Gas Chromatographic Presumptive Test for Coliform Bacteria in Water

A gas chromatographic procedure which shows promise as a presumptive test for coliform bacteria in water is described. Total coliform bacteria concentrations were determined from the incubation times at 37 C required for ethanol to be produced. Fecal coliform densities were determined in a similar manner at 44.5 C. The culture medium was filter sterilized M-9 salts supplemented with 1% lactose, 0.1% Casamino Acids, and 0.1% yeast extract. Best results were obtained when the initial total coliform concentrations were 5 per ml or higher and when fecal coliform concentrations were 50 per ml or higher. Minimum detection times at these concentrations were 9 and 12 h, respectively.     

Effects of Varying Concentrations of Novobiocin Incorporated into Two Salmonella Plating Media on the Recovery of Four Enterobacteriaceae

Hydrogen sulfide-producing strains of salmonellae, Escherichia coli, Citrobacter freundii, and Proteus mirabilis were isolated from fresh pork sausage. All the strains produced black-centered colonies on Hektoen enteric agar (HE). On xylose lysine deoxycholate agar (XLD), C. freundii produced yellow colonies, and the strains of the other three genera formed black-centered colonies. The selectivity of HE and XLD for salmonellae was improved by the addition of novobiocin to both media. With increasing concentrations of novobiocin, the degree of growth inhibition for the four genera was less on HE than on XLD. Novobiocin concentrations of 80 μg/ml in HE and 5 μg/ml in XLD did not affect the growth or colonial morphology of salmonellae. When 80 μg of novobiocin per ml was incorporated into HE, P. mirabilis strains were not recovered, 40% of C. freundii strains failed to form black-centered colonies, and growth of E. coli strains was not affected but colonies were altered without eliminating the black centers. When novobiocin at 5 μg/ml was incorporated into XLD, colonies of P. mirabilis strains were not recovered, C. freundii formed yellow colonies, and the colonies of the H₂S-producing E. coli strains were unaffected.     

New Method of Isolating Salmonellae from Milk

The use of a cotton gauze swab and subsequent culture of the swab was found to be a more sensitive method for isolating Salmonella from liquid milk than the revised procedure of North. The swab method was found to be as sensitive as the North procedure for recovering Salmonella when incubated at 37 C but more sensitive when incubated at 43 C. Incubation of the swab cultures at the elevated temperature of 43 C gave good results when Salmonella was present at levels as low as one per liter. Swabs exposed to milk contaminated with 100 Salmonella per liter remained positive even when subsequently washed for 2 hr in noncontaminated milk. Bismuth sulfite agar and Brilliant Green sulfadiazine agar were equally effective for isolating Salmonella from broth cultures; use of both media resulted in maximal isolations.     

Variation in Plating Efficiency of Salmonellae on Eight Lots of Brilliant Green Agar

The plating efficiency of Salmonella anatum, S. cubana, S. dublin, S. tennessee, and S. typhimurium was determined for eight lots of Brilliant Green Agar made by two manufacturers. Washed cells were used as the inoculum and cultures were incubated at 41.5 C. All lots of Brilliant Green Agar were supplemented with 12 mg of sulfadiazine per 100 ml of medium. Of the eight lots of Brilliant Green Agar tested, average recovery of the test salmonellae in three did not differ from recoveries with Trypticase Soy Agar, which was used as a control to indicate the number of viable salmonellae in the test suspension capable of growth on a nonselective medium. Two lots of Brilliant Green Agar gave salmonellae recoveries with geometric means about 25% lower than, and significantly different from, those of the control agar. The remaining three lots of Brilliant Green Agar were generally unproductive.     

Growth of Heterotrophic Bacteria and Algal Extracellular Products in Oligotrophic Waters

The unexpected observation of 200 to 400 coliform bacteria per 100 ml in an unpolluted pristine stream was studied within Grand Teton National Park, Wyo. The high numbers of waterborne bacteria occurred in mid- to late summer at a location where there was a coincidental bloom of an algal mat community. Periphyton samplers were used to measure the algal growth that coincided with the increase in number of bacteria. Laboratory studies followed the growth of various coliform bacteria in the supernatant obtained from a Chlorella culture isolated from the mat community. Mixed natural bacterial populations from the stream and pure cultures of water-isolated fecal and nonfecal coliforms increased by two to three orders of magnitude at 13°C when grown in the algal supernatant. Radioactive algal products were obtained by feeding an axenic Chlorella culture ¹⁴C-labeled bicarbonate under laboratory cultivation at 13°C with illumination. Radioactive organic material from the algae became incorporated into the particulate fraction of pure cultures of coliform bacteria as they reproduced and was later released as they died.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 μm to remove large particles and of 0.22 μm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 × 10² CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.     

Bacteriological water quality effects of hydraulically dredging contaminated upper Mississippi River bottom sediment.

Bacteriological effects of hydraulically dredging polluted bottom sediment in the navigation channel of the Upper Mississippi River (river mile 827.5 [about 1,332 km] to 828.1 [about 1,333 km]) were investigated. Bottom sediment in the dredging site contained high total coliform densities (about 6,800 most-probable-number total coliform index per g [dry weight] and 3,800 membrane filter total coliforms per g [dry weight]), and fecal coliforms comprised an average 32% of each total coliform count. Total coliform and fecal coliform densities in water samples taken immediately below the dredge discharge pipe were each approximately four times corresponding upstream values; fecal streptococcus densities were approximately 50 times corresponding upstream values. Correlation analysis indicated that mean turbidity values downstream to the dredging operation were directly and significantly (r greater than 0.94) related to corresponding total coliform, fecal coliform, and fecal streptococcus densities. Salmonellae and shigellae were not recovered from either upstream or downstream water samples. Turbidity and indicator bacteria levels had returned to predredge values within less than 2 km below the dredge spoil discharge area at the prevailing current velocity (about 0.15 m/s).     

Microbiological Quality of Protein Feed Supplements Produced by Rendering Plants

Samples of protein feed supplements produced by rendering plants were examined for salmonellae, total aerobic bacterial counts, coliform counts, and enterococci. Isolations of salmonellae were more frequent from products with high counts; however, 6% of the samples with total counts of less than 1,000 per gram and 14% of the samples with coliform counts of less than 1 per gram contained salmonellae. Serotypes of Escherichia coli which have been associated with disease in domestic animals and poultry were also isolated from products. Although the distribution of serotypes of salmonellae isolated from environmental swabs and flies was similar to that isolated from products, the isolation of several serotypes from flies which had not been isolated in plants suggested that flies may be a potential source of contamination.     

Presence of Culturable Bacteria in Cocoons of the Earthworm Eisenia fetida

Viable bacteria were found to coexist with developing embryos in egg capsules (cocoons) of the earthworm Eisenia fetida. Earthworms were reared under standardized conditions, and bacterial densities were measured in distinct batches of cocoons collected weekly for 10 weeks. Cocoons weighing 12 mg contained a mean viable bacterial population of approximately 10⁸ CFU/g of cocoons. No difference was found in viable counts obtained from cocoons incubated at 15°C and cocoons incubated at 24°C. Viable bacterial numbers increased with cocoon age, while acridine orange direct counts of microbial cells were stable at approximately 10⁹ cells per g of cocoons. Bacteria isolated from cocoons were used to develop antisera in rabbits for the production of strain-specific fluorescent antibodies. Fluorescent antibody and selective plating techniques were used to monitor populations of these bacteria in earthworm bedding and to determine whether cocoons acquire bacteria from the environment in which they are formed. Cocoon isolates were readily recovered from cocoons formed in inoculated bedding at densities of 10⁸ CFU/g of cocoons. Bradyrhizobium japonicum USDA 110 and UMR 161 added to bedding were also recovered from cocoons, but at lower densities than cocoon isolates. Escherichia coli K-12(pJP4) inoculum was recovered from bedding but not from cocoons. The bacterial complement of Eisenia fetida cocoons is affected by inoculation of selected bacterial isolates in the worm growth environment.     

Delayed secondary enrichment for the isolation of salmonellae from broiler chickens and their environment.

Specimens collected from six broiler flocks were cultured for salmonellae by three methods. (i) For direct enrichment, the specimen was homogenized, and 1 ml of the homogenate was inoculated into tetrathionate-brillant green broth; (ii) for preenrichment, liquid specimens and homogenates were incubated at 37 degrees C, and on the next day 1 ml was inoculated into tetrathionate-brillant green broth; and (iii) for delayed secondary enrichment, incubated preenrichment cultures were held at room temperature for 7 to 10 days and then subcultured to fresh tetrathionate-brilliant green broth. All tetrathionate-brilliant green broth cultures were incubated at 42 degrees C for 24 to 48 h before plating. Significantly more isolations of salmonellae were obtained by delayed secondary enrichment than by direct enrichment or preenrichment. Salmonellae were isolated from 417 of 2,283 (18.3%) samples of litter, intestinal contents, and feces cultured by all three methods. Of these positive specimens, direct enrichment detected 208 (49.9%), preenrichment detected 282 (67.6%), and delayed secondary enrichment detected 373 (89.4%). Of 896 specimens of swabs and rinse fluids that were cultured by preenrichment and delayed secondary enrichment, 259 (28.9%) yielded salmonellae. Delayed secondary enrichment detected 254 (98.1%) of these, and preenrichment detected 147 (56.8%). A total of 23 serotypes of salmonellae were identified. The greater effectiveness of delayed secondary enrichment for the isolation of salmonellae was not likely due to the selection of certain serotypes or to an increased inhibition of competing flora.     

Destruction of Enteric Bacteria in Liquid Egg with β-Propiolactone

Liquid whole egg or egg white, inoculated with Escherichia coli 1485, Salmonella senftenberg ATCC 8400, or Salmonella typhimurium 84-I, was treated with concentrations of β-propiolactone ranging from 0.05 to 0.3%. Egg white containing 1 × 10³ to 1 × 10⁶ cells of E. coli 1485 per ml was sterilized in 1 hr at 27 C by lactone concentrations of 0.2 and 0.3%. Egg white containing 1 × 10⁵ cells of S. senftenberg ATCC 8400 per ml was sterilized in 12 hr at 10 C by 0.1% lactone and in 2 to 3 hr by 0.3% lactone at the same temperature.

Liquid whole egg inoculated with 1 × 10⁵ cells of either species of Salmonella was sterilized in 4 to 5 hr at 10 C with 0.2% lactone or in 2 to 3 hr by 0.3% lactone at this temperature. A mild heat treatment of either 15 min at 37 C or 1 min at 55 C markedly shortened the exposure times required for sterilization by β-propiolactone at 10 C.

After disinfection was complete, the lactone-treated liquid whole egg was reinoculated with low cell numbers of either species of Salmonella to determine the presence of residual lactone or toxic products. Liquid whole egg treated with 0.2% lactone would support the growth of salmonellae after 13 to 14 hr at 10 C. A heat treatment of 45 min at 37 C or 10 min at 55 C immediately after addition of 0.2% lactone allowed growth of the salmonellae in the lactone-treated liquid whole egg. No evidence of residual toxicity from the lactone treatment was found.

The amount of lactone needed to prevent the outgrowth of low cell numbers of either strain of Salmonella in liquid whole egg was quantitated. Liquid whole egg containing 0.06 to 0.07% lactone would not support salmonellae growth from inocula of 1 to 10 cells per ml of egg. Lactone concentrations above 0.08% prevented outgrowth of salmonellae inocula of 10 to 200 cells per ml of liquid whole egg.

     

Effect of Type of Enrichment and Duration of Incubation on Salmonella Recovery from Meat-and-Bone Meal

Twenty-five meat-and-bone meal samples were enriched with either selenite-cystine or tetrathionate and incubated for 1 and 2 days. Seven were previously found to be positive; of the other 18, 16 were positive for salmonella. The number of somatic serogroups per sample ranged from 1 to 11 with a mean of 3.8. Significantly more (P < 0.01) group C₁ salmonellae were isolated using tetrathionate than selenite, whereas significantly more of groups G, 35, and Difco poly-valent D were isolated from selenite than tetrathionate. Seventy-six percent of the presumptive colonies from Brilliant Green agar showed a positive lysine decarboxylase reaction, and there were no differences between media or times of incubation. Ninety-four per cent of the lysine decarboxylase-positive cultures showed a positive somatic antiserum response; again there were no differences between times or enrichments although there were significantly more total positive serogroups at 2 days than at 1 day from tetrathionate but not from selenite. There were indications that certain serogroups preferred either one or the other enrichment. There were no differences in total positive samples with the two enrichments although neither alone was sufficient to identify all positives. Several lactose-positive salmonellae were recovered.     

Thermal Resistance of Salmonellae Isolated from Dry Milk

Salmonella anatum, S. binza, S. cubana, S. meleagridis, S. newbrunswick, and S. tennessee isolated from dry milk, and S. senftenberg 775W were studied for heat resistance to determine whether these organisms would survive pasteurization as recommended by the 1965 Pasteurized Milk Ordinance of the U.S. Public Health Service. Thermal inactivation determinations were made on washed cells of the test microorganisms suspended in sterile whole milk. Excluding S. senftenberg, D values ranged from 3.6 to 5.7 sec at 62.8 C, from 1.1 to 1.8 sec at 65.6 C, and from 0.28 to 0.52 sec at 68.3 C. Corresponding values for S. senftenberg were 34.0, 10.0, 1.2, and 0.55 sec for respective exposure temperatures of 65.5, 68.3, 71.7, and 73.9 C. The present milk pasteurization processes as recommended by the Public Health Service will inactivate all seven strains of salmonellae studied, provided that the initial concentration does not exceed a calculated 3 × 10¹² salmonellae per ml of milk.     

Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay.

The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).     

Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay.

The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).     

Frequency distribution of coliforms in water distribution systems.

Nine small water distribution systems were sampled intensively to determine the patterns of dispersion of coliforms. The frequency distributions of confirmed coliform counts were compatible with either the negative-binomial or the lognormal distribution. They were not compatible with either the Poisson or Poisson-plus-added zeroes distribution. The implications of the use of the lognormal distributional model were further evaluated because of its previous use in water quality studies. The geometric means from 14 data sets ranged from 10(-6) to 0.2 coliforms per 100 ml, and the geometric standard deviations were between 10 and 100, with one exception. If the lognormal model is representative of the coliform distribution; the arithmetic mean sample count is a poor estimator of the true mean coliform density, and the probability of water in a distribution system containing small patches with large coliform densities without detection by routine monitoring is finite. These conclusions have direct bearing on the interpretation of microbiological quality standards for drinking water.     

Evaluating the Membrane Fecal Coliform Test by Using Escherichia coli as the Indicator Organism

The fecal coliform membrane filter method (MFC) currently used in water pollution analysis was evaluated by using two strains of Escherichia coli, a known fecal coliform, as the indicator organism. A large relative error in the results obtained with this method was found to be dependent upon the brand of membrane filter employed, the medium, and the temperature of incubation. MFC densities varied between 10 and 60% of the densities determined by means of total bacteria counts and total coliform counts performed at 35 C. Due to the large relative error encountered, the MFC method cannot be recommended as an analytical tool for the laboratory enumeration of E. coli. The results do show that the MFC method can be used at 35 C for enumeration of E. coli and for differential counts of E. coli and Enterobacter aerogenes.     

Yeasts from Marine and Estuarine Waters with Different Levels of Pollution in the State of Rio de Janeiro, Brazil

Yeast counts were made at 24 marine and estuarine sites in the vicinity of Rio de Janeiro, Brazil. Mean salinities of estuarine sites ranged from 14.2 to 27.4‰, and mean temperatures ranged from 25 to 28°C. Total coliform counts varied from 80% above 100,000 colony-forming units (CFU)/100 ml at heavily polluted sites to 100% below 100 CFU/100 ml at unpolluted sites. Total yeast counts above 100 CFU/100 ml were typical of heavily and moderately polluted water but atypical of lightly polluted and unpolluted water. Mean total yeast counts were 2,880 CFU/100 ml for heavily polluted sites, 202 CFU/100 ml for moderately polluted sites, and 3 CFU/100 ml for lightly polluted and unpolluted sites. Total yeast counts had a positive response to increased pollution levels, and Candida krusei and phenotypically similar yeasts as a group were prevalent in polluted estuarine water but rare in unpolluted seawater. The 549 strains of yeasts and yeast-like organisms isolated were grouped into 67 species, of which the 21 most prevalent made up 86% of the total yeast population. The prevalent genera in the polluted estuary were Candida, Rhodotorula, Torulopsis, Hanseniaspora, Debaryomyces, and Trichosporon.     

Salmonellae Associated with Further-processed Turkey Products

"Further-processed" turkey products, prepared from chilled, eviscerated, and thawed carcasses at two commercial turkey-processing plants, were evaluated, for the presence of salmonellae. These organisms were isolated from swab samples from 12% of chilled, eviscerated turkey carcasses, 27% of finished products, and 24% of processing equipment. The same serotypes as those found throughout a plant on any one visit were recovered from 31% of rinse-samples taken from hands and gloves of processing personnel. Salmonellae were found in samples taken on 37 of 48 visits; a greater number of recoveries were made on days when freshly killed turkeys were processed (87%) than when frozen-defrosted carcasses were processed (59%). The predominant serotype isolated from meat and environment usually changed from visit to visit. Salmonella sandiego and Salmonella anatum were the most frequent among 23 serotypes recovered. Most of the isolated serotypes are commonly associated with turkeys and have been incriminated as causative agents of human salmonellosis. The implication is that further-processed turkey products, if inadequately cooked by the consumer and if improperly refrigerated between the time of manufacture and consumption, could directly transmit salmonellae. These same products might also contaminate other foods by introducing salmonellae into food-preparation areas.