Study of soil algae

Summary 1. A number of criticisms can be levelled at the existing methods of microbiological assay of available soil nutrients. These criticisms are examined in some detail.2. A new method for the microbiological assay of available soil nutrient is described using algae as the test organisms.3. The new technique overcomes the criticisms levelled at previous methods.4. The simplicity of the method makes it suitable for general use.     

Comparative studies to determine the efficiency of 6 methylpurine deoxyriboside to detect cell culture mycoplasmas

Summary Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were:Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection withSpiroplasma mirum when this organisms was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98. 8, and 97.1%, respectively. The work was supported by grant AI-15748 from the National Institutes of Health, Bethesda, MD     

Molecular identification of Salmonella isolates from poultry and meat productsin Irbid City,Jordan

The sensitivity and accuracy of molecular diagnosis of Salmonella from meat and poultry products using polymerase chain reaction (PCR) was compared with conventional microbiological methods. A total of 212 samples representing the most frequently used fresh and frozen meat and poultry products (whole, cut, ground, and processed) were collected from different locations within the city of Irbid. DNA was extracted directly from each food sample and amplified using Salmonella-specific primers. Samples were also analysed using conventional microbiological methods for the presence of Salmonella spp. Results showed that Salmonella was detected in 185 samples out of 212 (87%) by PCR technique, while 172 (81%) samples were detected Salmonella positive by conventional microbiological methods. On the other hand, 27 (12.7%) samples were negative by PCR and 40 (18.8%) samples were negative by conventional microbiological methods. PCR assay proved to be an effective method for Salmonella detection in meat and poultry products with high specificity and sensitivity and more importantly a less time-consuming procedure. Using PCR, Salmonella spp. detection could be achieved within 24–36 h compared to 3–8 days for the conventional microbiological methods.     

Treatment of streptococcal pharyngitis with amoxycillin once a day.

OBJECTIVE--To evaluate treatment of group A beta haemolytic streptococcal pharyngitis with amoxycillin once daily compared with phenoxymethylpenicillin three or four times a day. DESIGN--Randomised controlled study of consecutive patients presenting with symptoms suggestive of group A beta haemolytic streptococcal pharyngitis in whom culture of a throat swab yielded positive results. SETTING--Five family medicine practices. SUBJECTS--157 patients aged over 3 years who required treatment with antibiotics. MAIN OUTCOME MEASURES--Clinical response, bacteriological response, days at work and school lost, and compliance. RESULTS--During the period of the study 393 patients presented with symptoms suggesting streptococcal pharyngitis; 157 of them had throat swabs that yielded positive results on culture. Eighty two were treated with phenoxymethylpenicillin and 75 with amoxycillin. No difference was observed in the clinical response, days at work and school lost (139 days for 64 patients taking phenoxymethylpenicillin v 100 days for 57 patients taking amoxycillin; p > 0.2), or residual positive cultures after two days (6 (7.3%) v 3 (4%); p > 0.5). A significant difference in the bacteriological response was found after 14 days (5 (6.1%) v 0; p < 0.04) with no positive cultures observed in the amoxycillin group. CONCLUSIONS--These findings support the hypothesis that amoxycillin once daily is as effective as phenoxymethylpenicillin in the treatment of group A beta haemolytic streptococcal pharyngitis.     

Microbiological analysis of 5-formyltetrahydrofolic acid and other folates using an automatic 96-well plate reader

The growth of auxotrophic bacteria remains the method of choice for the determination of biologically active folate metabolites in plasma. This report describes a microbiological assay for folates adapted to use disposable 96-well plates and an automatic plate reader. The modifications in the assay decreased reagent costs and made the analysis of hundreds of samples per day possible with a sensitivity limit of 10 fmol of (6S)-5-formyltetrahydrofolic acid. This limit compares favorably with that of previously reported, more laborious methods. The unnatural 6R diastereomer of 5-formyltetrahydrofolic acid did not interfere with the microbiological assay of the natural 6S diastereomer.     

Comparative studies on the microbiological vitamin B12 assay at two laboratories.

The turbidimetric methods in routine use at two laboratories for the microbiological assay of vitamin B12 have been compared. Attempts were made to standardize some major parts of the method, i.e., assay design, test strain (Lactobacillus leichmannii), test medium, and reference standard. The laboratories used different approaches to achieve efficient assay procedures. During a 6-year period four comparative experiments were carried out. In these experiments the vitamin B12 content of five different products was determined in a series of independent assays at each laboratory. A satisfactory degree of agreement (difference less than 5%) was found for four of these products.     

Comparative studies on the microbiological vitamin B12 assay at two laboratories.

The turbidimetric methods in routine use at two laboratories for the microbiological assay of vitamin B12 have been compared. Attempts were made to standardize some major parts of the method, i.e., assay design, test strain (Lactobacillus leichmannii), test medium, and reference standard. The laboratories used different approaches to achieve efficient assay procedures. During a 6-year period four comparative experiments were carried out. In these experiments the vitamin B12 content of five different products was determined in a series of independent assays at each laboratory. A satisfactory degree of agreement (difference less than 5%) was found for four of these products.     

Simple screening method for isolation of penicillin acylase-producing bacteria.

A new screening method for bacteria capable of producing penicillin acylase is described. The method is based on the use of Serratia marcescens sensitive to 6-aminopenicillanic acid but comparatively resistant to benzylpenicillin. It is simple, quite specific, and requires no special equipment. It can also be used to screen for phenoxymethylpenicillin acylase activity. We also suggest an acidimetric method for rapid detection of cloned genes in genetic engineering studies of penicillin acylase.     

Simple screening method for isolation of penicillin acylase-producing bacteria

A new screening method for bacteria capable of producing penicillin acylase is described. The method is based on the use of Serratia marcescens sensitive to 6-aminopenicillanic acid but comparatively resistant to benzylpenicillin. It is simple, quite specific, and requires no special equipment. It can also be used to screen for phenoxymethylpenicillin acylase activity. We also suggest an acidimetric method for rapid detection of cloned genes in genetic engineering studies of penicillin acylase.     

Synthesis and SAR study of pyrrolo[3,4-b]pyridin-7(6H)-one derivatives as melanin concentrating hormone receptor 1 (MCH-R1) antagonists

The discovery and optimization of novel pyrrolo[3,4-b]pyridin-7(6H)-one MCH-R1 antagonists are described. A systematic SAR study probing the effects of aryl-, benzyl- and arylthio-substituents at the 2-position of the pyrrolo[3,4-b]pyridin-7(6H)-ones led to identification of the 2-[(4-fluorophenyl)thio] derivative 7b as a highly potent MCH-R1 antagonist. This compound also has favorable pharmacokinetic properties along with a high metabolic stability and a minimal impact on CYP isoforms and hERG.     

Factors influencing microbiological assay of cell-culture mycoplasmas

Summary A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar and broth media. Mycoplasmas were detected in 375 cultures (5.8%).M. orale andA. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aerobic incubation detected 45.8%. Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains ofM. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas. These studies were supported in part by Contracts N01-AG-4-2865 and N01-AG-8-2117 from the National Institute on Aging and N01-GM-6-2119 from the National Institute of General Medical Sciences.     

Interval estimation of the density of organisms using a serial-dilution experiment

The serial-dilution assay is a standard microbiological method for estimating the density of organisms in a solution. The commonly used interval-estimation procedures of Woodward and deMan are described and compared. A new method for the calculation of two-sided confidence intervals, which is superior to the standard procedures, is presented. The computations are illustrated for a decimal dilution assay with three samples at each of three dilutions.     

Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

A sensitive procedure for screening microorganisms for the presence of penicillin amidase

A procedure is described for screening bacteria for the presence of penicillin amidase. Cells, grown in the presence of phenylacetic acid, are incubated with phenoxymethylpenicillin (type I), benzylpenicillin (type II) or ampicillin and the 6-aminopenicillanic acid formed is detected and quantitatively estimated by its strong reaction with fluorescamine at pH 4. There is no requirement for separation of the penicillin substrate from the product but when alpha-aminobenzylpenicillin derivatives are used as enzyme substrates the amount of 6-aminopenicillanic acid formed must be determined by calculation. The procedure allowed positive and reliable identification of penicillin amidases in six organisms known to produce the enzyme and indicated that some of these enzymes had different properties in reactivity towards alpha-aminobenzylpenicillin derivatives.     

Development of a method to detect and quantify <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> conidia by quantitative PCR for environmental air samples

Exposure to Aspergillus fumigatus is linked with respiratory diseases such as asthma, invasive aspergillosis, hypersensitivity pneumonitis, and allergic bronchopulmonary aspergillosis. Molecular methods using quantitative PCR (qPCR) offer advantages over culture and optical methods for estimating human exposures to microbiological agents such as fungi. We describe an assay that uses lyticase to digest A. fumigatus conidia followed by TaqMan™ qPCR to quantify released DNA. This method will allow analysis of airborne A. fumigatus samples collected over extended time periods and provide a more representative assessment of chronic exposure. The method was optimized for environmental samples and incorporates: single tube sample preparation to reduce sample loss, maintain simplicity, and avoid contamination; hot start amplification to reduce non-specific primer/probe annealing; and uracil-N-glycosylase to prevent carryover contamination. An A. fumigatus internal standard was developed and used to detect PCR inhibitors potentially found in air samples. The assay detected fewer than 10 A. fumigatus conidia per qPCR reaction and quantified conidia over a 4−log₁₀ range with high linearity (R ² > 0.99) and low variability among replicate standards (CV=2.0%) in less than 4 h. The sensitivity and linearity of qPCR for conidia deposited on filters was equivalent to conidia calibration standards. A. fumigatus DNA from 8 isolates was consistently quantified using this method, while non-specific DNA from 14 common environmental fungi, including 6 other Aspergillus species, was not detected. This method provides a means of analyzing long term air samples collected on filters which may enable investigators to correlate airborne environmental A. fumigatus conidia concentrations with adverse health effects.     

In Vitro Management of Hospital <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Biofilm Using Indigenous T7-Like Lytic Phage

Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1–HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.     

5-Azacytidine: Microbiological Assay in Mouse Blood

A 6-chloropurine-resistant strain of Escherichia coli has been used to develop a microbiological assay for the estimation of 5-azacytidine distribution in mouse tissues.     

Liquid Nitrogen Preservation of Saccharomyces carlsbergensis and its Use in a Rapid Biological Assay of Vitamin B6 (Pyridoxine)

Growth medium as well as freezing menstruum greatly influenced the recovery of Saccharomyces carlsbergensis when it was quickly frozen in liquid nitrogen at - 196 C and quickly thawed at 40 C. Nearly 90% recovery in viability was obtained when S. carlsbergensis was grown in Trypticase Soy Broth and frozen in vitamin B(6) basal assay medium. The growth phase of S. carlsbergensis also influenced recovery after freezing. When S. carlsbergensis was grown in Trypticase Soy Broth and frozen in the broth at the logarithmic-growth phase, only 7% viability was retained; the recovery rate increased to 81% when the culture was frozen in the maximal stationary phase. To have the least possible lag period of growth after thawing, a technique called growth-phase conditioning was introduced. After 1 hr of growth-phase conditioning, S. carlsbergensis was clearly out of lag phase, and budding was observed. A vitamin B(6) microbiological assay with a 6-hr incubation period and with the use of liquid nitrogen-frozen S. carlsbergensis is described.     

Polysaccharide from Dry Navy Beans, Phaseolus vulgaris: Its Isolation and Stimulation of Clostridium perfringens

A microbiological assay is described for determining gas produced by Clostridium perfringens (Veilon and Zuber) Holland from materials believed to be flatulent. A rationale for its use is given and analyzed. The fractionation and the results of the assay for several components of lima beans, Phaseolus lunatus L., and navy beans, P. vulgaris L., are given. In particular, a polysaccharide was isolated from dried P. vulgaris seeds. Only about one-seventh as much could be isolated from green P. lunatus seeds as from P. vulgaris seeds. The polysaccharide stimulates voluminous gas production by C. perfringens and meets all criteria of the rationale for flatulent sus pects. It has not been tested on humans; however, data from the microbiological assay correlate well with human results for green and dry lima beans and navy beans.     

Production of the Monamycins, Novel Depsipeptide Antibiotics

Methods are described for the production of the monamycins by Streptomyces jamaicensis in shake flasks and jar fermentors. The effects on the fermentation of variations in pH, temperature, medium composition, volume of inoculum, and strain of the organism are discussed. The methods employed for the extraction and for the microbiological assay of the antibiotics are outlined.