developed up to the twentieth day, when 38 per cent. was present, and at this point coagulation started in the form of a very fine precipitation of the casein, which was followed by a coarse granulation that later became streaked by the subsequent contraction of the mass. At the end of thirty-seven days a total of 48 per cent. of gas had collected. The final acidity of the fluid was only 2 per cent. acid in terms of a normal solution, and marks a rise of but 0.3 per cent. above the original reaction of the milk. It is supposed that the acidity alone was not responsible for the precipitation of the casein, but that some enzyme was probably present that facilitated coagulation. There was no peptonization of the casein, no indol or H2S formed either in milk or in sugar-free bouillon. The odors given off were of a sour and cheesy nature and of the nonputrefactive class. Acid albumin was present. Anaërobe J causes a heavy clouding of gelatin, but does not liquefy the medium. Blood serum was not liquefied. The bacillus stains well with aniline dyes but is negative with Gram's stain. Anaërobe J was isolated from two samples of milk, and has since been found in the rectal contents of young, milk-fed calves. It is nonpathogenic for guinea pigs.

Anaërobe K.

Anaërobe K is merely a variety of the type represented in anaërobe H, from which it differs in a few minor characters. The colonies on blood agar are hemolytic, biconvex in outline and are fringed by short radiating hairlike processes. The bacillus is motile and acts on sugars, producing 60 per cent. to 70 per cent. of gas in twenty-four hours, and 100 per cent. of gas is produced in milk in the same time. The relation of H to CO2 is as 3:1. The reactions of the sugar broths varied from 4 per cent. to 6.5 per cent. acid, and milk was raised to 4 per cent. acid at the end of the incubation period. Gelatin was not liquefied. Blood serum was not Indol was formed in The anaerobe

liquefied. There was no peptonization of the casein.
sugar-free broth in eleven days, but H2S was never found.
is Gram-positive. It is nonpathogenic for guinea pigs.

Anaërobe L. Anaërobe L is a motile bacillus, producing terminally placed ovoid spores. It grows on blood agar in the form of arborescent hemolytic colonies. It moves actively from place to place, in undulatory, rotatory or oscillatory paths, whether it occurs singly or in the form of chains. Chain formation is common and the length of the chain may reach 120u. The original surface colonies on solid media were extensively spreading arborescent growths, composed of a complex, matted network of threads or chains but with no branchings, and these chains were found to be encased in a sheath-like tubule of a cellulose structure. It was at first supposed that the enveloping sheath was a contaminating fungus, but neither anaërobic nor aerobic conditions were successful in developing anything of that nature. In later subcultures this unusual form disappeared, but was observed at another time when anaërobe L was isolated from another sample of milk. Bacilli without spores measure 2.1p to 5.9u long by 0.6μ to 0.74 broad. Spore formation was less rapid than with other bacilli. Bacilli with spores range from 2.7μ

1 When incubation is prolonged over four or five days it is necessary to watch the cotton plug carefully, and occasionally to replace the same with a dry sterile one, to prevent fungi from working into the culture.

to 4.6 long by 0.6u to 0.74u broad by 1.0up to 1.2p. broad at the spore, which measure 1.2 to 1.5p long by 0.9μ to 1.0μ broad. Free spores measure 1.2μ to 1.5 long by 0.9p. to 1.0p. broad. Anaërobe L acts energetically on dextrose, producing 95 per cent. of gas, composed of hydrogen or explosive mixture and carbon dioxide in the relation to each other as 4:7. The reactions of dextrose broth at the end of the period of incubation ranged from 4 to 4.5 per cent. acid. In lactose and saccharose bouillon only small quantities of gas were produced, 2 to 5 per cent., and the reaction at the end of the growth of the cultures ranged from 2.5 to 3 per cent. acid, showing that some fermentation had gone on. The odors given off were of an offensive and putrefactive character.

Milk in the fermentation tube was completely coagulated in about fortyeight hours, after which the coagulum was streaked by irregular lines of shrinkage, the margins of these lines of fracture furnishing a starting point for the process of peptonization, which begins on the third day, or twentyfour hours after the complete coagulation of the milk. The casein mass was almost completely dissolved, leaving a faintly clouded, yellowish fluid, giving a strong biuret reaction and a deep cherry-red color reaction with the addition of NaOH solution. This fluid contained acid albumin, H2S, acetic acid and some sugar. Tests for indol were negative. The reactions for the bulb and branch fluids from the fermentation tube were 7.2 per cent. and 7.55 per cent. acid respectively.

The colonies in gelatin were characteristic in that they assumed a thistle ball appearance because of the many radiating, thread-like processes starting from the original opaque colony. Liquefaction of the medium was slow but complete. Indol was produced only in sugar-free broth plus tissue after ten to twelve days' incubation. Blood serum was liquefied. Anaërobe L was nonpathogenic for guinea pigs. The anaërobe is Gram-positive.

Anaërobe M (Bacillus pseudo-tetani n. sp.). — Anaërobe M is a long, thin, actively motile bacillus, bearing in its vegetative condition terminally placed spores that are spherical in form. It grows on blood agar as hemolytic biconvex colonies. Bacilli without spores measure 2.7 to 3.0u long by 0.3u broad by 0.44 to 1.0u broad at the spores, that in turn measure 0.44 to 1.0μ in diameter. Free spores were of the same dimensions as the enclosed spores. The anaërobe acted on dextrose, lactose and saccharose, producing as high as 40 per cent. of gas in the first and 20 per cent. and 25 per cent. of gas in the last two sugars; the relation of H to CO2 in the gas from dextrose bouillon was as 17:1, from lactose broth, as 7:1, and from saccharose bouillon, as 10:1, all corresponding in the small proportion of CO2 and the large quantity of H present. The rise in acidity of the sugar broths varies from 0.2 per cent. to 0.4 per cent. acid in terms of a normal solution. The odors given off were sour and nonputrefactive in character. The growth in milk was unusual, for no coagulation occurred, even after thirty-six days' incubation at 36° C., during which time 7 per cent. of gas was produced and the acidity was raised from 1.7 per cent. to 1.9 per cent. There was no peptonization of the casein. There was no growth in nutrient gelatin and

blood serum was not liquefied. The anaërobe was Gram-negative. Indol was formed in sugar-free and in ordinary bouillon. The anaërobe is nonpathogenic for guinea pigs.

Anaërobe N.-Anaërobe N is an actively motile spore-bearing bacillus that grows on blood agar in the form of arborescent hemolytic colonies. Bacilli without spores measure 1.6μ to 4.7μ long by 0.6μ to 0.9μ broad. Bacilli with spores measure 1.9p to 3.3μ long by 0.6μ to 1.0μ broad at the position of the spore, that ranges from 1.5 to 1.8μ long by 0.74u to 0.9p broad. Free spores showed the same variation as the enclosed spores. Sugars were decomposed, but practically no gas was formed; the products of growth were lactic and acetic acids, and the reactions were as follows: in dextrose broth the reaction was raised from 1.1 per cent. acid to 2.25 per cent. in the bulb and 8.4 per cent. in the branch; in lactose broth, to 6.2 per cent. in the bulb and to 6.4 per cent. in the branch; in saccharose broth, to 2.25 per cent. acid for both bulb and branch. Milk was slowly changed by the microscopic precipitation of the casein during the first twenty-four hours, though the appearance of the bulk was the same as that of normal milk. After six days the coagulum became streaked by longitudinal lines of shrinkage. The reaction of the milk was raised to 5.7 per cent. acid. No gas was formed. Biuret tests were negative. H2S was absent. Butyric acid was present in small quantities. The odors given off from the cultures were offensive and putrefactive in character. The anaërobe is Gram-positive. Gelatin was liquefied. Blood serum was not liquefied. Indol and H2S were found in elevenday cultures grown in sugar-free broth plus tissue. Anaërobe N was found to be nonpathogenic for guinea pigs.

Anaërobe 0.- Anaërobe O is a long, broad, spore-bearing, club-shaped, motile bacillus that grows on blood agar in the form of biconvex hemolytic colonies. The motility is confined to the nonspore-bearing bacilli, which move in either direction in undulatory paths. Bacilli bearing spores were occasionally observed making spasmodic efforts to lunge forward, or were seen rotating slowly, without any material change in position. Spore development was rapid. Bacilli without spores measure 1.8u to 3.1u long by 0.6u broad. Bacilli with spores measure 3.0μ to 4.4u long by 0.44p to 0.6p broad by 0.9p. to 1.2 broad at the spores, which in turn measure 1.0p to 1.5μ long by 0.74u to 1.0 broad. Free spores measure 1.5 to 1.8u long by 1.0 to 1.2u broad. Sugars were attacked, but with practically no gas formation. The reactions of dextrose, lactose and saccharose bouillon in the branches of the fermentation tube were raised, respectively, to 3.7 per cent., 2.4 per cent. and 3.4 per cent. acid, and for the bulbs of the tubes to 3.3 per cent., 4.6 per cent. and 3.6 per cent. acid. Lactic and acetic acids were present. Milk was coagulated, streaked by lines of shrinkage, etc., in much the same way as the anaërobe just described, but there was formed 57 per cent. of gas, composed of H and CO2, in a relation to each other as 2:1. The reaction of the milk was raised to 5.2 per cent. acid. Biuret tests were negative. H2S and indol were found in sugar-free broth cultures after eleven days' growth at 36° C. The growth in gelatin was good, but there was no liquefaction of the medium. Blood

serum was not liquefied. The anaërobe is Gram-positive. Anaërobe O was nonpathogenic for guinea pigs.

Anaërobe P.- Anaërobe P is a motile, spore-bearing bacillus forming arborescent colonies on blood agar. The motility is confined chiefly to bacilli without spores, and these move sluggishly and in undulatory paths. Spore formation is slow. Bacilli without spores measure 2.1p. to 4.8μ long by 0.6μ to 0.9p. broad. Free spores measure 1.5p. long by 0.9μ to 1.2u broad. The anaërobe decomposed dextrose with ease, forming 88 per cent. of gas, composed of H and CO2 in a relation to each other as 1:4. Only 3 per cent. to 4 per cent. of gas was formed from lactose or saccharose broth. Dextrose broth gave a reaction of 3.83 per cent. acid at the end of the growth, and the two other sugars in bouillon gave 2.5 per cent. and 3 per cent. acid, respectively. A sour odor was given off from the sugar cultures. H2S was present. The effect on milk was the production of a very fine precipitation of the casein, without any gross indication of coagulation. About 31 per cent. of gas was produced. The odors given off were very offensive, and the intensity increased with the development and diminished as the growth stopped, indicating the formation of volatile intermediate chemical products. The reaction of the bulb fluid was 8.38 per cent. acid and that of the branch was 6.9 per cent. acid to phenolphthalein. H2S was present. Indol was absent. Acid albumin was present. Biuret tests were positive, as were color reactions with the addition of NaOH solution. Sugar was still present in the fluid. The cream in the bulb was emulsified, but that of the branch was unaffected. Indol and H2S were formed in sugar-free bouillon after eleven days' incubation. Blood serum and gelatin were liquefied. The anaërobe stains well with aniline dyes and is positive with Gram stain. The bacillus is nonpathogenic for guinea pigs.

Anaërobe Q.-Anaërobe Q morphologically resembles anaërobe H and like types, but is culturally quite different. The anaërobe grows on blood agar in the form of biconvex hemolytic colonies. It is a motile bacillus, going from place to place in undulatory paths. Spore formation is rapid. Bacilli without spores measure 2.5p to 5.0μ long by 0.9p to 1.0p. broad. Bacilli with spores range from 2.4μ to 4.4μ long by 0.6u to 1.0μ broad by 1.2μ to 1.3μ broad at the greatest diameter. The contained spores measure 1.3 to 2.1u long by 0.9 to 1.0μ broad. Free spores measure 1.5 to 1.8u. long by 0.9p to 1.0μ broad. Anaërobe Q acts on dextrose, but slightly on lactose and not at all on saccharose, so far as gas production is concerned. The reaction of the dextrose bouillon from the bulb of the fermentation tube at the end of the incubation was 3.73 per cent. acid, while the broth from bulb and branch of the lactose bouillon was 3 per cent. acid, and that of the saccharose bouillon was 2.5 per cent. acid in terms of a normal solution. Twenty-five per cent. of gas was formed from dextrose broth, and this was composed of H and CO2 in the relation to each other as 4:1. Milk was coagulated into the reticular type of coagulation in twenty-four hours. A total of 21 per cent. of gas was produced. The reaction of the available fluid at the end of the growth was 6.9 per cent. acid for the bulb and 6.3 per cent. acid

for the branch. Indol was absent. H2S was present in the branch fluids but not in the bulbs of the fermentation tubes. Acid albumin was present. Sour odors were given off from the sugar cultures and offensive putrefactive odors were noticeable in milk and tissue tube cultures. Beyond an emulsification of the cream in the bulb it was unaffected by the bacillus. Biuret tests were positive, as were the NaOH color reactions. Sugar was still present at the completion of the growth in milk. Indol and H2S were found in eleven-day-old cultures in sugar-free bouillon. Blood serum and gelatin were liquefied. The anaërobe stains well with aniline dyes and with Gram's stains. Anaërobe Q was nonpathogenic for guinea pigs.

OTHER INVESTIGATIONS ON THE RELATION OF ANAEROBES TO MILK. In the discussion of the literature I have selected those papers having a general bearing on the above report, and also those of a biological character that seem to have been the result of work with pure cultures of anaerobes. Much work, chiefly from a chemical standpoint, has been done with mixed cultures, and the data tell us but little of the specific action of different bacilli.

Toch, Hofmeister, Dogiel and others have shown that normal milk contains no peptones, and it follows that in cases where delicate reactions were obtained by the application of the biuret test the results indicated slight decomposition of the proteids present. Control tests made subsequent to the sterilization of the milk were invariably negative to the biuret tests in all the milk media used in the experiments described in this paper.

The action of bacteria on milk has been studied by chemists to a considerable extent, though they have in the majority of cases probably worked with mixed cultures; Duclaux 10 in 1900 reported on the influence of micro-organisms on milk inoculated with aërobes and tyrothrix tenuis and found that the former increased the soluble casein from 0.4 to 1.89 per cent., and the latter rendered in one case 2.57 per cent. of the total 3.9 per cent. of the casein soluble, and at another time dissolved the whole of the casein. Much research has been directed towards the putrefaction of proteids by bacteria that act in mixed rather than in pure cultures. Emmerling 11 in 1896 and 1897 used sugar-free wheat bran, and found putrefactive changes that he decided were produced by proteus vulgaris, and later he found that staphylococcus and streptococcus pyogenes were capable of decomposing egg albumin and blood fibrin. These results were later contradicted by Buchner, Leber, Frankel, Brieger and Lambert, and Rettger believes that Emmerling was unknowingly working with anaërobes. Rettger 12, 13 worked with well-known pathogenic and nonpathogenic anaërobes, and he holds that strict putrefaction.