Microanalytical methods: latex agglutination and immunoenzyme analysis in the diagnosis of meningococcal infection

Modified polystyrene latexes with high adsorption capacity, comparable to that of latexes produced by Difco Laboratories (USA), have been developed in the USSR. Diagnostic latex preparations for the detection of meningococci of serogroups A, C, Y and Haemophilus influenza, type b, prepared on the basis of these new latexes, have shown high specificity and sensitivity in experimental and clinical tests.The latex preparations for the detection of serogroup B meningococci requires further improvement. The use of latex preparations, together with other laboratory methods, in the diagnosis of meningococcal infection has promoted the etiological confirmation of the disease in 84% of cases; this method has proved to be 1.5 times more effective than the bacteriological one and not less sensitive than the enzyme immunoassay, while being more specific.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

Development and experimental study of a dried nutrient medium for the microbiological diagnosis of streptococcal infections

The comparative study of different protein bases has shown that the combined base containing animal blood hydrolysate (amino peptide) and acidic casein hydrolysate, moderately cleaved, in the proportion 1:1 is a good source of nitrogen and ensures the intensive growth of streptococci. As determined by the study of the physiological parameters and growth of streptococci, the presence of fodder yeast extract, glutamine, glucose and phosphates in media containing blood hydrolysate and casein hydrolysate has been found to render a stimulating effect on the growth and multiplication of these organisms. The data thus obtained have been used as the basis for developing the formula of a dried culture medium, capable of ensuring the growth of streptococci without blood or serum added and not inferior in its quality to Todd-Hewitt Broth manufactured by Oxoid Ltd. (Great Britain) and Difco Laboratories (USA). The physico-chemical and physiological characteristics of the proposed medium have been determined. The use of the new dried culture medium in medical practice will make it possible to improve the microbiological diagnosis of streptococcal infections.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

Extracellular acid proteases produced by Saccharomycopsis lipolytica.

Saccharomycopsis lipolytica CX161-1B produced at least three extracellular acid proteases during exponential growth in medium containing glycerol, Difco Proteose Peptone, and mineral salts at pH 3.4 (Difco Laboratories, Detroit, Mich.). Little extracellular acid protease activity was produced with glutamic acid as the sole nitrogen source, somewhat higher levels were obtained with peptone, and much higher levels were obtained with Difco Proteose Peptone. The relative amounts of the three proteases varied during growth on Difco Proteose Peptone, which suggested that the proteases were not coordinately regulated. The proteases were purified to near homogeneity (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) by use of ultrafiltration, gel filtration, and DEAE-Sephacel and hydroxylapatite chromatography. Protease I had a molecular weight near 28,000, an isoelectric point of pH 4.9, and a pH optimum of 3.5. Protease II had a molecular weight near 32,000 and a pH optimum of 4.2. Protease III had a molecular weight near 36,000, an isoelectric point of 3.8, and a pH optimum of 3.1. All three proteases were glycoproteins; proteases I, II, and III contained 25, 12, and 1.2% carbohydrate, respectively. The proteases were inhibited by pepstatin and 1,2-epoxy-3-(4-nitrophenoxy) propane and were largely insensitive to diazoacetyl-DL-norleucine methylester and to compounds which inhibit the serine, sulfhydryl, or metallo-proteases.     

A new medium for the enumeration and subculture of bacteria from potable water

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new medium for the enumeration and subculture of bacteria from potable water.

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of Undefined Medium and Its Dialyzable Fraction for Growth of Mycoplasma

In examining the medium used in cultivation of Mycoplasma for deoxyribonucleic acid isolation, it was found that an aggregate was present which sedimented with the organisms and which was ethyl alcohol-precipitated during deoxyribonucleic acid purification. To eliminate the contaminating material, a method was devised to obtain only the dialyzable constituents of the medium. This report describes the preparation of a dialysate of soy peptone-yeast extract. The medium, obtained by immersion of the encased dehydrated ingredients in sodium chloride solution for 5.5 hr at approximately 80 C, has been employed as the basal medium for cultivation of a number of Mycoplasma species. Comparative growth curves of two saprophytic strains and two parasitic species indicated that multiplication in dialysate, with suitable supplement, followed the pattern typical of the common eubacteria. Thus, by elimination of the sediment which occurred in nondialyzed medium, Mycoplasma could be concentrated without concomitant accumulation of contaminating macromolecules.     

Comparison of skeletal muscle monolayer cultures initiated with cells dissociated by the vortex and trypsin methods.

Monolayer cultures of 12-day embryonic chick skeletal muscle were prepared from cells dissociated with crude trypsin (Difco 1:250) and by a mechanical method that utilizes shearing forces obtained in a vortex of flowing medium. For each technique, experiments were performed in which the feeding schedule and density of cells planted were varied. Culture growoth was observed microscopically and with time-lapse cinematography. Regardless of the parameter varied, the time of onset of fusion and the extent of myotube formation were greatly improved in cultures initiated with the vortex-dissociated muscle cells.     

Comparison of skeletal muscle monolayer cultures initiated with cells dissociated by the vortex and trypsin methods

Summary Monolayer cultures of 12-day embryonic chick skeletal muscle were prepared from cells dissociated with crude trypsin (Difco 1:250) and by a mechanical method that utilizes shearing forces obtained in a vortex of flowing medium. For each technique, experiments were performed in which the feeding schedule and density of cells planted were varied. Culture growth was observed microscopically and with time-lapse cinematography. Regardless of the parameter varied, the time of onset of fusion and the extent of myotube formation were greatly improved in cultures initiated with the vortex-dissociated muscle cells.     

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii.

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

The growth and protein A accumulation of Staphylococcus aureus A-676 on a medium made from nonfood raw material

In this research the experimental analytical method of balancing culture media, based on the determination of the yield of the target product, i.e. staphylococcal protein A, has been used. The medium, developed in the course of this research work on the basis of a hydrolysate of protein-containing waste products of the tanning industry, makes it possible to achieve the growth of producer strains similar to that achieved in peptone-yeast medium. The parameters of the growth of S. aureus strain A-676 and the biological activity of protein A obtained in the newly developed medium have been studied.     

Mapping of mutations in Pseudomonas aeruginosa defective in pyoverdin production.

Twelve mutants of Pseudomonas aeruginosa PAO defective in pyoverdin production were isolated (after chemical and transposon mutagenesis) that were nonfluorescent and unable to grow on medium containing 400 microM ethylenediaminedi(o-hydroxyphenylacetic acid). Four mutants were unable to produce hydroxamate, six were hydroxamate positive, one was temperature sensitive for pyoverdin production, and another was unable to synthesize pyoverdin on succinate minimal medium but was capable of synthesizing pyoverdin when grown on Casamino Acids medium (Difco Laboratories, Detroit, Mich.). The mutations were mapped on the PAO chromosome. All the mutations affecting pyoverdin production were located at 65 to 70 min, between catA1 and mtu-9002.     

Serological identification of group D streptococci using commercial antisera

Three commercial group D streptococcal antisera were tested for the serological identification of 100 group D enterococci; 20 Streptococcus bovis; 5 isolated from each of the following streptococcal groups: A, B, C and G; and 3 isolates from serological group F. Antisera from Difco Laboratories, BBL, and Wellcome Reagents Limited were used in the classic capillary tube precipitin test on extracts prepared using the Rantz and Randall procedure. No false positive precipitin reactions were observed. Of the enterococcal isolates, all 100 reacted with the Wellcome, 99 reacted with the BBL, and 96 reacted with the Difco group D antisera. However, of the 20 S. bovis isolates, only 2 reacted with the BBL, and 1 reacted with both the Difco and the Wellcome antisera. Each antiserum was then used to prepare staphylococcal coagglutination (CoA) reagents and each isolate was subsequently tested. A simple extraction procedure was performed by suspending colonies of an isolate in a loopful of salin on a microscope slide and gently heating the slide directly in the flame of a Bunsen burner. All 100 enterococci and all 20 S. bovis gave positive results with the BBL and the Wellcome CoA reagents. Using the Difco reagent, 2 S. bovis isolates failed to produce postitive results. No false positive results were observed with the non-Group D isolates. Our results indicate that the CoA technique using commercial group D antisera may provide faster and sometimes more sensitive serological identification than the classic capillary tube precipitin test.     

Blood-Free Medium for the Rapid Growth of Pasteurella tularensis

A medium composed of (in g/100 ml) Tryptose broth with thiamine (Difco), 2.6; cysteine-HCl, 0.12; glucose, 1; FeSO₄, 7H₂O, 0.005; KCl, 0.02; histidine, 0.1; tris(hydroxymethyl)aminomethane (tris) buffer, 0.3; and agar, 1; will support rapid growth of the fully virulent SCHU-S4 strain of Pasteurella tularensis. Although the test organism grew rapidly on medium from which KCl and tris buffer were omitted, these two components increased the stability of the medium upon storage at 4 C. It was necessary to (i) control carefully the relative concentration of the ferrous iron and cysteine-HCl, (ii) incubate the prepared medium overnight prior to use, and (iii) incubate the inoculated plates in an atmosphere of high relative humidity. Rapid growth of the organism was obtained also from very small inocula in the liquid form of the medium. Biochemical studies designed to elucidate the mechanisms involved in the enhancement of growth of P. tularensis in this relatively simple blood-free medium were initiated.     

An efficient system for catalyzing ester synthesis using a lipase from a newly isolated Burkholderia cepacia strain

The synthesis of ethyl-oleate by the lipase from the newly isolated strain Burkholderia cepacia LTEB11 in three different systems has been studied - immobilization on a hydrophobic support (Accurel EP 100®), encapsulation in reverse micelles, and direct addition of powdered free enzyme to the reaction medium. The immobilized enzyme performed best, giving a 70% ester yield in 10 h, this yield being five-fold greater than that obtained for reversed micelles, and two and a half times greater than that obtained for direct addition. An increase in the amount of immobilized enzyme preparation added gave a 100% ester yield in 3 h. The immobilized preparation was quite stable, giving a 100% yield of ethyl-oleate during 11 repeated reactions, and 50% yield after 24 reactions. These results suggest that the lipase of our strain of B. cepacia LTEB11 immobilized on Accurel has good potential for application in biocatalysis in organic media.     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis.

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.     

Isolation and purification of a new protease from Aspergillus niger LCF no 9

Summary Aspergillus niger LCF N ^o 9 synthesizes large amounts of a semi-alkaline protease highly active on Benzoyl arginine p-nitroanilide (BAPNA) that is likely to find industrial use. An industrial-grade enzyme of around 80% purity was prepared and a reference enzyme was obtained in high yield from the industrial product by ultrafiltration and HPIEC on a mono Q column. The industrial-grade enzyme was produced on a pilot scale with a yield of 0.52% of medium solid starting material by adsorption on CM-Sephadex A₅₀, precipitation with acetone at –30°C, ion-exchange chromatography on DEAE-Sepharose and diafiltration.     

Isolation and Characterization of Aminopeptidase-Deficient Lactobacillus bulgaricus Mutants

Lactobacillus bulgaricus CNRZ 397 is able to hydrolyze many amino-acyl- and dipeptidyl-β-naphthylamides. Analysis of heat inactivation kinetics, protease inhibitor effects, and the subcellular location of aminopeptidase (AP) activities from the parental strain and mutant derivatives dificient in alanyl- or leucyl-β-naphthylamide hydrolysis pointed out the existence of four APs. All mutants isolated were totally deficient in AP II, a cell wall metallo-enzyme with a broad substrate specificity but that is specifically responsible for lysyl-AP activity and is characterized by a molecular mass of 95,000 daltons. AP I and AP III are cytoplasmic enzymes that exhibit arginyl-AP activity; both enzymes are inducible during growth in rich peptide MRS medium (Difco Laboratories, Detroit, Mich.). The existence of a fourth AP (AP IV) that is involved in leucyl-AP activity was suggested. Moreover, we showed that X-prolyl-dipeptidyl-AP activity, which was not catalyzed by an AP, involved an enzyme(s) that is controlled by a regulatory mechanism that is common to that of AP II.