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In the previous article,' experiments were recorded which demonstrated that an active fat-splitting ferment was present in solution in broth cultures of a variety of acid-fast bacilli. These experiments were carried out in such a manner as to be roughly quantitative, and the activity of the lipase, as determined by these experiments, was found to be very considerable. It is a matter of importance to determine whether these lipases are excreted by these acid-fast bacilli as exoferments, or whether they are bound up in the bodies of the bacteria, and appear in solution only when the bacteria have autolyzed (endoferments). Carrikre2 has described a thermolabile lipase in the organisms of a six-months-old culture of the tubercle bacillus; his observations suggest that this lipase was an endo-ferment and that even minute amounts of culture medium destroy or inactivate it. This observation is not in harmony with the results presented above, for the lipase of the acid-fast bacteria described there was present and active in the culture medium, and it was thermostabile. It is possible, however, that the lipase, which is demonstrable in younger cultures of tubercle bacilli, may have disappeared, as the cultures were incubated for a longer period, without affecting the activity of a second thermolabile endo-lipase which is locked up within the bacteria themselves. The following observations were undertaken with a view of determining the relative amounts of lipase, respectively, in the culture medium and in the bodies of the bacteria grown in the culture medium. The cultures (various acid-fast bacilli) were of unequal ages, as indicated in Table 1. Various degrees of autolysis of the bacteria, therefore, must have taken place, depending on the strain of organism studied and the length of incubation.

N-methyl-N-nitroso-N′-nitroguanidine was used for the induction of two types of mutations in the PN strain ofMycobacterium phlei. Nineteen auxotrophic, 136 scotochromogenic and 50 achromogenic mutants were isolated after of treatment with nitrosoguanidine at a concentration of 1,000 μg/ml. Auxotrophic mutants required primarily amino acids and vitamins and half of them may be used for further genetic work due to their low frequency of spontaneous reversions. Colonies of scotochromogenic mutants were orange with the exception of one, which was strawberry red. Most scotochromogenic mutants were detected on a streptomycin containing medium. Roughly two thirds of the scotochromogenic mutants and one half of achromogenic mutants did not revert to the original photochromogenic character.

All the four types of acid-fast bacilli used in this investigation have proved virulent for the golden hamster. Of the four types the bovine is the most virulent for the species and, after subcutaneous inoculation, gives rise to severe generalized progressive tuberculosis, the lesions containing enormous numbers of tubercle bacilli. The human type is also highly virulent for the hamster and produces general progressive tuberculosis closely resembling that set up by bovine bacilli. Caseation of the lesions in the organs was, however, less extensive in the human than in the bovine infections but there was little difference in this respect between the glandular lesions of the two infections, and multiplication of bacilli was as profuse in one as in the other. Avian bacilli can readily multiply in the tissues of the hamster and cause death, but appear unable to give rise to macroscopic tuberculous lesions. Only one hamster was injected with avian bacilli and the result of this experiment is not in accord with those of Balfour-Jones already quoted. The vole bacillus is less virulent for the hamster than either the bovine or the human type of bacillus. Of the two hamsters injected with this type of bacillus one was killed 152 days later, its general condition then being good and its body showing plenty of subcutaneous fat. Nevertheless the organs were found to contain tuberculosis-like lesions, numerous in the spleen and liver, and the lymphatic glands generally were enlarged. The remaining hamster died 188 days after inoculation and showed generalized disease very similar to that in the last hamster. The glandular and visceral lesions of both hamsters contained very numerous acid-fast bacilli but, in striking contrast to those produced by bovine and human bacilli, showed no evidence of caseation.Acid-fast bacilli were present in the faeces of all the hamsters; they were numerous in those of hamsters 3, 4, 5 and 2 and relatively sparse in those of hamsters 1 and 6.

Clinical, post-mortem and histological findings are described for an African fish eagle (Haliaeetus vocifer), which was naturally infected with acid-fast organisms. In addition to this infection, there was an invasion of the air sacs and, to a lesser extent the lungs, by an Aspergillus sp. The predominant clinical sign was dyspnoea and there were acid-fast organisms in lesions in the liver and kidney in addition to the respiratory tract.

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A strain of Mycococcus luteus was cultivated in 5 per cent horse serum-saline for 52 weeks. After incubation for about 6 weeks, Gram-positive, but not acid-fast, intracellular granules developed in a few of the cocci. At about 14 weeks these cocci lysed and liberated granules, which had become strongly acid-fast. Between 22 and 42 weeks the free granules were replaced by strongly acid-fast, slightly bent, slender rods which could not be grown. Repeated subcultivation of the parent mycoccus strain on nutrient agar prevented the development of acid-fast rods in serum-saline.

‘Flue muature of thse acid-fast reaction luas been tlue sunhject of msuunueromus stmudies in thue l)a st, ansd sonsse excelleust re\’i(’w-s of tise literature exist(7, 14, 16) uuuakinsg a (‘onusl)r(’hsemssiv(’ re”s’iew mnuuns(’(’essarv Isere. ‘Fhme stua lies mel )t)!’t(’d ins thsis I ) l er \V(’!e unidertakems 5l)(’(’ifi(tibl ’ to (bet(’rnsin(’ tIutrole of h)hl(nol ins tlue r(’actioms aus(l tise cisensical status of the solutions of fmichusius ins thse acid-fast stains Pr(l)smmatiomssm. Ihs(’ muhtinssate ainsu is to defuse elueuusical I lifferent’es 1)(’tti(’efl mssvcobacteria and nsons-aci(ifast bacteria, if l)ossil)b(’, ansd Is) (l(’\elOl) “acidfastisess” fronus its unsatisfactory status as a stainuinug reactions insto a sensil)l(’ biochuensuical equation. lhi(’ use of h)hs(m50b ins the acid-fast stains is (lefinsit(’ly necessary, for stainiusg will not occur if it, or auuihiuseas t)rigiusallyPnol)osetl i ’ Ehrlich, is usot prt’seust. ‘Flue I)hsemsob nsust influensce tlue (13-c or thse smubstammee ‘,vhsich insparts tlse aci(l-fast character; and tlu(’ natmure of tisis influensce is thue

A large number of attempts have been made to cultivate the organism of leprosy, ever since Hansen in 1872 discovered the presence of acid-fast rods in the tissues of patients suffering from the disease. At the present time it is doubtful whether any one has succeeded in bringing forth unquestionable evidence as to the cultivation of the true etiologic agent. Many positive cultivation experiments have been reported in the literature. Some investigators have isolated acid-fast rods only, while others have cultivated pleomorphic organisms showing the presence of an acid-fast stage. From the descriptions given it is doubtful whether two workers have cultivated the same organism. The experimental work to be reported in this article deals with the isolation of a diphtheroid having an acid-fast as well as a nonacid-fast stage. No attempt will be made to cover the literature thoroughly, merely a brief review of the contributions having a bearing on this problem and of some of the more recent reports will be included.1

The actinomycetes are a group of fungi, usually classified as hypomycetes, probably bearing a relationship to the acid fast and diphtheroid groups of bacteria. The organisms are commonly found in the soil, and certain types may produce lesions in animals and man. In man the type most often isolated from lesions is Actinomycetes bovis with primary lesions about the head and neck in approximately half the cases studied. The infection in man is commonly called "lumpy jaw," characterized by a purulent discharge containing "sulfur granules" and the formation of sinuses and granulation tissue. At the periphery of the lobulated granules are radiating clubs, composed of mycelia slightly thicker at the tip, surrounded by sheaths which take the acid rather than basic dyes. The name "ray fungus" has been given to the actinomycetes because of the appearance of the granules, when examined under the microscope. The club forms are usually seen in tissues, although a few workers have reported formation of clubs in artificial media (1, 2). The granules when crushed and stained are seen to be made up of extremely fine, branching gram positive filaments. When first isolated, the organisms grow best under anaerobic or partially anaerobic conditions: but upon further cultivation most strains will grow aerobically on suitable media. The mode of infection of the disease is not exactly clear. It is more common among farmers and those who handle grains, straw, etc. For this reason it is thought that the organisms are carried on grains and other vegetable matter to the mouth. The infection in cattle and in man is believed to be caused by the same organism. Wright (3) suggested that Actinomycetes bovis was a normal inhabitant of the secretions of the buccal cavity and intestinal tract, both

SUMMARY: About three hundred strains of proactinomycetes were isolated from Rothamsted soils and examined together with certain strains from the National Collection of Type Cultures and other sources. Although little more than 9% were partially acid-fast on immediate isolation, subcultivation on rich media such as milk or nutrient glucose broth increased the percentage to 31%. The strains showed a range of features, from the soft mycobacterial type of growth with transient vegetative mycelium and very sparse aerial mycelium, if any, to the harder, more actinomycete-like variety. Of the acid-fast species Proactinomyces opacus, Proactinomyces salmoni-color, and Proactinomyces paraffinae predominated. In all, ninety-two strains were observed continuously for nearly two years on a variety of complex and simple media, and were stained at frequent intervals. Acid-fast cell elements occurred more often in complex protein- and fat-containing media, and in chemically defined media containing paraffin or large quantities of glycerol. They varied in shape and size according to the species and the age and quality of the growth. It is thought that differences of permeability of the cytoplasmic membrane in different environments may account for these variations. No evidence was obtained of any “resting spores” or “ chlamydospores” in the vegetative mycelium. Since there are no true spores in the aerial mycelium when it is present, the proactinomycetes in general may be regarded as asporogenous.

The recent advances in the study of the other bacteria with application to the dissociation phenomenon, have been applied in the study of acid-fast organisms. For some time, we have realized that the term "dissociation" as employed at present, is not adequate to explain the instability and subsequent variation which occur in cultures. But for uniformity of bacteriological nomenclature, we have adopted the term until a better one is coined. In describing the "R" and "S" colonies, we have had to depart somewhat from the general usage of these terms, that is the "R" meaning rough, and "S" smooth. The colonies of acid-fast organisms are relatively varied and complex. It seems better to employ the letter "R" to indicate greater resistance to environment and relative avirulence; and "S" to indicate colonies which are more sensitive to environment while possessing for certain species relatively great virulence. The terms "rough" and "smooth" apply directly only to avian tubercle bacillus, when cultivated on plain gentian-violet-egg medium. The avirulent colony isolated from this culture is flat and somewhat rough in appearance. The virulent is perfectly smooth, round and resembling a moth-ball. The physical properties are different. They have been fully described elsewhere. When the bovine "R" and "S" are cultivated on plain gentian-violet-egg medium, differentiation is very difficult. At times they are almost indistinguishable, but the addition of 0.25 per cent sodium taurocholate to the medium, alters completely the topography of the colony. The "S" appears in perfectly round smooth moth-balls, and the "R" in larger, spreading and somewhat rough colonies. Lacking suitable media, the human tubercle bacillus H37 has been more difficult to dissociate. After 2 years' study, using various media, we have been able to dissociate two types of colonies; but as the animal experiments are not yet completed, very little more than that can be said at present. We have dissociated two extreme types of colonies from four BCG cultures obtained from various sources. Each of these four cultures has revealed the same types of colonies. For details the reader is referred to a recent paper (10). In this publication we have included photographs taken from time to time in order to keep a record of our observations. When studying the photographs, the reader will notice considerable variation in some of the colonies. Unquestionably, there are more than two types of colonies developing during the life cycle of the organism, but at present we have considered and confined ourselves to only the two extreme types, one which can produce progressive disease, leading to the death of the animal, and the other which is but slightly virulent, and sometimes not at all so for susceptible animals. Full details of the technique employed by us have been described in the test. Anyone attempting to duplicate the work must strictly adhere to the technique described. Departures from it may lead to failure. The underlying factors favoring dissociation are not yet clearly understood. We believe that every single bacillus contains the two components, "R" and "S." If the environment is favorable for the development of the "R" component, the offspring will be "R's," although the original organism may be "S." Conversely, if the environment is favorable for the "S" and not for the "R" component the "S" will develop. For example, if an avirulent "R" colony obtained from the avian bacilli is cultivated on egg medium, which is favorable for the organism, the offspring after a suitable length of time will develop "S" colonies.

The cellular reactions to defatted tubercle bacilli are complex and like those to heat-killed whole tubercle bacilli. The firmly bound lipid, when removed from these organisms, is non-acid-fast; it contains an hydroxy acid which is acid-fast and a polysaccharide, which is not. This hydroxy acid gives rise to foreign body giant cells and the tissues eventually become infiltrated with eosinophiles. The polysaccharides, both from the defatted bacilli and from the unfilterable lipid, call neutrophiles from the blood stream. The reactions to the unfilterable lipid include those of both its constituents.

It has been shown that 158 patients, about one per cent of 15,180 patients, admitted during the past 10 years had demonstrable pulmonary lesions associated with non-photochromogenic mycobacteria in the sputum. At one time or another, 81 per cent of them had lived on a farm. The ratio of patients per 100,000 population ranged from 4 to 15 times higher in rural and small town areas than in the large, urban centers. The incidence of the disease was higher in the southern than in the northern part of the state. There was no history or evidence that the spouses of 151 married or widowed patients had ever had tuberculosis. Pretreatment susceptibility of these organisms to streptomycin showed that 58 patient strains of 81 examined were susceptible, 23 partially resistant and none resistant. The strains of 80 patients were examined for susceptibility to isoniazid and all of whom were resistant. Response to streptomycin plus PAS, INH plus PAS, or, to all three drugs given simultaneously, was poor. The sputum of about 70 per cent remained positive after many months and even years of treatment. Only 68 per cent were positive to OT or PPD. Of 51 tested with PPD-S and PPD-B, 54 per cent were positive to S, and 85 per cent were positive to B. Among 458 household contacts, 27 per cent were positive to OT or PPD. Among 296 household contacts tested with PPD-S and PPD-B, only 15 per cent were positive to S and only 35 per cent were positive to B. The percentage of positive reactors to B was twice as high in men as in women. The percentage of reactors of all contacts was only 6 per cent greater than that found among a group of 140 non-contacts. The incidence of positive reactors to PPD-B has been found to be greater in the Negro, in all groups tested, than in the white race. However, the incidence of disease in the Negro was found to be only 11.7 per cent of that found in the two races combined. Yet, of the 15,180 patients admitted, 41 per cent were Negroes. If a 6 mm. or greater induration to PPD-B is accepted as being specific, the incidence of infection is high. However, the incidence of known disease is rare. From the evidence at hand, we conclude that the source of infection may well be environmental, and that the organisms may be avirulent to man except under certain circumstances or predisposing conditions.