 Bacillus subtilis var. niger. Stability of the dissociants. 9 Bacillus subtilis-Bacillus pum- Dissociation of Bacillus praiissnitzii 9 ilus intermediates Dissociation of Bacillus aterrimus. 9 Bacillus pumilus 67 Dissociation of Bacillus nigcr 11 Bacillus coagulans 73 Factors affecting the production of Bacillus firm us 75 acetylmethylcarbinol 12 Bacillus lentus 77 Use of the Voges-Proskauer test. 12 Group 2. Sporangia definitely Steaming the medium 13 swollen by oval spores 78 Length and temperature of incu- Bacillus polymyxa 78 bation 15 Bacillus macerans 79 Kind of peptone 16 Bacillus circulans 81 Reaction of medium 17 Bacillus macerans-Bacillus cir- Aeration 17 culans intermediates 86 Test reagents for acetoin 18 Bacillus alvei 87 Conclusions on production of acetyl- Bacillus laterosporus 88 methylcarbinol 18 Bacillus brevis 90 Storage of fat 19 Group 3. Sporangia swollen by Fermentation studies 20 round spores 94 Induced utilization of sucrose .... 20 Bacillus sphaericus 94 Induced utilization of other carbo- Bacillus sphaericus var. rotans 96 hydrates 23 Bacillus sphaericus var. fusi- Value of fermentation tests 23 formis 97 Agglutination responses 24 Bacillus sphaericus var. loeh- Bacteriophagy 28 nisii 98 Methods and mediums 31 Bacillus pasteurii 98 Microscopic examination 31 Unclassified cultures 99 Macroscopic examination 32 Bacillus aminovorans 99 Physiological examination 33 Bacillus badius 100 Key to species of the genus 35 Bacillus freudenreichii 100 Descriptions of species studied 39 Bacillus rarerepertus 100 Group 1. Sporangia not definitely Literature cited 102 swollen 39 Index to mesophilic members of the Bacillus megatherium 39 genus Bacillus ...,,,,.,. 109 1 On military leave. 2 The preliminary work was done by F. E. Clark and N. R. Smith. Owing to a change in the former's assignment, the work was continued and completed with the help of R. E. Gordon. The writers wish to express their deep appreciation to Charles Thorn formerly principal mycologist, in charge of the then Division of Soil Microbiology for his encouragement and helpful suggestions during the course of the work : to R. S. Breed of the New York State Agricultural Experiment Station ; and to others who have shown so much interest in the subject. The writers also wish to acknowledge the great assistance given by J. Roger Porter. University of Iowa, who turned over to them a collection of sporeforming bacteria that he had collected from American and European laboratories They also wish to thank the American Type Culture Collection, the National Collection of Type Cultures (London), and the many individuals who donated named cultures 2 MISC. PUBLICATION 559, U. S. DEPT. OF AGRICULTURE SUMMARY Aerobic mesophilic sporeforming bacteria are usually considered as inhabitants of the soil. Because of the formation of heat-resisting spores, they may be found in practically any location. They may cause the spoilage of food and other materials and may be found even in medical work, where they are sometimes suspected of being associated with certain pathological conditions. During the past few years much interest in this genus has arisen because of the discovery of antibiotic substances produced by certain species. Ford and his coworkers attempted to classify this genus in 1916, but lack of knowledge of the different stages of growth and the varia- tion in bacterial cultures, as well as lack of adequate differential tests, left their work incomplete. The result was that bacteriologists—medi- cal and agricultural—often attached a new name to a culture that they had isolated, believing it to be a new species. This, combined with the distribution of mislabeled cultures, led to such confusion that no one could identify more than a very few of the species. A total of 621 cultures of aerobic mesophilic sporeforming bacteria were studied and placed in 3 groups on the basis of morphology and physiology. Of this number, 363 bore specific names ; the remaining 258 were either received unnamed or were isolated by the writers. Of the three groups the first is the largest, comprising about 77 percent of the cultures studied. It includes Bacillus megatherium, B. cereus and varieties, B. subtilis and varieties, B. pumilus (formerly called B. mesentericus in America), and the lesser known species B. coagulans, B. firmus, and B. lentus. Studies on dissociation were made on B. mycoides and its biotype B. praussnitzii, which resulted in placing B. mycoides as a variety of B. cereus. Comparative studies indicated that B. anthracis differed from B. cereus in being path- ogenic and nonmotile. Since both are mutable characters, B. an- thracis has been placed as a variety of B. cereus. Dissociation of the black B. niger and the black B. aterrimus to colorless forms indistin- guishable from B. subtilis proved that they were only varieties of the latter. Group 2 contains about 19 percent of the cultures. It includes B. polymyxa, B. macerans, B. alvei, B. circulans, B. brevis, and B. laterosporus. Group 3, the round-spored group, with only about 4 percent of the cultures, contains B. sphaericus and varieties and B. pasteurii. B. fu-siformis has been placed as a variety of B. sphaericus because of its production of urease. B. rotans and B. loehnisii are also tentatively placed there as varieties. The apparent unimportance of groups 2 and 3 because of the small number of cultures studied may bear no relation to the occurrence of the species of these groups in nature. The Voges-Proskauer reaction proved to be an important and a reliable test for separating some species if certain modifications of the usual method were followed. The storage of fat and the fermenta- tion of carbon compounds were studied extensively and were found valuable, provided that liberal interpretations were made of the observations. Of special interest was the ability of certain strains to produce adaptive enzymes so that carbohydrates not regularly at- tacked could be utilized. In manv cases these cultures lost this abilitv AEROBIC MESOPH1LIC SPOREFORMING BACTERIA 3 after a few months' storage on nutrient agar in the absence of the carbohydrate. Agglutination by antiserums produced with whole cells as antigens was too specific for taxonomic use. Bacteriophage, although too specific in some instances, was very useful in others, especially in the study of B. megatherium, B. cereus and varieties, B. pumilus, and B. brevis. It was found that many species previously described and separated from others because of a rough, folded, mucoid, rhizoid, or pigmented growth were merely growth phases or varieties of established species. It was also found that still other species, characterized by some special physiological trait were only biotypes of the ''parent species." Con- sequently, a large number of adequately described named cultures have been placed in the synonymy of the various species. If the orig- inal description was not adequate to characterize the organism, it was assigned to a species on the basis of the culture as received. This resulted in a reduction from about 120 to 15 specific names. A dichotomous key for use in identifying cultures of the mesophilic aerobic sporeforming bacteria has been prepared in which two charac- ters, so far as possible, are used at each separation instead of one. A variation or misinterpretation of one of these should not, there- fore, lead to a wrong conclusion. NEED FOR CLASSIFICATION During the 27 years since Ford and his associates (60, 61) 3 published their work on the sporeforming bacteria, very little progress has been made in the classification of this group. In the meantime new species have been described briefly without comparing them with those already existing in the literature. Often there was only a simple isolation to which was ascribed some special character, as the ability to grow upon a certain medium or to produce a certain compound, and no attempt was made to observe closely related strains for that same property. The futility of attempting to arrange species so badly described is illustrated by the classification of the genus Bacillus in the fifth edition of Bergey's Manual (6). For a number of years it had been obvious that this genus was poorly arranged and needed thorough study. The stimulus to attempt this came when a culture of B. siamensis Sirabaed (94-) was studied and was identified as a strain of B. cereus (17), the most common of the sporeformers occurring in soil. Since the best method of approach seemed to