SUMMARY: T w o hundred and ninety-six strains of mesophilic species of the genus Bacillus were isolated from soil and examined for the characters described principally by Smith, Gordon & Clark (1fkW) as well as for some additional characters. T w o hundred and forty-six belonged clearly to named species in the classification of Smith et al., thirty-two strains were clearly intermediate between two species and eight strains remained unallocated. In addition, eleven other strains appeared to represent a previously undescribed species (Proom & Knight, 1950 The strains of B. pasteurii were the most heterogeneous in their nutritional requirements, the components ammonium ion, amino-acids, aneurin, biotin and nicotinic acid being involved. All strains required amino-acids and aneurin ; in addition, biotin or nicotinic acid and sometimes ammonium ion were required, depending on the particular strain.There are few records of wide comparative surveys of bacterial genera which have made use not only of the classical bacteriological distinguishing tests and methods, but which have also included determinations of exact nutritional requirements, and of metabolic and serological-characters. Intensive studies of particular species from these aspects have been somewhat more common; but even here comparative studies of numerous strains or clones of a species, when made, have only seldom included all the features noted above, so that well-rounded pictures of their biological properties are infrequent. The Culture Collection at these Laboratories, with the staff and services necessary for its maintenance and development, offered an opportunity to make comparative It was hoped that such comparative surveys might yield information which would help to improve the diagnosis, differentiation and classification of bacteria as they came to hand. It was further hoped that from this material might come indications of natural relationship, thus helping to make classifications somewhat less arbitrary than they tend to be at present. A long-term objective is to accumulate information which might contribute to the study of taxonomic and evolutionary problems. This is not the place to discuss the problem of species differentiation; some pertinent examples will appear in the sequel. Indeed it might be well at this point to state our standpoint in this respect. We believe that the question of species definition and differentiation among bacteria is best not argued as a metaphysical question, apriori as it were, but that the question of classification will be better worth discussion only when much more pertinent material is available. It is part of the purpose of the present work to supply some of this material. It is, of course, true that for practical purposes distinguishing characters, however empirically determined, are invaluable and that a t least empirical classifications do have to be made for current use. But this does not preclude a desire to achieve better classifications, which will presumably be the better the more closely they reflec...

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SUMMARY : Eight strains of bronchiseptica, six strains of parapertussis and fifteen strains of pertussis were examined for their minimal nutritional requirements in defined media. All strains showed an absolute requirement for nicotinic acid and no other vitamin was required for growth. Amino acids were essential for parapertussis and pertussis, but bronchiseptica would grow in either a mixture of amino acids, or lactate or citrate. Two old laboratory strains were exceptional in that they could utilize either glutamic acid, a-ketoglutaric acid, citrate, lactate, succinate or pyruvate. The amino acid requirements of the three species were relatively simple and showed some similarities. Bronchiseptica would grow in a mixture of glutamic acid, proline and leucine, while parapertussis required added cystine and methionine, and pertussis required in addition alanine, asparagine and serine. In simple amino acid mixtures glutamic acid was essential, but was replaceable by a-ketoglutaric acid. Nutritionally the species are very similar, but are quite different from the Haemophilus injiuenxae-parainjluenxae-canis, or the Brucella abortus-suis-melitensis groups of organisms. The nutritional evidence supports the already impressive evidence on other grounds that these three groups should be classified separately. If generic status is given to one group it should be given to all three. L6pez (1952) has proposed a new genus Bordetella, consisting of the species B . pertussis, B . parapertussis and B. bronchiseptica, and this seems a reasonable solution to the present anomalous position. The three species can be differentiated by tests for inhibition. Thus B . bronchiseptica is the only species not inhibited by 2.0 yo citrate and B. pertussis the only species inhibited by colloidal copper sulphide.The Parvobacteriaceae have been subdivided into a multiplicity of genera, primarily on grounds of animal pathogenicity. Many of the genera consist of only a few pathogenic species; in some instances all the species in a genus produce similar diseases in the field or in experimental animals. Since pathogens are highly specialized species it has usually been possible to devise a number of positive or negative diagnostic tests for their identity, tests which are not so readily available for the whole range of bacteria. To some extent these tests have obscured the underlying basis of pathogenicity used for their classification. This classification is, of course, a reflexion of the historical development of bacteriology and has been evolved to satisfy the practical needs of human and veterinary medicine. Even if comparative studies made it possible to present a more aesthetically satisfying classification on general biological lines it would almost certainly be unacceptable at the present time to those workers most interested in this group of organisms. However, the existing system of classification introduces two difficulties that are pertinent here. First, it is difficult to introduce a species into such an arbitrary classification. Th...

SUMMARY: Working details are given for keeping as desiccates a collection of some 1500 strains of bacteria. The efficacy of drying, the various methods of freezing, and the effect of storage were tested by viable counts, using a spinning bottle modification of the roll-tube method. The survival rate of bacteria suspended in broth or other protective colloids, and subjected to freezing at -78", varied with the species from about 100 yo with the resistant Staphylococcus aureus to about 10 yo with the sensitive Neisserz'a gonorrhoeae, and from about 100 to 1.5 yo or less, respectively, when the organisms were suspended in saline. The percentage of organisms which survive the freeze-drying process was found to vary with the species, from 100% to less than 1.0 yo when the organisms were suspended in broth and from 100 yo to no survivors when the organisms were suspended in saline. The storage loss of dried cultures was found to be a function of the storage temperature ; however, suitably dried cultures could be kept a t room temperature for very long periods. By far the most important factor influencing loss on storage was the presence of traces of moisture, and to ensure optimal survival the cultures must be as dry as possible. It appears that even with adequately dried cultures the presence of oxygen is deleterious.The survival rate of bacteria in a sample of dried culture after heating to 80' for 1 hr., determined by plating a suspension of the heated sample in broth, provides a simple measure of the capacity of the particular batch of the dried culture to remain viable on storage at ordinary temperatures.The drying of bacteria arouses most general interest as a solution of the utilitarian problem of avoiding the continuous subcultivation of strains and of maintaining them in any particular metabolic state. The essential requirements of a collection of dried cultures is that, with the routine employed, all strains should be viable as required, and not that particular batches of dried cultures should survive for very long periods. On this point, rather strangely in view of the spate of published methods for the preservation of bacteria, there is almost no information. We have maintained as desiccates a collection of some 1500 strains of different organisms, mainly pathogenic bacteria, for several years and have examined quantitatively the effect of freezing, drying, and storing dried bacteria under different conditions. METHODSRoutine for drying bacterial cultures. The method used is that of Swift (1937) with minor modification%, and the routine we adopted is as follows. Most aerobic organisms are grown for 24-48 hr. on agar slopes of suitable media, and the growth emulsified with about 2 ml. of nutrient broth. The broth, containing c. 3 g.N/l., is made by extracting fresh horse muscle with a papain digest of horse muscle containing c. 1.5 g.N/1.Most anaerobic organisms are grown in 250 ml. amounts of nutrient broth, containing 0.01 yo thiolacetic acid, incubated for 24-48 hr., and the culture

The characteristic nutritional patterns were determined for seven species or varieties of the genus Bacillus. Six strains of Bacillus cereus var. anthracis grew in a complex but defined amino acid medium+thiamine. Three strains of the insect pathogen B. cereus var. thuringiensis had the same nutritional pattern as the parent species B. cereus and grew in a seven amino acid medium. Of twenty strains of B. Jimnus, ten grew on the seven amino acid medium +biotin while the growth of five other strains was markedly affected by the addition of thiamine. Two of the remaining five strains required thiamine on first isolation, but soon dispensed with it on subculture, while the other three strains continued to require thiamine. Six strains of B. lentus grew in the seven amino acid medium + biotin +thiamine + urea.When the seven amino acid medium was replaced by the complex amino acid medium three of these strains of B. lentus grew without the addition of urea, and the remaining three strains required either urea or ammonium chloride. The sixteen strains of