Showing papers in "Applied and Environmental Microbiology in 1971"
TL;DR: Bacteriocin activity can be detected and assayed by a modification of the punchhole method.
Abstract: Bacteriocin activity can be detected and assayed by a modification of the punchhole method.
696 citations
TL;DR: Three, convenient agar-diffusion methods have been developed that enable detection of the nuclease of Staphylococcus aureus at concentrations as low as 0.005 mug/ml in agar and broth cultures.
Abstract: Based on the metachromatic property of Toluidine Blue O, three, convenient agar-diffusion methods have been developed that enable detection of the nuclease of Staphylococcus aureus at concentrations as low as 0.005 mug/ml in agar and broth cultures. The interactions of agar and deoxyribonucleic acid with Toluidine Blue O are discussed.
362 citations
TL;DR: The semi-micro method for interferon assay was based on quantitation of inhibition of cytopathic effects in 6-mm wells and was more sensitive than the plaque-inhibition method and was extremely economical.
Abstract: Mostmethods ofinterferon assay, suchas plaque inhibition andyield reduction, areexpensive interms oftimeandmaterials. Lesscomplex methods, suchasinhibition ofcytopathic effects (CPE), tendtobesubjective anddifficult to quantitate. Finter (1)hasdescribed howcytopathology maybequantitated bytheuptake and elution ofavital dyeandhasapplied this technique totheassay ofinterferon. A micro-method fortitration ofhumanandchick interferon was described byTiles andFinland (4)whobased interferon activity on inhibition ofCPE,as observed inthemicroscope oron metabolic inhibition. This paper describes theassay ofrabbit interferon byasemi-micro, dye-binding method whichisextremely economical, especially in respect tocell culture materials andinterferon samples.
303 citations
TL;DR: L. monocytogenes was isolated from vegetation or soil taken from 11 of the 12 farms and from 6 of the 7 nonagricultural sites and a total of 27 strains were isolated from the 19 sites.
Abstract: Samples from 12 farms were examined during two successive spring and early autumn seasons. L. monocytogenes was isolated from vegetation or soil taken from 11 of the 12 farms and from 6 of the 7 nonagricultural sites. A total of 27 strains were isolated from the 19 sites. The organism was not isolated from any of the autumn collections.
280 citations
TL;DR: A medium was developed which permitted isolation, apparently for the first time, of the bacteria responsible for the acid production in the 100-year-old San Francisco sour dough French bread process, raising a question as to whether they should be properly grouped with the heterofermentative lactobacilli.
Abstract: A medium was developed which permitted isolation, apparently for the first time, of the bacteria responsible for the acid production in the 100-year-old San Francisco sour dough French bread process. Some of the essential ingredients of this medium included a specific requirement for maltose at a high level, Tween 80, freshly prepared yeast extractives, and an initial pH of not over 6.0. The bacteria were gram-positive, nonmotile, catalase-negative, short to medium slender rods, indifferent to oxygen, and producers of lactic and acetic acids with the latter varying from 3 to 26% of the total. Carbon dioxide was also produced. Their requirement for maltose for rapid and heavy growth and a proclivity for forming involuted, filamentous, and pleomorphic forms raises a question as to whether they should be properly grouped with the heterofermentative lactobacilli.
240 citations
TL;DR: Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B and B, kojic acid, zearalenone, T-2 toxin, or ochratoxin A.
Abstract: Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.
232 citations
TL;DR: A mutant strain that secretes twice as much cellulase as its parent was obtained by irradiating conidia of Trichoderma viride QM 6a with a linear accelerator.
Abstract: A mutant strain that secretes twice as much cellulase as its parent was obtained by irradiating conidia of Trichoderma viride QM 6a with a linear accelerator.
219 citations
TL;DR: This study investigates factors that normally cause variation in zone diameters in conventional disc plate diffusion assay procedures and forms the basis for a test protocol which is presented in a following paper.
Abstract: Several factors are investigated that normally cause variation in zone diameters in conventional disc plate diffusion assay procedures. Of these factors the most serious is the unequal exposure of the individual plates at top or bottom of stacks to temperatures above and below room temperature. This unequal temperature exposure is avoided by novel handling and incubation procedures. A major variable, but one which can be controlled, is the varying time interval between pouring seeded agar and the time of applying the pads with antibiotic to the plates. This influence of time of setting and the effects of several other sequential operations are combined into a composite variable. This variable is then accounted for and normalized by interposing “external” reference plates set with a reference solution in the sequence of approximately 100 plates. No “internal” reference zones are employed. Such factors as volume of agar poured, wedge shape of agar in a dish, volumetric errors in dilutions, and timing considerations are studied and discussed. The results of this study form the basis for a test protocol which is presented in a following paper.
214 citations
TL;DR: A survey of the NaCl tolerance of species of terrestrial fungi selected from the major taxonomic classes suggests that uniformity of tolerance by multiple strains of various species suggests that this may provide a useful taxonomic criterion.
Abstract: A survey was made of the NaCl tolerance of 975 species of terrestrial fungi selected from the major taxonomic classes. The penicillia and aspergilli were notably the most resistant with the majority of their species able to grow in the presence of 20% or more of NaCl. The Basidiomycetes, as a class, were decidedly the least tolerant with over half the species unable to withstand more than 2% NaCl. Uniformity of tolerance by multiple strains of various species suggests that this may provide a useful taxonomic criterion.
179 citations
TL;DR: An improved solid agar medium (MP medium) has been developed which allows detection of pectolytic activity in bacteria and has successfully been used to detect pECTolytic organisms in soil, forest litter, and rotting vegetable samples.
Abstract: An improved solid agar medium (MP medium) has been developed which allows detection of pectolytic activity in bacteria. Organisms tested exhibited a variety of regulatory controls governing pectate lyase synthesis. The medium contains mineral salts, pectin, and yeast extract. After growth of the organisms, the agar plate is flooded with a polysaccharide precipitant, and pectolytic activity is shown by clear zones around active colonies. High concentrations of phosphate are shown to be necessary for pectic enzyme formation on solid media. The medium has successfully been used to detect pectolytic organisms in soil, forest litter, and rotting vegetable samples.
175 citations
TL;DR: The effects of temperature and length of incubation on ochratoxin A production in various substrates were studied and the toxin was found to be very stable over prolonged storage and even to autoclaving for 3 hr.
Abstract: The effects of temperature and length of incubation on ochratoxin A production in various substrates were studied. The optimal temperature for toxin production by Aspergillus ochraceus NRRL-3174 was found to be around 28 C. Very low levels of ochratoxin A are produced in corn, rice, and wheat bran at 4 C. The optimal time for ochratoxin A production depends on the substrate, ranging from 7 to 14 days at 28 C. Ochratoxin B and dihydroisocoumaric acid, i.e., one of the hydrolysis products of ochratoxin A, were produced in rice but at levels considerably lower than ochratoxin A. No ochratoxin C was produced in rice at 28 C. When added to rice cereal or oatmeal, the toxin was found to be very stable over prolonged storage and even to autoclaving for 3 hr.
TL;DR: In the absence of asparagine in the medium, the toxin yields fell drastically, and the thin-layer chromatograms of the chloroform extracts of the cultures indicated the total absence of aflatoxin G(1) and the presence of new intense blue and green fluorescent bands having R(F) values lower than aflatoxins.
Abstract: Aspergillus parasiticus ATCC 15517 produced 28 to 30 mg of aflatoxin per 100 ml of a medium containing sucrose, asparagine, and salts in stationary and shaken cultures. In the absence of asparagine in the medium, the toxin yields fell drastically, and the thin-layer chromatograms of the chloroform extracts of the cultures indicated the total absence of aflatoxin G(1) and the presence of new intense blue and green fluorescent bands having R(F) values lower than aflatoxins. Initial pH was critical and had to be around 4.5 for good growth and high toxin production on this medium. Optimum concentrations of KH(2)PO(4) and MgSO(4).7H(2)O in the medium were much lower than those normally used in fungal growth media.
TL;DR: Bacillus subtilis was most commonly employed, but other organisms may be employed in the present procedure, which is economical, uses simple facilities, and provides good accuracy of test results.
Abstract: A detailed disc plate procedure is introduced for assay of antibiotics. The procedure is based on a previous study by the authors and deviates from conventional procedures in several respects: selected plastic petri dishes are employed; critical temperature control is simply provided at all stages of the test with refrigeration of the plates never used; all dilution is done with displacement microburettes; six pads (6.3 mm diameter) per dish are employed, all filled with the same unknown or reference solution; the sequence of all plates handled on 1 day is made a part of the protocol which allows accounting for the influence of the order of pouring and setting the plates; external reference plates are set at specified locations in the sequence; and, by averaging the diameters of all zones on a plate, most of the consequence of wedge shape of agar in plates, which is common and almost unavoidable, is removed. The present method is economical, uses simple facilities, and provides good accuracy of test results. Bacillus subtilis was most commonly employed, but other organisms may be employed in the present procedure.
TL;DR: A reproducible test system requiring small amounts of test compound was developed for evaluating antiviral and interferon-inducing activity and known active compounds evaluated in this microplate system had activity similar to that seen in macro in vitro systems.
Abstract: A reproducible test system requiring small amounts of test compound was developed for evaluating antiviral and interferon-inducing activity. In the antiviral experiments, KB cells were grown in disposable polystyrene microplates covered with a standard domestic plastic wrap. Viruses used in the system were types 1 and 2 herpes simplex virus, vaccinia virus, type 3 adenovirus, myxoma virus, pseudorabies virus, type 3 parainfluenza virus, types 1A and 13 rhinovirus, vesicular stomatitis virus, coxsackievirus B, and type 2 poliovirus. Inhibition of viral cytopathogenic effect was the primary criterion of evaluation of antiviral activity. Reduction in cell and supernatant fluid virus titers was used as a secondary means of evaluation. The microplate system was adaptable for determining prophylactic, therapeutic, and inactivating effects against viruses. Mouse L-929 cells were used for the interferon induction studies, with vesicular stomatitis virus utilized as the indicator of interferon activity. Known active compounds evaluated in this microplate system had activity similar to that seen in macro in vitro systems.
TL;DR: Two hundred isolates from San Francisco sour dough French bread fermentations were screened by fermentation tests and for their ability to grow in the presence of cycloheximide, finding strong evidence on the role of S. exiguus in the sour dough system.
Abstract: Two hundred isolates from San Francisco sour dough French bread fermentations (40 from each of five different bakeries) were screened by fermentation tests and for their ability to grow in the presence of cycloheximide (Actidione). All of the isolates from four of the bakeries and 70% of those from the fifth were unable to utilize maltose but grew well on other sugars, even in the presence of cycloheximide. The remaining few isolates from the fifth bakery utilized maltose but not galactose and were inhibited by cycloheximide. No bakers' yeast types were found. Sixteen of the maltose-negative and five of the galactose-negative isolates were subjected to more rigorous taxonomic procedures. All of the maltose-negative isolates were identified as asporogenous strains of Saccharomyces exiguus (Torulopsis holmii) and the galactose-negative ones, as S. inusitatus. The predominance of S. exiguus, its vigor in the particular acidic environment of the sour dough, and the correlation of its numbers with the leavening function constitute strong evidence on the role of this organism in the sour dough system.
TL;DR: Ochratoxins A and B were given to 1-day-old Babcock B-300 cockerels to evaluate acute toxic effects and both appeared to have similar pathological effects.
Abstract: Ochratoxins A and B were given to 1-day-old Babcock B-300 cockerels to evaluate acute toxic effects. Two trials with ochratoxin A gave 7-day oral median lethal dose estimates of 116 μg (3.3 mg/kg) and 135 μg (3.9 mg/kg) per chick. Chicks given daily oral doses of 100 μg of ochratoxin A died on the second day. Single subcutaneous doses of 400 μg of ochratoxin A were also lethal. The 7-day oral median lethal dose of B was estimated at 1,890 μg (54 mg/kg) per chick. Chicks given oral doses of 100 μg of ochratoxin B daily for 10 days survived. Sublethal doses of both ochratoxins A and B resulted in growth suppression which was proportional to the amount of ochratoxin given. Visceral gout was the principal gross finding. Microscopic examinations revealed acute nephrosis, hepatic degeneration or focal necrosis, and enteritis. Suppression of hematopoiesis in the bone marrow and depletion of lymphoid elements from the spleen and bursa of Fabricius were frequently seen. Both ochratoxins appeared to have similar pathological effects. This is the first report on the toxicity of ochratoxin B.
TL;DR: Decimal reduction values (D value) for 30 viruses were determined and it was necessary to increase the radiation dose by a factor of >3 to inactivate virus suspended in Eagle's minimum essential medium as compared to the sameirus suspended in distilled water.
Abstract: Decimal reduction values (D value) for 30 viruses were determined. The weighted D values of the viruses suspended in Eagle's minimum essential medium ranged from 0.39 to 0.53 Mrads. It was necessary to increase the radiation dose by a factor of >3 to inactivate virus suspended in Eagle's minimum essential medium as compared to the same virus suspended in distilled water. The destruction rate curves were of a first-order reaction.
TL;DR: The susceptibility tests are rapid and simple to perform and are helpful in characterizing gram-negative anaerobic bacilli and are not intended for use in predicting clinical effectiveness of the drugs utilized.
Abstract: A method for identification of gram-negative anaerobic bacilli is described. Based on differences in susceptibility to paper discs containing 10 μg of colistin, 60 μg of erythromycin, 1,000 μg of kanamycin, 1,000 μg of neomycin, 2 units of penicillin, and 15 μg of rifampin, these bacteria may be placed into five groups. Other tests such as colony morphology, production of pigment, growth in bile, esculin hydrolysis, and reaction on egg yolk-agar may be used for further identification. The susceptibility tests are rapid and simple to perform and are helpful in characterizing gram-negative anaerobic bacilli. They are not intended for use in predicting clinical effectiveness of the drugs utilized.
TL;DR: In this paper, a cellulolytic, thermophilic actinomycete (previously isolated from municipal refuse compost samples) was identified as Thermomonospora curvata.
Abstract: A cellulolytic, thermophilic actinomycete (previously isolated from municipal refuse compost samples) was identified as Thermomonospora curvata. A determination was made of the optimal conditions for cellulase production by T. curvata when grown at 55 C in a medium containing mineral salts, cellulose, and yeast extract. The pH and temperature optima (pH 6.0 and 65 C) for the cellulase produced by T. curvata were identical to those previously observed for the cellulase extracted from crude compost samples. Such similarities, together with the prevalence of T. curvata in compost samples and its ability to grow at composting temperatures, indicate that this actinomycete could possibly be considered as a major cellulose decomposer in the municipal refuse composting process.
TL;DR: Salmonella recovery rates for stream bottom sediments were observed to be higher than those from surface water.
Abstract: Salmonella recovery rates for stream bottom sediments were observed to be higher than those from surface water.
TL;DR: A method is described for measuring the mineralization of an organic solute by the heterotrophic indigenous bacteria in lake sediments and may serve as a base for a more definitive procedure.
Abstract: A method is described for measuring the mineralization of an organic solute ((14)C-glucose) by the heterotrophic indigenous bacteria in lake sediments. Since there is no suitable procedure for the determination of in situ microbial activities in sediments, the procedure described is probably the best devised so far and may serve as a base for a more definitive procedure.
TL;DR: An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described and showed that TSC allowed virtually complete recovery of most of the C. perfringen strains while inhibiting practically all facultative anaerobes tested.
Abstract: An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 mug of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective.
TL;DR: In a number of commercially prepared media, the inhibitory activity of trimethoprim correlated inversely with the amount of thymidine found to be present by microbiological assay, and the significance for the routine testing of new, synthetic antibacterial agents is discussed.
Abstract: The ability of a potent dihydrofolate reductase inhibitor, trimethoprim, to inhibit the growth of Escherichia coli B in vitro is dependent on the composition of the medium in which the cells are grown. The inhibition observed in minimal broth could be partially reversed by the addition of thymidine, ribonucleosides, amino acids, and vitamins. No reversal occurred in the absence of thymidine. In a number of commercially prepared media, the inhibitory activity of trimethoprim correlated inversely with the amount of thymidine found to be present by microbiological assay. The significance of these findings for the routine testing of new, synthetic antibacterial agents is discussed.
TL;DR: A method has been developed to produce and purify gram quantities of T-2 toxin, a mycotoxin elaborated by a strain of Fusarium tricinctum isolated from toxic corn, and the amount of toxin measured in white corn grits declined as the incubation temperature was raised to 20, 25, and 32 C.
Abstract: A method has been developed to produce and purify gram quantities of T-2 toxin [4beta, 15-diacetoxy-8alpha-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3alpha-ol], a mycotoxin elaborated by a strain of Fusarium tricinctum isolated from toxic corn. After growing for 3 weeks at 15 C on 1,200 g of white corn grits, F. tricinctum NRRL 3299 elaborated at least 9.0 g of T-2 toxin, and 2.3 g of crystalline product was recovered. A lesser amount of toxin was produced on rice, but none was detected in wheat incubated at 20 C. The amount of toxin measured in white corn grits declined as the incubation temperature was raised to 20, 25, and 32 C.
TL;DR: When compared at similar levels of water activity, glycerol was more inhibitory than sodium chloride to relatively salt-tolerant bacteria and less inhibitorythan salt to salt-sensitive species.
Abstract: When compared at similar levels of water activity, glycerol was more inhibitory than sodium chloride to relatively salt-tolerant bacteria and less inhibitory than salt to salt-sensitive species.
TL;DR: The Analytab system of 20 biochemical tests for identification of Enterobacteriaceae was evaluated in parallel with conventional tests on 128 Enterobacteriaiaceae, 5 Aeromonas, and 1 Yersinia enterocolitica and showed almost complete agreement.
Abstract: The Analytab system of 20 biochemical tests for identification of Enterobacteriaceae was evaluated in parallel with conventional tests on 128 Enterobacteriaceae, 5 Aeromonas, and 1 Yersinia enterocolitica. The results of tests for H2S and indole production, citrate utilization, lysine and ornithine decarboxylase, arginine dihydrolase, nitrate reduction, β-galactosidase, and fermentation of arabinose, rhamnose, mannitol, and glucose showed almost complete agreement between the two systems. Eighty-eight per cent of Enterobacteriaceae were correctly speciated with the Analytab system; on repeat testing with heavier inocula of organisms failing to ferment glucose initially, the proportion of Enterobacteriaceae correctly speciated became 93%.
TL;DR: The yeastlike fungus Pullularia pullulans utilizes simple mono- and disaccharides both in the production of cell mass and the elaboration of extracellular polysaccharide.
Abstract: The yeastlike fungus Pullularia pullulans utilizes simple mono- and disaccharides both in the production of cell mass and the elaboration of extracellular polysaccharide. The utilization pattern of these sugars and the effect obtained by varying the pH of the medium are studied, and the ability of the organism to utilize and elaborate extracellular polysaccharides from noncarbohydrate sources is explored.
TL;DR: Staphylococcus aureus C-243, an enterotoxin B-producing strain, was cultured on media adjusted to various water activity (a(w)) levels by means of two different solute systems, and total numbers and rate of growth were diminished at low a(w) levels, and enteringotoxin synthesis was extremely sensitive to reduction.
Abstract: Previous studies indicated that enterotoxin B production by staphylococci was strongly inhibited by slight reductions in water activity (aw) levels. Similar studies reported herein, employing an enterotoxin A-producing strain, indicated that this organism was capable of producing enterotoxin at a much lower aw level than that required for enterotoxin B production. Staphylococcal growth rates were slowed by decreased aw levels in all media tested; however, final cell counts did not drop below 108/ml in the media with the lowest aw levels.
TL;DR: Arguments are presented to support the view that dry matter and some of the microbes, chiefly the protozoa, do not leave the rumen at the PEG rate and a rumination pool must be postulated.
Abstract: The feed and feces of a continuously fed sheep were analyzed for carbon, hydrogen, and nitrogen, with oxygen as the remainder. The daily feed-feces weight difference was used as the reactant in an equation representing the rumen fermentation. The measured products were the daily production of volatile fatty acids (VFA), CH(4), CO(2), and ammonia. The carbon unaccounted for was assumed to be in the microbial cell material produced in the rumen and absorbed before reaching the feces. The ratio of C to H, O, and N in bacteria was used to represent the elemental composition of the microbes formed in the rumen fermentation, completing the following equation:C(20.03)H(36.99)O(17.406)N(1.345) + 5.65 H(2)O --> C(12)H(24)O(10.1) + 0.83 CH(4) VFA + 2.76 CO(2) + 0.50 NH(3) + C(4.44)H(8.88)O(2.35)N(0.785) microbial cells absorbed With C arbitrarily balanced and O balanced by appropriate addition of water, any error is reflected in the H. The H recovery was 98.5%. The turnover rate constant for rumen liquid equilibrating with polyethylene glycol (PEG) was 2.27 per day. Direct counts and volume measurements of the individual types of bacteria and protozoa in the rumen were used to calculate the total microbial cell volume in the rumen, not equilibrating with it. The dry matter in the rumen (582 g) and the nitrogen content (12.05) of the microbes in the rumen were estimated, the latter constituting 85% of the measured N in the rumen. Calculations for rumen dry matter and nitrogen turning over at the PEG rate introduce big discrepancies with other parameters; a rumination pool must be postulated. Its size and composition are estimated. Arguments are presented to support the view that dry matter and some of the microbes, chiefly the protozoa, do not leave the rumen at the PEG rate. One experiment with the same sheep fed twice daily showed significantly less production of microbial cells than did the continuous (each 2 hr) feeding. Analysis of the microbial cell yield suggests that, on the basis of 11 mg of cells per adenosine triphosphate molecule, a maximum of six adenosine triphosphate molecules could have been formed from each molecule of hexose fermented.