Optimal growth temperature for the isolation of Plesiomonas shigelloides, using various selective and differential agars.

The growth characteristics of known strains of Plesiomonas shigelloides were compared with those of Aeromonas species (the major competing species in environmental waters) on plesiomonas differential agar, inositol brilliant green bile salt, and modified salmonella-shigella agar at incubation temperatures of 37, 42, and 44 degrees C. Using local isolates from clinical and environmental sources, optimal growth conditions, as determined by colony counts and the colony characteristics, plesiomonas differential agar proved to be ideal when incubated at 44 degrees C. Contrary to earlier recommendations for 48 h incubation, the colonies could be recognized readily after an incubation of 24 h.     

USE of an ELECTRONIC HGMF INTERPRETER SYSTEM FOR ENUMERATION of NALIDIXIC ACID RESISTANT SALMONELLAE IN CHICKEN CAECA

An electronic counting system using hydrophobic grid-membrane filters (HGMF) and the HGMF Interpreter was evaluated for its usefulness in enumerating nalidixic acid resistant Salmonella in frozen chicken caeca. Salmonella recovery was equivalent on both Hektoen Enteric and EF-18 agars. However, the color of the Salmonella growth on EF-18 agar was more easily differentiated by the HGMF Interpreter electronic counting system. the study showed that a 4 h resuscitation on a nonselective medium was required in order to maximize the subsequent recovery on Hektoen Enteric agar, though not on EF-18 agar. Using the EF-18 agar as the Salmonella selective medium, a method was established that recorded counts of nalidixic acid resistant Salmonella rapidly and easily in electronic data files, for subsequent retrieval and manipulation.     

pH gradients through colonies of Bacillus cereus and the surrounding agar.

pH-sensitive microelectrodes, constructed with a tip diameter of about 4 microns, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 microns into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1.45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.     

ENUMERATING 3MTM PETRIFILMTM AEROBIC COUNT PLATES USING THE PETRISCAN® AUTOMATED COLONY COUNTER

In the food and dairy industries, aerobic plate counts are determined by a time-consuming and laborious hand-counting method. The PetriScan ® automated colony counter was developed to improve efficiency in the microbiology laboratory. In this study, colony counts of food, dairy, and milk products plated on 3M^TM Petrifilm^TM Aerobic Count Plates were compared using both automated and manual count plate methods. For sample variation, 16 different food, dairy, and milk products were used. Samples were prepared and serially diluted using Butterfield's diluent according to approved AOAC methods and APHA's Standard Methods. Plates were inoculated, incubated, and counted according to AOAC methods. For data collection, plates with counts between 5 and 300 colonies were included. A total of 55 low (5–30), 29 medium (31–100), and 23 high (101–300) count plates were used. Duplicate results were recorded for both methods; hand counts were tallied by two scientists. The duplicates of the mean log values for manual counts varied by 0.0005 and 0.0007, and the duplicates for the automated counts varied by 0.0011. The mean log value difference between the automated and manual counts for pooled data was 0.035. The correlation coefficient for the regression line comparing the automated and manual count methods for pooled data was 0.98. The regression equation was y = 0.9257x + 0.0781. These results demonstrate that the PetriScan® automated colony counter is a comparable and practical alternative to the standard method of manually counting plates.     

Evaluation of Media, Time and Temperature of Incubation, and Method of Enumeration of Several Strains of Clostridium perfringens Spores

Two basal media, containing the ingredients found in common in both SPS (BBL) and TSN (BBL) media and in the previously described media of Schaedler et al. (1965) and Starr et al (1971), but minus antibiotics, were selected as the most suitable for the enumeration of Clostridium perfringens spores in a model system. These media were also used to study the influence of the presence of glucose, xylose, or ribose in various concentrations (0, 0.01, 0.1, and 1.0%) on colony morphology and spore recovery. As the sugar concentration in the basal agar medium increased, the colonies of all the test organisms also increased in size, and more of the black colonies became white in color. At the 1.0% sugar level, glucose permitted only white colony development, whereas the pentoses were completely inhibitory. Both pour plates and most-probable-number tubes were inoculated with the spores of several strains of C. perfringens and incubated at 20, 30, 37, and 45 C for 24, 48, and 72 h. Statistical analyses of the enumeration data indicated, at the 99% confidence level, that a Trypticase(BBL)-yeast extract-glucose-sulfite-iron agar gave maximal population estimates at 37 C in 72 h.     

Stimulation of the growth of cutaneous strains of Peptococcus saccharolyticus by iron, haematin and blood

The growth of nine strains of Peptococcus saccharolyticus was assessed quantitatively by culture Trypticase Soy/yeast extract/Tween 80 agar (TSY-TW) with and without supplementation with iron or haematin and on blood agar, in aerobic, reduced 02 (3% O2 with 8% CO2, 8% H2 and 81% N2) and anaerobic atmospheres. All strains grew better anaerobically and under reduced O2 conditions than aerobically on supplemented or unsupplemented TSY-TW.Supplementation of TSY-TW with iron or haematin resulted in an average 4.4-fold increase in bacterial count in a reduced O2 atmosphere and an average 4.2-fold increase under anaerobic conditions. Under aerobic conditions the increase in count ranged from O to greater than 5000-fold, as some strains failed to grow on unsupplemented TSY-TW but responded well to the supplements of iron or haematin. The highest bacterial counts were obtained on Columbia blood agar incubated anaerobically. However, P. saccharolyticus failed to grow aerobically on plain or heated Columbia blood agar with or without supplements. TSY-TW blood agar supported the growth of the one strain tested under all three atmospheric conditions. The type strain (ATCC 14953) differed from all others in its failure to grow aerobically or in a reduced O2 atmosphere on supplement or unsupplemented media. Colony size varied greatly on different media, in different atmospheres and from strain to strain, being greatest in a reduced O2 atmosphere on Columbia blood agar. There was no correlation between the viable bacterial count and colony size.     

Anaerobic Roll Tube Media for Nonselective Enumeration and Isolation of Bacteria in Human Feces

Medium 10 (M10), developed for rumen bacteria and containing small amounts of sugars, starch, volatile fatty acids, hemin, Trypticase, yeast extract, cysteine, and sulfide, plus agar, minerals and CO(2)-HCO(3)-buffer, was used with the Hungate anaerobic method as a basal medium to evaluate the efficacy of various ingredients. Three-day-old colony counts from adults on normal diets (17 samples) were 0.55 x 10(11) to 1.7 x 10(11) per g (mean, 1.15 x 10(11)) for M10. Single deletion of volatile fatty acids, Trypticase, yeast extract, or sulfide did not reduce counts. Deletion of hemin or both Trypticase and yeast extract significantly lowered counts. Addition of fecal extract, rumen fluid, 1% dehydrated Brain Heart Infusion (BHI) or 2 to 6% liver infusion did not increase counts; 1% dehydrated bile or 3.7% BHI markedly depressed them. Decreasing the gas-phase CO(2) concentration from 100 to 5% with N(2) and correspondingly lowering the HCO(3) had little effect. Counts in supplemented Brewer Thioglycollate (Difco), BHI, and Trypticase soy agar were similar or lower than in M10; ease in counting was best in M10. Comparison of features of 88 predominant strains of fecal bacteria randomly isolated indicated that M10 supported growth of as many or more species of bacteria as compared to supplemented BHI. The results suggest that predominant bacteria of human feces, in general, are not as nutritionally fastidious as rumen bacteria and indicate that media for counts or isolation containing large amounts of rich organic materials are neither necessary nor desirable when adequate anaerobic techniques are used.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37 degrees C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37 degrees C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37 degrees C for 48 h is recommended.     

Spiral Plate Method for Bacterial Determination

A method is described for determining the number of bacteria in a solution by the use of a machine which deposits a known volume of sample on a rotating agar plate in an ever decreasing amount in the form of an Archimedes spiral. After the sample is incubated, different colony densities are apparent on the surface of the plate. A modified counting grid is described which relates area of the plate of volume of sample. By counting an appropriate area of the plate, the number of bacteria in the sample is estimated. This method was compared to the pour plate procedure with the use of pure and mixed cultures in water and milk. The results did not demonstrate a significant difference in variance between duplicates at the α = 0.01 level when concentrations of 600 to 12 × 10⁵ bacteria per ml were used, but the spiral plate method gave counts that were higher than counts obtained by the pour plate method. The time and materials required for this method are substantially less than those required for the conventional aerobic pour plate procedure.     

Evaluation of a chromogenic medium supplemented with glucose for detecting Enterobacter sakazakii

A commercial chromogenic agar medium (DFI) was supplemented with glucose (mDFI) to enhance the specificity of Enterobacter sakazakii (E. sakazakii) detection. Escherichia vulneris (E. vulneris), a putative false-positive strain on the DFI medium, produces alpha-glucosidase. The enzyme alpha- glucosidase hydrolyzes a substrate, 5-bromo-4-chloro-3- indolyl-alpha,D-glucopyranoside (XalphaGlc), producing green colonies. E. sakazakii strains produced green colonies on both DFI and mDFI agar, whereas E. vulneris produced green colonies on DFI agar but small white colonies on mDFI agar. E. sakazakii and E. vulneris were also readily differentiated by colony color when the mixed culture of the two strains was plated on mDFI agar and incubated for 24 h at 37 degrees C. The results indicate that the selectivity of the commercial chromogenic agar medium could be improved by a simple supplementation with glucose.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and Group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37°C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37°C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37°C for 48 h is recommended.     

Gentamicin-based medium for the isolation of group D streptococci and application of the medium to water analysis.

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH(2)PO(4), 2.0 g of NaHCO(2), 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO(3) (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Comparison of respiratory activity and culturability during monochloramine disinfection of binary population biofilms.

Biofilm bacteria challenged with monochloramine retained significant respiratory activity, even though they could not be cultured on agar plates. Microbial colony counts on agar media declined by approximately 99.9% after 1 h of disinfection, whereas the number of bacteria stained by a fluorescent redox dye experienced a 93% reduction. Integrated measures of biofilm respiratory activity, including net oxygen and glucose utilization rates, showed only a 10 to 15% reduction. In this biofilm system, measures of microbial respiratory activity and culturability yielded widely differing estimates of biocide efficacy.     

Inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> and coliform bacteria in traditional brass and earthernware water storage vessels

The detection and enumeration of indicator bacteria such as Escherichia coli is used to assess the extent of faecal contamination of drinking water. On the basis of this approach, the effectiveness of storing water contaminated with faecal indicator bacteria in brass or earthern vessels (mutkas) of the type used in rural India have been investigated. Suspensions of bacteria in sterile distilled water were maintained for up to 48 h in each vessel and enumerated by surface plate counts on nutrient agar (non-selective) and several selective coliform media at 37 °C either under standard aerobic conditions, or under conditions designed to neutralise reactive oxygen species (ROS), e.g. using an anaerobic cabinet to prepare plates of pre-reduced growth medium or by inclusion of sodium pyruvate in the growth medium, with incubation of aerobically-prepared plates in an anaerobic jar. The counts obtained for E. coli decreased on short-term storage in a brass mutka; counts for selective media were lower than for equivalent counts for non-selective medium, with ROS-neutralised conditions giving consistently higher counts than aerobic incubation. However, after 48 h, no bacteria were cultivable under any conditions. Similar results were obtained using water from environmental sources in the Panjab, and from rural households where brass and earthern mutkas are used for storage of drinking water, with enumeration on selective coliform media (presumptive total coliforms). In all cases results indicated that, while storage of water in a brass mutka can inactivate E. coli and coliforms over a 48 h period, standard aerobic plate counting using selective media may not be fully effective in enumerating sub-lethally damaged bacteria.     

Gentamicin-Based Medium for the Isolation of Group D Streptococci and Application of the Medium to Water Analysis

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH₂PO₄, 2.0 g of NaHCO₂, 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO₃ (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Modified microdilution antimicrobial susceptibility test for anaerobic bacteria

A modified microdilution antimicrobial susceptibility test is described for anaerobic bacteria. The microdilution procedure at 24 and 48 h of incubation was compared with agar dilution using Wilkins-Chalgren (WC) broth and agar respectively. Results showed a total of 12.2% minor and major discrepancies with the microdilution test at 24 h incubation and 7.8% at 48 h. If anaerobic isolates are to be tested for antimicrobial susceptibility, then the 24-h microdilution procedure is an acceptable alternative to agar dilution.     

Agar Concentration in Counting Clostridium Colonies

Decreasing the agar concentration of a counting medium from the usual 1.5% resulted in larger colonies with less interference from gas in Clostridium botulinum 115B and C. sporogenes PA 3679. Optimal agar concentration was 0.65% for C. botulinum with 24-hr incubation and 0.50% for C. sporogenes with 48-hr incubation. Lower concentrations yielded growth too diffuse for counting. Motility was considered the explanation for increased colony size in softer agar. The greater the degree of motility, the greater would be the diffusibility expected, and thus the higher the agar concentration required to insure discrete colonies. For quantitating motility, evaluations were made by use of microscopic examination of liquid cultures and rate of diffusion in a semisolid medium. With both criteria, the degree of motility of C. botulinum 115B clearly exceeded that of C. sporogenes PA 3679. Small-colony variants of C. botulinum in 0.65% agar yielded only small colonies on subculture, with a corresponding decrease in degree of motility of the cells by both criteria. Colony size of the nonmotile C. perfringens ATCC 3624 was unaffected by lowered agar concentrations.     

Rapid high-throughput assessment of aerobic bacteria in complex samples by fluorescence-based oxygen respirometry

A simple method has been developed for the analysis of aerobic bacteria in complex samples such as broth and food homogenates. It employs commercial phosphorescent oxygen-sensitive probes to monitor oxygen consumption of samples containing bacteria using standard microtiter plates and fluorescence plate readers. As bacteria grow in aqueous medium, at certain points they begin to deplete dissolved oxygen, which is seen as an increase in probe fluorescence above baseline signal. The time required to reach threshold signal is used to either enumerate bacteria based on a predetermined calibration or to assess the effects of various effectors on the growth of test bacteria by comparison with an untreated control. This method allows for the sensitive (down to a single cell), rapid (0.5 to 12 h) enumeration of aerobic bacteria without the need to conduct lengthy (48 to 72 h) and tedious colony counts on agar plates. It also allows for screening a wide range of chemical and environmental samples for their toxicity. These assays have been validated with different bacteria, including Escherichia coli, Micrococcus luteus, and Pseudomonas fluorescens, with the enumeration of total viable counts in broth and industrial food samples (packaged ham, chicken, and mince meat), and comparison with established agar plating and optical-density-at-600-nm assays has been given.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.