Comparison of methods for enumeration of selected coliforms exposed to ozone.

mT7 medium performed no better than m-Endo medium in enumerating cells of Escherichia coli and Citrobacter freundii exposed to ozone. Also, there was no difference in the plate count of heterotrophic bacteria in ozonated raw water determined on modified Henrici agar or R2A agar. Statistically significant differences were seen between bacteria and the type of water in which they were suspended during ozonation.     

Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies

A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.     

Monensin-based medium for determination of total gram-negative bacteria and Escherichia coli

Plate count-monensin-KCl (PMK) agar, for enumeration of both gram-negative bacteria and Escherichia coli, is composed of (per liter) 23.5 g of plate count agar, 35 mg of monensin, 7.5 g of KCl, and 75 mg of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Monensin was added after the medium was sterilized. The diluent of choice for use with PMK agar was 0.1% peptone (pH 6.8); other diluents were unsatisfactory. Gram-negative bacteria (selected for by the ionophore monensin) can be used to judge the general quality or sanitary history of a commodity. E. coli (differentiated by its ability to hydrolyze the fluorogenic compound MUG) can be used to assess the safety of a commodity in regard to the possible presence of enteric pathogens. Pure-culture studies demonstrated that monensin completely inhibited gram-positive bacteria and had little or no effect on gram-negative bacteria. When gram-negative bacteria were injured by one of several methods, a few species (including E. coli) became sensitive to monensin; this sensitivity was completely reversed in most instances by the inclusion of KCl in the medium. When PMK agar was tested with food and environmental samples, 96% of 535 isolates were gram negative; approximately 68% of colonies from nonselective medium were gram negative. PMK agar was more selective than two other media against gram-positive bacteria and was less inhibitory for gram-negative bacteria. However, with water samples, KCl had an inhibitory effect on gram-negative bacteria, and it should therefore be deleted from monensin-containing medium for water analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monensin-based medium for determination of total gram-negative bacteria and Escherichia coli.

Plate count-monensin-KCl (PMK) agar, for enumeration of both gram-negative bacteria and Escherichia coli, is composed of (per liter) 23.5 g of plate count agar, 35 mg of monensin, 7.5 g of KCl, and 75 mg of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Monensin was added after the medium was sterilized. The diluent of choice for use with PMK agar was 0.1% peptone (pH 6.8); other diluents were unsatisfactory. Gram-negative bacteria (selected for by the ionophore monensin) can be used to judge the general quality or sanitary history of a commodity. E. coli (differentiated by its ability to hydrolyze the fluorogenic compound MUG) can be used to assess the safety of a commodity in regard to the possible presence of enteric pathogens. Pure-culture studies demonstrated that monensin completely inhibited gram-positive bacteria and had little or no effect on gram-negative bacteria. When gram-negative bacteria were injured by one of several methods, a few species (including E. coli) became sensitive to monensin; this sensitivity was completely reversed in most instances by the inclusion of KCl in the medium. When PMK agar was tested with food and environmental samples, 96% of 535 isolates were gram negative; approximately 68% of colonies from nonselective medium were gram negative. PMK agar was more selective than two other media against gram-positive bacteria and was less inhibitory for gram-negative bacteria. However, with water samples, KCl had an inhibitory effect on gram-negative bacteria, and it should therefore be deleted from monensin-containing medium for water analysis.(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)     

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 media for enumeration of coliform bacteria and Escherichia coli in non-disinfected water

In this work alternative media for detection and enumeration of E. coli and coliform bacteria were compared to the reference method ISO 9308-1 (LTTC) using non-disinfected water samples with background flora. The alternative media included LES Endo agar medium (LES Endo), Colilert-18 with 51-well Quanti-tray (Colilert), Chromocult Coliform agar (CC), Harlequin E. coli/Coliform medium (HECM) and Chromogenic Escherichia coli/Coliform medium (CECM). A total of 110 samples of groundwater, bathing water and spiked water was used. Our results revealed that confirmation of coliform bacteria counts is necessary, not only on lactose-based LTTC and LES Endo media, but also on the chromogenic agar media tested, due to the growth of oxidase positive colonies. LTTC and CC media also allowed the growth of some morphologically typical coliform colonies containing gram-positive bacteria. The recovery of coliform bacteria was lower on LES Endo than on LTTC. In most cases Colilert, CC, HECM and CECM gave higher coliform counts than LTTC. The use of the LTTC medium led to higher E. coli counts than obtained with any of the alternative mediums. There are three explanations for this: (1) high sensitivity of LTTC, (2) false positives on LTTC or (3) false negatives especially with Colilert, but also with chromogenic agar media. Although LTTC was found to be a very sensitive medium, the high degree of background growth of non-disinfected waters disturbed substantially the use of it. In conclusion, our results suggest that Colilert, CC and CECM are potential alternative media for detection of coliform bacteria and E. coli from non-disinfected water.     

The antibacterial effect of river water onShigella Shigae in connection with the presence of corresponding antagonists and bacteriophages

Summary and conclusions It is emphasized that in the Tjiliwung river water no antagonists against the occurring typhoid bacteria are to be detected. Bacterial antagonists against dysentery bacteria can, however, easily be isolated, whereas the latter could not be isolated from the water by means of S.S. medium orLeifson plates. This elicited the question whether antibiotic substances in nature might cause a detrimental effect onShigella shigae.From 2054 colonies of water bacteria on broth agar 294 strains with an antagonistic effect on shigae bacteria were cultivated with methods based on the agar streak, the perforated Petri dish and that ofKelner. Among 3419 other colonies isolated from the same water samples no antagonists against typhoid bacteria could be detected. The method ofKelner proved 14% of the water bacteria growing on broth agar to be antagonistic against shigae bacteria. Some of these antagonists lost their activity soon after subinoculation on broth agar.In 14 out of 16 samples of Tjiliwung water a filtrable thermolabile agent could be detected, detrimental toSh. shigae when inoculated fairly profusely and in the presence of organic material (extract broth 1 : 10). The filtrated agent reduced the survival period ofSh. shigae to a maximum of 1/360 of the test period and inhibited the growth of these bacteria up to a dilution of 1 : 256. Heating at 100°C. destroyed the agent. In all cases this thermolabile anti-bacterial factor was associated with the presence of shigae phage and did not occur in two samples lacking the latter. The agent could not be separated from the phage particles by means of phage-tight glacial acid collodion filters through which human hemoglobine passes easily. The anti-shigae factor acted as a bacteriophage, not as an antibiotic. Propagation of the phage in three successive cultures could be demonstrated.No anti-typhoid agent was found in the filtrates lacking typhoid phages. It is concluded that the unheated river water filtrates owe their anti-shigae action mainly to the presence of corresponding phages. It is possible that in nature other factors may change the influence of the shigae-phage. Anti-bacterial substances produced by the numerous antagonists present are of no importance for the reduction in number of shigae bacteria in the river water.     

A STUDY OF THE PECTOLYTIC BACTERIAL POPULATION IN SOME FARM WATER SUPPLIES

SUMMARY: Pectolytic bacteria in considerable numbers were detected in samples from farm water supplies. Two media were employed, and quantitative data were obtained, in both cases, but colony counts on sodium pectate-calcium agar poured plates gave higher numbers than a presumptive count in sodium pectate solution. The agar medium also permitted the growth of non-pectolytic bacteria, and the total colony count was higher than that obtained on standard nutrient agar. Presumptive coliaerogenes counts in broth tubes showed that these organisms were, on the average, about four times as numerous as the pectolytic organisms.     

Evaluation of Standard and Modified M-FC, MacConkey, and Teepol Media for Membrane Filtration Counting of Fecal Coliforms in Water

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined.     

Oligotrophic bacteria in ultra-pure water systems: Media selection and process component evaluations

Summary Presently, tryptic soy agar (TSA) medium is used in the semiconductor industry to determine the concentration of viable oligotrophic bacteria in ultra-pure water systems. Deionized water from an ultra-pure water pilot plant was evaluated for bacterial growth at specific locations, using a non-selective medium (R2A) designed to detect injured heterotrophic as well as oligotrophic bacteria. Results were compared to those obtained using Tryptic Soy Agar. Statistically greater numbers of bacteria were observed when R2A was used as the growth medium. Total viable bacterial numbers were compared both before and after each treatment step of the recirculating loop to determine their effectiveness in removing bacteria. The reduction in bacterial numbers for the reverse osmosis unit, the ion exchange bed, and the ultraviolet sterilizer were 97.4%, 31.3%, and 72.8%, respectively, using TSA medium, and 98.4%, 78.4%, and 35.8% using R2A medium. The number of viable bacteria increased by 60.7% based on TSA medium and 15.7% based on R2A medium after passage of the water through an in-line 0.2-m pore size nylon filter, probably because of the growth of bacteria on the filter. Our results suggest that R2A medium may give a better representation of the microbial water quality in ultra-pure water systems and therefore a better idea of the effectiveness of the various treatment processes in the control of bacteria.     

Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay.

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.     

New medium for the simultaneous detection of total coliforms and Escherichia coli in water.

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.     

A method to determine protein of bacteria grown in agar

A method is described to determine the amount of protein of bacteria grown in agar. Amino acid contents of hydrolysates of agar cultures are compared with the amino acids in hydrolysates of the same bacteria grown in a liquid medium. In the latter medium also the amount of bacterial protein is determined. From these data the protein content of bacteria grown in agar can be calculated.     

Novel filamentous spore-forming iron bacteria causes bulking in activated sludge

Several Gram-positive iron bacteria were isolated from bulking sludge. They were filamentous and had false branching. They had sheaths with iron deposits and formed spores on modified sucrose casitone yeast extract agar. They did not grow on influsion agar for longer than 1 month but could withstand 80°C for 1 h on the same medium. Adding them to sewage before aeration increased the biochemical oxygen demand of waste water and caused poor settling properties of activated sludges. They were the predominant filamentous micro-organisms in bulking activated sludges. At present, these strains cannot be assigned to recognized taxa of Bacillus spp. or sheathed iron bacteria.     

Complex medium toxicity to some DNA repair-deficient strains of Salmonella typhimurium.

The recovery of several strains of Salmonella typhimurium LT-2 which had first been grown in minimal medium varies when the organisms are grown on minimal medium agar and complex medium agar. The strains tested included mutants with deficiencies in DNA-repair systems (uvrB-and rec-), a deep rough (rfa-) mutant, and a double mutant carrying both the uvrB- and the rfa-mutation. The uvrB- and rec-mutations imparted sensitivity to complex medium agar. The rfa-mutation suppressed the sensitivity of the uvrB-mutant to complex medium agar. Differences in colony-forming ability were not observed when the bacteria were first grown in the complex medium broth.     

国家标准测定食品细菌总数培养基的改进研究

应用我国国家标准营养琼脂（GB4789 2－94简称NA）与美国食品药品管理局（FDA）标准平析以（简称SA）两种培养基对动植物食品中细菌总数进行检测对比,结果表明FDA标准平板比GＢ营养琼脂效果较好,前比后的检出率高出２３．９％,且菌落大而明显,为此,对这两种培养基进行优化筛选试验,并优选出Ｃ８培养基,扩大试验结果表明C8培养基的检出率较GB营养琼脂及FDA标准平板分别高出35．8％和9．5％。     

Agar Plate Method for Detection and Enumeration of Alkylbenzenesulfonate-Degrading Microorganisms

A simple method for detection and enumeration of alkylbenzenesulfonate (ABS)-degrading microorganisms by using agar plates was developed and used in microbiological studies of coastal marine and polluted river waters. The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution were sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appeared around the colonies of ABS-degrading microorganisms against a pink background. Viable counts of ABS-degrading bacteria isolated from both seawater and freshwater environments were considerably higher in polluted waters than in less polluted areas. Viable counts of ABS-degrading bacteria averaged 1.5 x 105/ml in samples from the surface water of polluted Tokyo Bay and 3.0 x 104/ml in samples from the surface water of polluted Tamagawa River but were fewer in number in samples from less polluted waters.     

Detection and identification of groundwater bacteria capable of escaping entrapment on 0.45-micron-pore-size membrane filters.

Rural drinking water systems supplied by untreated groundwater were examined to determine whether coliform or heterotrophic plate count bacteria are capable of escaping entrapment on standard porosity (0.45-micron-pore-size) membrane filters. Filterable bacteria were present in 42% of the 24 groundwater sources examined by using nonselective media (R2A, full strength m-HPC, and 0.1x m-HPC agars). Pseudomonads were the most frequently identified group of filterable bacteria detected. Flavobacterium, Alcaligenes, Acinetobacter, and Achromobacter isolates were also identified. Total coliforms were not recovered from any of the 24 groundwater samples following filtration through 0.45-micron-pore-size membrane filters by using selective M-Endo LES agar or mT7 agar. In addition, none of the isolates identified from nonselective media were coliforms. Similarly, neither total coliforms nor specifically Escherichia coli were detected in these filtrates when Colilert P/A medium was used.     

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.