Liquid Shake Cultures Research Articles

Inhibition of the Growth of Peas by Tris-(2-Diethylaminoethyl)-Phosphate Trihydrochloride

The effects of Tris-(2-diethylaminoethyl)-phosphate trihydrochloride (SK&F 7997-A(3)) on the development of 4 varieties of Pisum sativum were investigated. The compound inhibited shoot elongation of all 4 varieties by as much as 50% or more when seeds were soaked in solutions of the inhibitor for 12 hours before planting. Seed treatment also affected flowering by causing an increase in the number of nodes to the first flower in the early varieties Little Marvel and Alaska. The number of nodes preceding the first flower in the late varieties Dwarf and Tall Telephone was not affected by high concentrations of SK&F 7997-A(3), but low concentrations appeared to cause a slight reduction in the number of nodes to flower.The inhibitor had little effect on growth when applied to established seedlings; some slight inhibition was noted when high doses were applied to the shoot tip.SK&F 7997-A(3) suppressed the growth response of dwarf and tall peas to exogenous GA(3). The compound did not inhibit biosynthesis of gibberellin by Fusarium moniliformc when present in shaken liquid cultures at concentrations as high as 10 mg/ml. The inhibitor suppressed the action of applied GA(3) on shoot elongation when the 2 chemicals were applied in 3 ways: 1) inhibitor on lowermost compound leaf and GA(3) on shoot tip; 2) GA(3) on lowermost leaf and inhibitor on shoot tip; and 3) soaking of seeds in the 2 compounds combined for 12 hours prior to planting. The third method of dual treatment yielded evidence that SK&F 7997-A(3) interacts noncompetitively with GA(3) in the regulation of shoot elongation.

THE ROLE OF THE EMULSIFIER IN THE FUNGITOXIC ACTION OF POLYBUTENE EMULSIONS.

Inhibition of growth of a wine yeast in shaken liquid culture by polybutene emulsions was shown to depend solely on the concentration and type of emulsifier used. The toxicity of various emulsifiers was examined, mainly at concentrations of 200–600 p.p.m. In general, the anionic alkyl aryl sulfonates were toxic, and the non-ionic alkyl aryl polyether alcohols were non-toxic. As the concentration was increased, the toxic materials were first fungistatic, then fungicidal. Two blended emulsifiers used in agricultural sprays gave very steep dosage–response curves.

THE EFFECT OF EXOGENOUS CATALASE ON THE AEROBIC GROWTH OF CLOSTRIDIA.

The results obtained by Holman (1955) indicating a marked protective effect of catalase on clostridia growing aerobically were shown to be due to impurities in the catalase preparation used. In aerobic liquid shake flask cultures, no stimulation of growth by crystalline catalase could be demonstrated, although withLactobacillus acidophilus andL. leichmannii a marked effect was observed.

DERMATOPHYTE VIABILITY AT MODESTLY RAISED TEMPERATURES.

In approximately three-fourths of 100 strains of dermatophytes tested, exposure for two weeks to even the modest heat of 39 C not only inhibited but was apparently lethal to inocula of the organisms on Sabouraud's dextrose agar. No strain survived similar exposure to 43 C. In most cases exposure to this latter temperature for 72 hours was sufficient to prevent growth of the inocula. In Sabouraud's liquid shake culture preparations inocula of 24 strains of dermatophytes failed to grow after exposures for one week to temperatures ranging from 39 to 43 C. In many cases exposure to 43 C for only 5½ hours completely inhibited established inocula in such preparations. Such 5½ hour daily exposures for four days proved lethal to all 12 strains of dermatophytes tested in this manner. The germination of spores ofMicrosporum gypseumwas completely inhibited by exposure to humid heating at 43 C for three days; spores ofTrichophyton mentagrophytesrequired ten days of such exposure for similar complete inhibition of germination. A technique is described for establishing infections withM. gypseumon the tails of rats. Such infections failed to develop if the tails were kept at 41 C for one week after inoculation. Similar infections established on rat tails for one week cleared after exposure for seven to ten days to 41 C. No spontaneous tendency to heal over a period of one month was observed in the case of controlM. gypseuminfections on rat tails kept at room temperature. The results of these studies might suggest clinical trial of prolonged wet heat in the treatment of stubborn dermatophytic infections. There are, however, hypothetical reasons for caution in such clinical trials.

RAPID QUANTITATIVE METHOD FOR MEASURING THE SENSITIVITY OF BLASTOMYCES DERMATITIDIS TO FUNGISTATIC AGENTS.

Guidry, D. J. (Louisiana State University School of Medicine, New Orleans) and Findlay Maier. Rapid quantitative method for measuring the sensitivity of Blastomyces dermatitidis to fungistatic agents. J. Bacteriol. 85:504-508. 1963.-Isolates of Blastomyces dermatitidis (yeast phase) were grown in continuous shake liquid culture. When sufficient cells were added to impart an initial optical density of 0.100 to the inoculated medium, growth was rapid, and the time required to assay fungistatic agents was reduced to 48 hr. Standard curves on light transmittance and direct cell count facilitated preparation of inoculum and measurement of growth. Generation time was determined for various growth phases of B. dermatitidis, and the data were applied to selection of the optimal period of growth for assaying fungistatic agents. Four isolates of B. dermatitidis were tested for their sensitivity to amphotericin and 2-hydroxystilbamidine. An amphotericin B concentration of 1.4 mug/ml was sufficient for maximal inhibition of all isolates tested; there were differences among isolates in sensitivity to lower concentrations of the drug. A 2-hydroxystilbamidine concentration of 62.5 mug/ml was sufficient for maximal inhibition of two isolates; the two remaining isolates required a concentration of 125 mug/ml. A latent period in inhibition of growth occurred when amphotericin B and 2-hydroxystilbamidine were added to cultures of B. dermatitidis. This latent period, together with generation time of the yeast cells, determined the maximal degree of inhibition which could be attained.

Isolation of antigenic fractions from Bordetella pertussis by means of lysozyme.

A CONSIDERABLE number of Gram-negative bacteria have been broken up in recent years by means of lysozyme1–4. The results of lysozyme treatment of Bordetella pertussis and the analysis of the resulting antigenic fractions form the subject of this communication. Suspensions (phase I), obtained from 24-h-old liquid shaken cultures, were used. After washing them with distilled water, we adjusted their germ count to 4 to 6 × 1010/ml. with reference to the International Standard for Opacity. We determined the conditions under which lysis would least impair antigenicity and found that our purpose would be best served by using a combined treatment with crystalline lysozyme (100 µg/ml.) and versene (200 µg/ml.) in a 0.03 M tris buffer of pH 8.0 under continuous stirring in a water bath of 37° C for 2–3 h.

Evaluation of a new method for the preparation of homogeneous mycelial suspensions.

Guidry, D. J. (Louisiana State University, New Orleans) and G. H. Trelles. Evaluation of a new method for the preparation of homogeneous mycelial suspensions. J. Bacteriol. 83:53-60. 1962.-An all-glass tissue homogenizer (providing both conical and cylindrical grinding surfaces) was employed in the preparation of homogeneous mycelial suspensions. Pellets of mycelium from constant shake liquid culture were submitted to grinding for various periods of time. The effect of grinding time on optical density of the homogenate, hyphal fragment size, and viability of the fungus was determined. A grinding time of 4 min was found to yield suspensions of mycelium in which 89% of the hyphal fragments contained four or less than four cells. Optical density was used as the basis for both preparation of standard samples of inoculum and measurement of growth. Preparation of a light transmittance curve facilitates the preparation of suspensions of any desired concentration. Although plate colony counts on samples from the same flask showed satisfactory agreement, there was appreciable variation in counts on samples from different flasks. The variation in growth from flasks receiving equal samples of inoculum was also determined. Results indicate that, at concentrations of mycelium equal to an optical density of 0.301, a difference of 15% or more in the optical density of two samples (10 flasks per sample) can be accepted as significant. Some of the factors influencing variation in viability of inoculum as well as variation in flask growth have been determined. Ways in which experimental errors can be kept to a minimum are discussed.