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The intravenous injection of horse serum, kept for 2 hours at 38°C. in the presence of one-fifth of its volume of a suspension of 0.5 per cent agar in physiological salt solution and then separated from the agar by centrifugalization and filtration, produces in normal rabbits in adequate doses a considerable and prolonged fall in the blood pressure, expulsion of feces, a diminished coagulability in the carotid blood, and at times accelerated respiration; that is, the various symptoms observed after the intravenous injection of horse serum in a seroanaphylactized rabbit. Horse serum previously kept for 30 minutes at 56°C. and then treated with agar in the manner described above, when injected intravenously into a normal rabbit will have no more effect on the arterial pressure, on the intestinal movement, on the respiratory rate, or the coagulation of arterial blood than the introduction of horse serum into the veins of a normal rabbit.

AREVIEW of the literature has failed to reveal any reports recommending bismuth sulfite agar for the isolation of the members of the Shigella group. It is generally recognized that no significant growth of these organisms occurs on this medium.' The Difco Manual, 1943, states that generally members of the dysentery groulp are inhibited, although some strains of Flexner and Sonne do develop. Some investigators indicate that they are completely inhibited. Sellers 2 regards this as one of the disadvantages of bismuth sulfite agar. In view of these facts it seems feasible to report our -observations. For the past 3 years bismuth sulfite agar, Difco (W. B.) and S. S. agar have been used for direct plating of all feces cultures in the Florida State Board of Health Laboratories. During the first 23/2 years we found an occasional culture of Shigella alkalescens on W. B. agar, but the number was so small it seemed of no significance. In December, 1944, a definite increase in the number of alkalescena isolations was noticed, the majority of which were found on W. B. agar, while the S. S. plates were negative. At this time pure cultures were plated on W. B. agar and the colonies closely observed. We found that they were quite varied in appearance, ranging from very pale green to a dark, brownish green, smooth and glistening. [273 Some are raised, ameboid in shape, and mucoid in appearance, while others are flat and round. Colonies with dark green center and lighter green periphery are also formed. The predominating type found almost consistently from directly plated feces cultures is a medium green colony, smooth, glistening, slightly raised, and frequently ameboid in shape.

Protein spots in agar gel of identical protein content but different in surface area are found to bind different amounts of dye upon staining with Amido Black B. The lower the protein concentration within the agar gel, the more the Amido Black B content of the spot falls short of the value expected from the amount of protein present, as measured by means of its ultraviolet-light absorption. If, however, pure agarose is used instead of agar, the relationship between the protein and dye contents of spots is linear down to protein concentrations of 0.05 μg per mm2 spot surface.

Agar has been shown to interfere with the activity of some antibiotics against Staphylococcus aureus . This interference has been observed as an increase in the minimal inhibitory concentration and in the diameter of the zone of inhibition. Purifying the agar with water extractions substantially reduced this adverse effect.

The in vitro activity of minocycline against 1,028 bacterial strains was determined in parallel in Mueller Hinton Agar and Trypticase Soy Agar. The broad antibacterial effect of minocycline against gram-positive cocci and gram-negative bacilli is confirmed. Minimal inhibitory concentrations for gram-positive bacteria in Mueller Hinton Agar were at least twofold less than in Trypticase Soy Agar. Minimal inhibitory concentrations for gram-negative bacilli in Mueller Hinton Agar were usually fourfold less than in Trypticase Soy Agar.

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated ( Desulfovibrio ) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.

The indication of H 2 S production by a modified Tergitol-7 agar combines the advantages of both selective and differential enteric media to provide a medium with broader coverage.

An alternative method to defibrinating sheep blood for use in bacteriological media is described. The new procedure incorporates sodium polyanethol sulfonate in a concentration of 0.05% (vol/vol). In testing 117 bacterial and fungal isolates, no significant differences were found with respect to adequate growth, pigment production, hemolytic reactions, and other physical attributes. Further tests demonstrate that the sodium polyanethol sulfonate in sheep blood agar plates does not cause any aberrations in zone sizes around disks used in antibiotic susceptibility tests. Consequently, the method represents a suitable alternative to the use of defibrinated sheep blood in the preparation of bacteriological media.

Microbiological assay of individual antibiotics in mixtures of antibiotics depends on the use of selective inactivation and/or of test bacteria with differential susceptibility. Controlled experiments revealed that streptomycin in concentrations of 20 and 40 μg/ml did not influence a disk diffusion assay of penicillin with Sarcina lutea (ATCC 9341) as the test organism. In the case of penicillin concentrations less than or equal to 1 IU/ml, addition of 80 μg of streptomycin per ml influenced the penicillin assay significantly. Clinical use of streptomycin resulting in levels above 40 μg/ml usually did not occur; therefore penicillin could be assayed as though streptomycin were not present. We observed additionally that S. lutea was unable to grow on agar plates prepared with semicarbazide hydrochloride.

The in vitro activity of five antibiotics against 368 strains of Escherichia coli was determined by use of Mueller-Hinton agar and Oxoid sensitivity test medium. Minimal inhibiting concentrations were essentially identical with both media.

l -Alanine-germinated spores of Bacillus subtilis developed a competence on agar plates after 10 h of incubation. Addition of marker amino acid to the plates was required for the transformation.

The solid agar overlay procedure used for viral plaquing was adapted to the study of Pseudomonas aeruginosa plaques in HeLa monolayers. After adsorption of the organism to the HeLa monolayers, an overlay consisting of 10% newborn calf serum (NBCS), 90% Eagles basal medium (EBM), and 1.5% agar containing neutral red was added. Plaques developed after 24 hr of incubation at 37 C which were indistinguishable from viral lesions. Optimal conditions for plaques included: serum in the adsorption fluid as well as the overlay medium; 1-hr adsorption time; healthy HeLa monolayers; and the presence of EBM. The formation of plaques instead of colonies in agar was due to the inhibitory effect of NBCS or EBM. By omitting both of these components from the overlay, colonies appeared in place of plaques. Adult bovine serum acted in a similar bacterial colony-suppressing fashion, but fetal or agamma calf serum did not. Furthermore, NBCS or adult bovine serum when incorporated in the overlay medium instead of fetal or agamma calf serum displayed a plaque-suppressing effect by reducing the number and size of plaques formed. The findings lend further evidence to support the hypothesis that P. aeruginosa may produce plaques from a protected intracellular site within HeLa cells.

A total of 232 isolates of Enterobacteriaceae and 19 Pseudomonas species were tested on bile-esculin-agar. Only the Klebsiella-Enterobacter-Serratia group produced blackening of the medium.

Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color.

Smith , P. B. (Communicable Disease Center, Atlanta, Ga.). Growth of staphylococci on mercuric chloride agar. J. Bacteriol. 84: 1016–1019. 1962.—An attempt has been made to corroborate a report that “epidemic” strains of staphylococci are more resistant to mercuric salts than are “nonepidemic” strains. A comparison of mercury resistance with coagulase production and phage type or pattern of 493 staphylococcal strains revealed that nearly half of the coagulase-negative strains tested were mercury-resistant, whereas many “epidemic” strains of certain phage types were sensitive to mercuric salts. Results obtained are compared with those from a previous investigation. The mercury resistance of some strains was found to vary significantly depending on the basal medium used; this could not be correlated with mercapto-group content of the medium. The data indicate that a medium containing mercuric chloride would have little practical value at the present time for the average laboratory engaged in isolating staphylococci.

An improved method for extraction of plant DNA is described. Quantitative species comparisons based on DNA-DNA hybridization are reported for several members of the family Leguminosae and for barley, wheat and rye. A maximum of about 10% homology in DNA polynucleotide sequences is found between monocotyledons and dicotyledons tested, whereas 20 to 90% homology is observed within a family. Species compared using a DNA fraction enriched for redundant polynucleotide sequences generally appear to be more closely related than when whole DNA is used. DNA-DNA hybridization may be useful in systematic and evolutionary study of plants, and also as a possible screening procedure for interfertility of species.

É proposta uma modificação da fórmula do agar fenil-alanina, com a finalidade de simplificar a prova da desaminação dêste aminoácido, que consiste na adição de citrato de ferro amoniacal ao meio.A modification of the phenylalanine agar medium is proposed. The objective is the simplification of the deamination test. The modification consist in adding ferric ammonium citrate to the medium.

1 - Anaerobic bacteria of the Clostridium genus acidify mineral media without when agar is added. 2 - Acidulation results from the attack on the agar as a source of carbon. 3 - The quantity of CO² produced by the decomposition of the agar is approximately that obtained with soil bacteria as shown by Waksmann and Diehm working with hemicelluloses. Although galactone is less atacked than the other hemicelluloses the acidity produced is sufficient to disturb the fermentation tests in semi-solid media with agar. 4 - The acidulation of Spray's sugar-free control medium is probably due to the decomposition of the agar by anaerobes. The acidity produced may interfere with the acidity of the fermentation of the sugar in Spray's test or may be added to it, thus giving a false indication of the real acidity.

Experiências foram realizadas com bactérias dos gêneros Listeria e Erysipelothrix, em meios líquido e sólido, utilizando o clorêto de 2, 3, 5 - trifeniltetrazólio. A atividade enzimática redutora das listérias para o TTC foi diferente da do E. rhusiopathiae, principalmente em meio líquido e nas horas iniciais de observação. Preparações feitas para microscopia ótica e electrônica dos germes tratados com TTC revelaram a presença de granulações polares, bipolares e centrais dentro do corpo das listérias. A evidenciação de granulações coradas de formazana, intracelulares nas listérias, confirma estudos anteriores quanto à possibilidade da existência de mitocôndrias nas bactérias.Based on a similarity of cultural and biochemical properties, WILSON and MILLES (6) compared the microorganisms of listeriosis with those of swine erysipelas including them in the genus Erysipelothrix with the species E. monocytogenes and E. rhusiopathiae. However, BARBER (1) and JULIANELLE (4), in a comparative study of both microorganisms, do not admit that possibility. Observing the morphological and citochemical characteristics of L. monocytogenes (N.º 7 973, 5 348, 5 105, 5 214, of the Seeliger collection) and E. ehusiopathiae cultures (N.º 1, 7, 11, 27, 37, of the WIX collection), both in phase "S", we were able to verify their comparative behavior when in contact with 2-3-5-triphenyltetrazolium chloride (TTC), "Synthetical Laboratories, Chicago, U.S.A". 1. To 1,0 ml of cultures of both microorganisms grown in plain broth plus 5% horse serum for 24 hours at 37ºC, 0.1 ml of sterilized 1% aqueous solution of TTC was added. The tubes were left at room temperature (± 25ºC) or in the incubator (37ºC). In the tubes with L. monocytogenes immediately appeared a red coloration (fig. 1) characteristic of formazan production, that became gradually more pronounced, from bottom to top (figs. 2, 3, 4, 5); at the and of 24 hours the color was intense the tube. In the tubes with E. rhusiopathiae (figs. 1, 2, 3, 4), only a faint pink coloration was seen after 2 hours; at the end of 24 hours, the intensity of color was not equal to that observed in tubes with L. monocytogenes. 2. Petri dishes with 15 ml of plain agar with 5% horse serum, containing 0.1 ml of 1% sterilized aqueous solution of TTC, were inoculated with L. monocytogenes and E. rhusiopathiae, and incubated for 24 hours at 37ºC. Colonies of L. monocytogenes that developed presented a red color, indicative of formazan, as previously described by GRAY et al. (3), while the E. rhusiopathiae colonies did not show any coloration. 3. Disks of filter paper, previously soaked with TTC and dried were placed over the growth of L. monocytogenes in Petri dishes with plain agar plus 5% horse serum form 24 hours. No change in the color of the colonies was observed. The same negative result was obtained when the porous clay cylinder technique for measuring antibiotic activity was employed, With E. rhusiopathiae also negative results were obtained with the disk technique. 4. Wet preparations of the cultures after 1 hour of contact with TTC in the same conditions of item 1, were observed at 900X magnification. L. monocytogenes presented normal shape and dimensions, but one to three intracellular polar, bipolar and or central, red-purple granules were noted. E. rhusiopathiae bacilli were morphologically normal…

The experiments reported above confirm the fact that lytic principle is distributed in active solution in a state of indivisible units. This permits its quantitative evaluation by serial dilution, as well as by plating on agar. The latter method, however, often gives readings considerably lower than those obtained by the broth dilution method of titration. By varying the concentration of agar it has been possible to show that the discrepancy is due to adsorption of the lytic agent on agar. When the concentration of the latter is increased from 0.3 per cent to 2.5 per cent the number of plaques of lysis is reduced more than 100 times. At the same time the average size of the plaques also decreases approximately to one-tenth of the original. The size, as well as the number of plaques, has been found to depend also on the condition of the culture employed in titration. Thus, when the culture exposed to the action of lytic agent is composed of young susceptible bacteria, the greater the concentration of bacteria, the smaller the plaques. When the culture is composed partly of young and partly of old susceptible bacteria, both the size and the number of the plaques are diminished with the increase in the relative concentration of old bacteria. On the other hand, presence in the culture of resistant bacteria does not affect either the size or the number of the plaques so long as the relative concentration of susceptible bacteria in the culture is sufficient to allow formation of them. The plaques appearing in the presence of a high concentration of resistant variants in the culture are relatively indistinct owing to overgrowth. Under carefully controlled conditions the size of plaques is found to be determined by the character of the lytic filtrate. Thus in the case of lytic agents which act upon more than one bacterial species the size of the plaques remains constant, irrespective of the bacterial substratum used for the production of the active filtrate.

Heterogeneity of the group-specific (Gc) components in normal human serum has been demonstrated by the use of a lithium borate buffer system in conventional vertical starch gel electrophoresis and by prolonged immunoelectrophoresis in agar gel. In both Gc 1-1 and Gc 2-2 phenotypes a protein component migrates ahead of the main band. Immunological evidence indicates that the faster migrating band contains Gc specificity. The possibility that the two electrophoretically distinct Gc components share a common polypeptide chain is discussed.

Cell multiplication and phage formation of lysogenic B. megatherium cultures have been determined under various conditions and in various culture media. 1. In general, the more rapid the growth of the culture, the more phage is produced. No conditions or culture media could be found which resulted in phage production without cell growth. 2. Cultures which produce phage grow normally, provided they are shaken. If they are allowed to stand, those which are producing phage undergo lysis. Less phage is produced by these cultures than by the ones which continue to grow. 3. Cells plated from such phage-producing cultures in liquid yeast extract medium grow normally on veal infusion broth agar or tryptose phosphate broth agar, which does not support phage formation, but will not grow on yeast extract agar. 4. Any amino acid except glycine, tyrosine, valine, leucine, and lysine can serve as a nitrogen source. Aspartic acid gives the most rapid cell growth. 5. The ribose nucleic acid content is higher in those cells which produce phage. 6. The organism requires higher concentrations of Mg, Ca, Sr, or Mn to produce phage than for growth. 7. The lysogenic culture can be grown indefinitely in media containing high phosphate concentrations. No phage is produced under these conditions, but the cells produce phage again in a short time after the addition of Mg. The potential ability to produce phage, therefore, is transmitted through cell division. 8. Colonies developed from spores which have been heated to 100°C. for 5 minutes produce phage and hence, infected cells must divide. 9. No phage can be detected after lysis of the cells by lysozyme.

Media for isolation of Haemophilus sp. from the conjuctiva were compared in an oasis in southern Tunisia where severe trachoma and seasonal epidemic purulent conjunctivitis are common. Of 89 children tested, IsoVitaleX-supplemented chocolate agar yielded Haemophilus in 87%, plain chocolate agar in 75%, sheep blood agar with a stab of Staphylococcus epidermidis in 74%, and Fildes medium in 58%. Since other microbial pathogens are easily identified in the modified blood agar, it was the most useful single medium.

Bacterial strains: The mutant strains were derived from wild-type S. typhimurium LT2, using the mutagen N-methyl-N-nitro-N-nitrosoguanidine and the procedure described by ADELBERG, MANDEL and CHEN (1965). The strains sensitive to valine were detected by replica plating from nutrient agar master plates onto minimal medium (ARMSTRONG and WAGNER 1964) containing 4 pg L-valine per ml. The abbreviation VS (valine sensitive) was arbitrarily selected to designate those strains whose growth is inhibited by the supplementation of minimal medium with valine. Another characteristic shared by the VS strains is a slow rate of growth on minimal medium. Of the 15 strains isolated, four (VS-7, 9, 10, and 13) were selected for the analyses presented in this report. Other S. typhimurium strains utilized in this study include: LT2; ilvAil8 (threonine dehydratase deficient) ; iluC8, 42, and 66 (reductoisomerase deficient) ; and ilvD6, 18, and 47 (dihydroxyacid dehydratase deficient) (ARMSTRONG and WAGNER 1964; ELLIOTT and ARMSTRONG 1968). Cotransduction tests: The procedures utilized can be found in ARMSTRONG and WAGNER (1964). For these studies four VS and two iluC strains were used as donors. In crosses with the VS strains, transduction mixtures wcre plated on minimal medium and incubated for a total of 48 hr (fist 24 hr at 37°C followed by 24 hr at room temperature). Donor recombinants were identified by their small colony size. To avoid feeding of the donors by the background growth in crosses with iluA and C strains, phage and cells were mixed in 2.5 ml of molten minimal-medium agar (0.75%) and spread as an overlay on minimal medium plates. This procedure allowed for a more accurate determination of donor recombinants. In crosses with iIvC42 and 66 as donors, use was made of partial revertants of these strains that can grow suboptimally on a valine supplement. Transduction mixtures were spread on minimal medium plates supplemented with 4 pg of L-valine per ml. After 4 days of incubation at 37"C, the wild-type and donor recombinants were easily identified by the difference in colony size. Growth studies: Duplicate assay tubes, each containing 3 ml of medium, were used. The supplementations to minimal medium are listed in Tables 3 and 4. Inocula were prepared from nutrient-broth cultures grown overnight at 37°C on a rotary shaker. These cultures were centri

Hart, Lewis T. (Louisiana State University, Baton Rouge), A. D. Larson, and C. S. McCleskey . Denitrification by Cornyebacterium nephridii . J. Bacteriol. 89: 1104–1108. 1965.— Corynebacterium nephridii was found to reduce nitrate (contrary to the original description) at a rapid rate. In the conventional 0.1% nitrate broth, neither nitrite nor nitrate was detected after 24 hr. There was no assimilation of nitrate nitrogen, and the final product of nitrate reduction was nitrous oxide. Manometric studies and growth experiments indicated that the organism is incapable of reducing nitrous oxide. C. nephridii is gram-negative, grows on bile salts (5%) agar, EMB Agar, and MacConkey Agar. It was proposed that this species be transferrrd to the genus Achromobacter and designated Achromobacter nephridii (Büsing, Döll, and Freytag) comb. nov.

Routine disc diffusion susceptibility tests (Bauer-Kirby technique), employing 5% sheep blood-Mueller-Hinton agar and 10-μg gentamicin sulfate discs, disclosed that a significant number of clinical enterococcal isolates were sensitive to the antibiotic, as also revealed by the agar dilution technique. With few exceptions, the isolates proved resistant to this antibiotic when tested for susceptibility in Brain Heart Infusion and Trypticase soy broth or agar. The addition of 5% sheep blood to Trypticase soy and Brain Heart Infusion agars resulted in markedly enhanced activity of the antibiotic, indicating medium-dependent activity of gentamicin against enterococci. Human serum and urine failed to support optimal growth of enterococci. Thus, it was not possible to correlate the activity of gentamicin in any of the media examined with that in serum or urine.

Ford, Denys K. (University of British Columbia, Vancouver, Canada). Culture of human genital “T-strain” pleuropneumonia-like organisms. J. Bacteriol. 84: 1028–1034. 1962.—The conditions under which “T-strain” pleuropneumonia-like organisms, as described by Shepard, are best cultured were investigated. The organisms were found to grow on several types of nutrient agar and broth, of which PPLO medium supplemented with yeast extract and horse serum was the simplest. Subculture was possible through broth cultures, provided the broths were not incubated longer than 16 hr. The organisms on agar required either Fortner's anaerobic atmosphere or 10% CO 2 , but broth cultures grew aerobically. “T-strains” grew over a pH range of 6.8 to 7.8, and a temperature range of 30 to 36 C. They were viable after storage for 16 days at 4 C and for 90 days at −20 C, and they resisted lyophilization. They were sensitive to 1.5 μg per ml of tetracycline and streptomycin, but were resistant to ampicillin and penicillin. Quantitative studies showed maximal concentration in broth of 10 6 to 10 7 organisms per ml, and logarithmic multiplication for the first 12 hr of broth culture, with a subsequent rapid decline in number. Colonial morphology was maintained after numerous subcultures.

Two commercially available media (nutrient agar and trypticase soy broth plus agar) induced sporulation of several yeast strains that sporulate with difficulty. In a comparison with the highly successful potassium acetate medium, the majority of 16 strains of Saccharomyces also sporulated. The two media appear useful because of their ease of preparation.

A two-layer plate consisting of a lower indicator layer of milk-agar and an upper nutrient agar layer was developed for isolating proteolytic marine bacteria.

The presence of oxytetracycline-resistant bacteria was investigated with commercially frozen chicken thighs and drumsticks. Bacterial flora were surveyed by means of total and coliform counts with Tryptone Glucose Extract Agar and Desoxycholate Agar, respectively. After counting, the Desoxycholate Agar plates were replicated on the same medium containing 25, 50, 75, and 100 ppm of oxytetracycline. Resistant colonies were found on all samples that were replicated. Of 2613 colonies isolated on Desoxycholate Agar, 47.8% grew in the presence of 25 ppm of oxytetracycline. From 50 to 100 ppm, the number of resistant isolates remained essentially the same, near 34%. Of 812 colonies of antibiotic-resistant bacteria identified with dulcitol-lactose-iron-agar, 82.5% were paracolons, 13.7% were pseudomonads, and 3.8% were Escherichia or Aerobacter . Bacteria resistant to oxytetracycline were shown to be present on commercially processed chicken. The origin of the resistance to oxytetracycline was not established; however, since the antibiotic was not used during processing, it appeared that these antibiotic-resistant bacteria arose in the intestines of the chickens as a result of feed which contained antibiotic. This is supported by a comparison with the antibiotic resistance of coliforms from chickens raised on feed both with and without oxytetracycline, for the percentages of resistant colonies are similar in both commercial chicken and chicken raised on feed containing the antibiotic.

1. Cooper's gelatin purified according to Northrop and Kunitz exhibited a minimum of osmotic pressure and a maximum of opacity at pH 5.05 ±0.05. The pH of solutions of this gelatin in water was also close to this value. It is inferred that such gelatin is isoelectric at this pH and not at pH 4.70. 2. Hydrogen electrode measurements with KCl-agar junctions were made with concentrated solutions of this gelatin in HCl up to 0.1 M. The combination curve calculated from these data is quite exactly horizontal between pH 2 and 1, indicating that 1 gm. of this gelatin can combine with a maximum of 9.35 x 10–4 equivalents of H+. 3. Conductivity titrations of this gelatin with HCl gave an endpoint at 9.41 (±0.05) x 10–4 equivalents of HCl per gram gelatin. 4. E.M.F. measurements of the cell without liquid junction, Ag, AgCl, HCl + gelatin, H2, lead to the conclusion that this gelatin in 0.1 M HCl combines with a maximum of 9.4 x 10–4 equivalents of H+ and 1.7 x 10–4 equivalents of Cl- per gram gelatin.

Eosin, if present in cultures containing tetanus spores, prevents the germination of these spores when its concentration (in glucose bouillon) reaches 0.2 per cent. When the concentration of the eosin sinks to 0.01 per cent., germination of the spores is no longer inhibited, but the vegetative bacilli developed from the spores execute a highly restrained form of multiplication. When the eosin concentration sinks to 0.001 per cent., vegetation and multiplication of the bacilli become more active, but no new spores are formed even after long periods of time. With glucose agar it is not until the concentration of the eosin in the cultures falls to .05 per cent. that sporulation again appears. At this concentration of the eosin, very few spores are formed ; but as the eosin sinks lower and lower, sporulation becomes more active, until with 0.001 per cent. it is essentially of normal degree. In concentrations of 0.003 per cent., eosin prevents perfect segmentation of the multiplying bacilli, with the result that, finally, long and convoluted threads of bacilli are produced. The spores which are formed in a medium containing 0.01 per cent. of eosin are often situated at the centre and not at one pole of the bacilli. Eosin in a strength of 2 per cent. is capable of destroying the vegetative bacilli, if the contact is prolonged to fifteen minutes, and in strength of 0.1 per cent., in twenty-four hours. Placing this latter mixture of bacilli and eosin in the sunlight greatly hastens the bactericidal effect, and the bacilli are found to be incapable of growth at the end of several hours. Eosin in high concentrations is not capable of killing the tetanus spores, even after long exposure to sunlight (thirty hours). The toxin production of tetanus bacilli grown in eosinized culture media diminishes as the concentration of the eosin increases. This effect is brought about partly by the restraining action of the dye on vegetation, and partly by its detoxicating action upon the poison., The toxin-producing power and the virulence of tetanus bacilli are not permanently modified by contact with eosin for a long period, or by successive cultivations in eosinized media. Eosin is likewise capable of restraining the vegetation of tetanus spores in the animal body. In spore threads inserted beneath the skin of rats, and surrounded with eosin in solution, a very restricted vegetation takes place. If the injections of eosin are repeated. vegetation soon ceases and the vegetated bacilli degenerate and disappear. The ungerminated tetanus spores remain alive in a latent condition indefinitely in the healed wound beneath the skin. These spores do not lose power to grow outside the body, or inside the body of animals under favorable conditions, or to produce toxin in a characteristic manner.