Pour-plate Method Research Articles

Antimicrobial and cytotoxic activity of hypochlorous acid: interactions with taurine and nitrite.

HOCl, a major bactericidal product of neutrophil MPO-halide system reacts with taurine to form taurine chloramine (TauCl), a less toxic anti-inflammatory mediator. Recently, it has been reported that HOCl may also react with nitrite (NO2-), a major end-product of nitric oxide (NO) metabolism, to form very active oxidant, nitryl chloride (NO2Cl). The present study was conducted to elucidate the effect of nitrite on bactericidal and some immunoregulatory properties of HOCl and TauCl. TauCl was prepared from NaOCl and taurine. The reaction was carried out at pH 5.0 and pH 7.4, in the presence or absence of nitrite. All reactions were monitored by UV absorption spectra. Bactericidal activity of HOCl and TauCl in the presence of nitrite was tested by incubation of E. coli with the compounds and determined by the pour-plate method. To test the effect of the compounds on activity of inflammatory cells, murine peritoneal neutrophils (PMN) and macrophages were used. The cells were activated in vitro with either LPS, IFN-gamma or zymosan and the production of following mediators was measured: reactive oxygen species using luminol-dependent chemiluminescence; nitric oxide by Griess reaction; TNF-alpha using capture ELISA. In addition, we tested the effect of HOCl and TauCl on activity of myeloperoxidase (MPO). At physiological pH nitrite reacts with HOCl but not with TauCl. This reaction was abolished in the presence of taurine. Nitrite prevented HOCl-mediated bacterial killing, inhibition of MPO activity, cellular cytotoxicity and inhibition of TNF-alpha production. Nitrite did not affect any activity of TauCl. We have shown that nitrite may react in vitro with HOCl but not with TauCl, to form new biologically active product(s). We did not confirm the hypothesis that a product of HOCl reaction with nitrite is more toxic than HOCl. To the contrary, we found that nitrite diminished bactericidal and immunoregulatory properties of HOCl. In vivo, nitrite will also compete with taurine for reaction with PMN-released HOCl. Nevertheless, due to high concentration of taurine in PMN cytosol, formation of TauCl will be a major regulatory mechanism of MPO-halide-system.

Multiregional Evaluation of the SimPlate Heterotrophic Plate Count Method Compared to the Standard Plate Count Agar Pour Plate Method in Water

A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35 degrees C for 48 h. Six laboratories tested a total of 632 water samples. The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (r = 0. 95; y = 0.99X + 0.06).

Short note on the effects of ethanolic extracts of selected South African seaweeds on the growth of commercially important plant pathogens, Rhizoctonia solani Kühn and Verticillium sp.

This paper describes the biological activity of ethanolic extracts from some commonly abundant seaweeds from KwaZulu-Natal, South Africa. The pour-plate method was used to determine the effects of selected seaweed extracts on the growth of two phytopathogens; Verticillium sp. and Rhizoctonia solani . Extracts from Caulerpa filiformis and Ulva rigida (Chlorophyta); Zonaria tournefortii (Phaeophyta); and Hypnea spicifera , Gelidium abottiorum and Osmundaria serrata (Rhodophyta) inhibited fungal growth by more than 50%. The extracts from the rhodophytes, Spyridia cupressina and Beckerella pinnatifida showed the weakest antifungal activity of the seaweeds tested. The pour plate method demonstrated both fungal growth promotion and inhibition, due to the seaweed extracts. At low concentrations (1:16 and 1:32 dilutions) the H spicifera extract promoted the growth of R . solani .

Distribution and activity of microorganisms in coastal waters off the Netherlands and Germany

Coastal waters up to 90 km off the coast of the Netherlands and Germany were investigated with the RV ‘Friedrich Heincke’ at 21 stations in July 1987. Hydrographic, chemical and biological parameters were measured simultaneously, such as temperature, salinity, oxygen saturation, adenosine triphosphate (ATP) concentrations and electron transport system (ETS) activities. The mean surface temperature was 16°C, salinity distribution showed a tongue of high salinity in the Southern Bight and coastal areas of low values by river input from the Rhine and Elbe. The mean oxygen concentration was 102.4%, with the highest percentages northwest of Helgoland and the lowest near Scheveningen and Cuxhaven. The ecosystem parameters of ATP-biomass and respiratory ETS-activity showed a distribution pattern corresponding to the inflow of clear Channel water and nutrient-rich river water. Highest biomass values and respiratory activities were found near Texel and northwest of Helgoland. The distribution patterns of microplankton species were more patchy. Distribution patterns are shown of phytoplankton species such as Ceratium fusus, Phaeocystis spec., Rhizosolenia shrubsolei, and Mesodinium rubrum and various groups of microplankton. The highest numbers of Ceratium were found near Helgoland, of Phaeocystis near Terschelling and Cuxhaven, of Rhizosolenia near Texel and Langeoog and of Mesodinium near the Dutch coast and north of Borkum. Concentrations of bacteria were determined both by cultivation and epifluorescence microscopy methods. The numbers obtained by the pour-plate method varied much more than those determined by the direct-count method. At all stations, oil-degrading bacteria were found. The numbers of bacteria able to degrade mineral oil were higher than the numbers of bacteria able to degrade a tetradecane-pentadecane mixture. Biological oxygen demand (BOD) was measured with and without oil additions after incubation of 5, 10 and 15 days and a potency to degrade oil was shown. Beside the mapping of respiratory activity by the ETS-method, the real oxygen uptake at in situ temperatures was measured in a few samples with short incubation. An ETS/R ratio of 4.1 was estimated.

Enumeration of bacteria in clinically significant blood cultures in neutropenic and non-neutropenic patients using a pour plate method

A 3-year review of clinically significant positive blood cultures was undertaken to assess any differences in the blood bacterial count between haematological neutropenic and other non-neutropenic patients. The pour-plate method was used. In Gram-positive infections the pour plate contained colonies in 61% of haematological patients and in 41% of others. In Gram-negative infection the figures were 54% and 25% respectively. The mean numbers of bacteria per ml of blood were increased in haematological patients compared with the others for both groups of organisms.

Bacteremia in Children Following Dental Extraction

The incidence and intensity of bacteremia following tooth extraction in children were measured by blood culture. The effects on bacteremia of the number and type of teeth extracted, oral hygiene, gingival health, presence of abscess, and antibiotic prophylaxis were assessed. Antibiotic prophylaxis reduced the incidence of bacteremia from 63% to 35%. The intensity of bacteremia was 2 cfu/mL of blood or less in 80% of the children. An agar pour-plate method of blood culture was significantly more effective than broth in culturing the small volumes of inoculum. Of 83 bacterial strains characterized, 39 were strict anaerobes or micro-aerophilic, and the remainder mainly streptococci. S. mitior and S. sanguis were most commonly isolated, often in pure culture, and were generally sensitive to antibiotics. No direct association was demonstrated between the plaque and gingival indices and incidence of bacteremia or between the number of teeth extracted and the incidence or intensity of bacteremia. It is concluded that all children at risk from infective endocarditis require antibiotic prophylaxis prior to tooth extraction, since it is impossible for the likelihood or intensity of transient bacteremia to be clinically predicted.

Evaluation of a modified dye pour-plate auxanographic method for the rapid identification of clinically significant yeasts

A modified dye pour-plate auxanographic (DPPA) method for the presumptive identification of medically important yeasts was evaluated, in a comparative study with a conventional procedure, the API 20C clinical yeast system (Analytab Products Inc.), and the Uni-Yeast-Tek (UYT) system. The 174 coded clinical isolates were members of the genera Candida, Cryptococcus, Rhodotorula, Saccharomyces, Torulopsis, and Trichosporon. The identification accuracies with DPPA, API, and UYT were 95%, 93%, and 99% respectively. DPPA and API required more time to inoculate but gave rapid identification profiles. UYT was simple to inoculate and both UYT and DPPA were easy to read. Cost analysis of the three rapid methods demonstrated DPPA to be the most economical making it a feasible alternative for small clinical laboratories as well as large laboratories possessing the facilities to make their own media.

A Simple and Safe Volumetric Alternative to the Method of Miles, Misra and Irwin for Counting Viable Bacteria

A rapid and simple method for counting viable bacteria is described. The technique involves the use of a semi-automatic micropipette and disposable glass-capillary tubes. Viable counts obtained by this method were not significantly different from those obtained by the Miles, Misra and Irwin method or by a pour-plate method. The micropipette procedure is less hazardous than techniques involving the use of calibrated dropping pipettes.

Enumeration of Clostridium botulinum spores in meats by a pour-plate procedure

Colonies of Clostridium botulinum could be easily distinguished from meat particles by supplementing Wynne agar with 0.4% egg yolk. The pour-plate method was suitable for enumeration of C. botulinum, provided the medium was covered with a layer of agar containing 0.01% dithiothreitol. Viable counts of heat-treated spores were consistently higher in Wynne agar supplemented with egg yolk (Wynne-EY agar) than in Wynne agar alone.

Coliform counts of polluted waters: a comparison of media and methods

Samples of sewage and polluted river water were used to compare various media for the enumeration of coliform organisms, including Escherichia coli, by the membrane filtration, pour-plate and MPN techniques. The pour-plate method with Lactose teepol agar was found to be at least as good as the other techniques, to which it is to be preferred as it is more economical, both in terms of time and materials.

Pour-plate method for the detection of coagulase production by Staphylococcus aureus.

A pour-plate method is described for the detection of coagulase production by Staphylococcus aureus. Either Brain Heart Infusion agar or yeast extract-Trypticase soy agar containing swine plasma was the best medium for the detection of coagulase by this method. The advantages of this method and its potential utilization in the clinical laboratory, in food microbiology, and in specialized studies with S. aureus are discussed.

Pour-Plate Method for the Detection of Coagulase Production by Staphylococcus aureus

A pour-plate method is described for the detection of coagulase production by Staphylococcus aureus. Either Brain Heart Infusion agar or yeast extract-Trypticase soy agar containing swine plasma was the best medium for the detection of coagulase by this method. The advantages of this method and its potential utilization in the clinical laboratory, in food microbiology, and in specialized studies with S. aureus are discussed.

DIAGNOSIS OF URINARY-TRACT INFECTION IN GENERAL PRACTICE

The diagnosis of urinary-tract infection in general practice has been assessed with the aid of suprapubic aspiration and the dip-slide technique. Using a laboratory pour-plate method as the criteria of infection it has been found that a combination of suprapubic aspiration and the dip-slide culture confirmed the presence of infection in 92% of cases, direct microscopy of this suprapubic aspirate in 73%, and culture of a single midstream sample of urine in 72%. Suprapubic aspiration was safe and reliable in general practice, and was tolerated well by patients. It is suggested that the diagnosis of a urinary infection in general practice can best be made by the dip-slide inoculation of a suprapubic aspirate of urine.

Agar medium for the selective enumeration of coagulase-positive Staphylococcus from the rat alimentary tract.

A selective agar medium (medium J(1)) is proposed for the quantitative enumeration of egg yolk-positive (EYP) and egg yolk-negative (EYN) Staphylococcus pyogenes from the digestive tract and feces of the rat. This medium, buffered at pH 5.0, is composed of acid casein hydrolysate and yeast extract with 7.5% sodium chloride, 1.6% sodium pyruvate, 0.0008% 2,3,5-triphenyltetrazolium chloride (TTC), and 6% egg yolk emulsion. Inoculation is by the pour-plate method and incubation is at 38 C in a water-jacketed incubator for 36 hr. Colonies of S. pyogenes reduce TTC; EYP strains are surrounded by a halo of opacity; and EYN strains may be surrounded by a red halo, but no opacity. Small, white colonies of S. epidermidis may develop, but Micrococcus, and all other groups of Staphylococcus recognized in the rat intestinal flora, are inhibited. Other bacterial genera, notably Bacillus, Corynebacterium, Proteus, and Streptococcus, are also inhibited.

Simplified quantitative methods for bacteriuria and pyuria

Although pyelonephritis is a common disease, it escapes clinical detection in an undesirably high proportion of patients. The present unsatisfactory diagnostic position would be much improved by widespread screening of patients by simple yet reasonably accurate methods. Bacterial counts by the pour-plate technique and estimates of the white cell excretion per hour or day, while undoubtedly of diagnostic value, are probably unsuitable for use on a wide scale. In an attempt to find more convenient procedures a simplified stroke-plate method of bacterial counting and a simplified quantitative white cell count method were devised and applied to over 1,000 mid-stream urine samples from 398 patients. Good correlation was obtained between the simpler stroke-plate method of bacterial counting and the more time-consuming pour-plate method. The quantitative white cell procedure was a much more sensitive index of pyuria than wet-film microscopy, and comparison with the bacterial count results showed that it gave a useful indication of urinary infection. It is suggested that a quantitative bacterial count should replace non-quantitative culture methods when urinary infection is suspected and that the quantitative white cell count should be performed as a routine part of the initial clinical and laboratory assessment of all patients, followed by a bacterial count if pyuria is revealed. Experience has shown that routine urine microscopy by a precise method leads to the detection of many cases of occult urinary infection.

Bacteriological examination of unbottled soft drinks.

A study of 300 samples, representing 14 different unbottled drinks, indicated that there are three vitally important criteria pertaining to their bacteriological examination. First, the total viable counts may be better accomplished by the pour-plate method, using enriched media, with incubation at either 30 or 37 C. Second, a comparative study of the coliaerogenes group and the enterococci as indices of pollution unquestionably favors the latter as the reliable indicator, owing to false interpretations of the presumptive test and to lack of accurate definition of fecal and nonfecal coliforms recovered from positive cases. The use of enterococci, however, did not provide as reliable an indicator as the pour-plate method. Third, the results with enterococci, in defining the probable source of pollution, are more precise. Experiments judiciously selected and simultaneously conducted revealed that the heat and heat-tellurite resistance tests, and the tetrazolium-reduction test, matched in relating 98.9% of available enterococci to an animal source. Negligible but vital discrepancies were obtained with the two odd strains which qualified as human-derived according to the heat and heat-tellurite resistance tests. The differential criterion of Skadhauge and Barnes, based on the failure of animal-derived enterococci to grow in the presence of a low concentration of potassium tellurite, did not apply to the other two methods, since 99.5% of the recovered strains were found tolerant to the specified tellurite concentration.

Bacteriological Examination of Unbottled Soft Drinks

A study of 300 samples, representing 14 different unbottled drinks, indicated that there are three vitally important criteria pertaining to their bacteriological examination. First, the total viable counts may be better accomplished by the pour-plate method, using enriched media, with incubation at either 30 or 37 C. Second, a comparative study of the coliaerogenes group and the enterococci as indices of pollution unquestionably favors the latter as the reliable indicator, owing to false interpretations of the presumptive test and to lack of accurate definition of fecal and nonfecal coliforms recovered from positive cases. The use of enterococci, however, did not provide as reliable an indicator as the pour-plate method. Third, the results with enterococci, in defining the probable source of pollution, are more precise. Experiments judiciously selected and simultaneously conducted revealed that the heat and heat-tellurite resistance tests, and the tetrazolium-reduction test, matched in relating 98.9% of available enterococci to an animal source. Negligible but vital discrepancies were obtained with the two odd strains which qualified as human-derived according to the heat and heat-tellurite resistance tests. The differential criterion of Skadhauge and Barnes, based on the failure of animal-derived enterococci to grow in the presence of a low concentration of potassium tellurite, did not apply to the other two methods, since 99.5% of the recovered strains were found tolerant to the specified tellurite concentration.

STUDIES ON THE CULTIVATION OF ACID FAST BACILLI BY POUR-PLATE METHOD

It is surely of importance to estimate the numbers of living bacilli in a suspension of tubercle bacilli. Neverthless, the accurate calculation method of living tubercle bacilli has not been developed, since there are much difficulties in culculating tubercle bacilli by pour-plate method, which is employed widely for culculation of the numbers of ordinary living bacilli. As is known, it is very difficult to let tubercle bacilli grow in the depth of solid media, and evaporation of solid medium poured in Petri dish is inevitable for a long period cultivation of tubercle bacilli.The purpose of the present report is to dissolve the difficulties above mentioned, by using the Kirchner agar medium containing some growth promoting substances for tubercle bacilli, and by employing scotch cellulose tape for prevention of drying up of the solid media.