Gentamicin-thallous-carbonate medium for isolation of fecal streptococci from foods.

Gentamicin-thallous-carbonate (GTC) agar was formulated by Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978) to select for fecal streptococci in sewage and water samples. The present study was conducted to determine the usefulness of GTC agar for the enumeration of fecal streptococci in foods. Comparisons were made with KF streptococcal (KF), Pfizer selective enterococcus (PSE), and thallous acetate (TA) agars. Samples of ground beef pork sausage, frozen broccoli, frozen fish, and ice cream were examined. Presumptive streptococcal counts obtained on GTC agar were significantly higher than those obtained on KF and PSE agars and were comparable to those obtained on TA agar. GTC was more sensitive than KF or PSE agars primarily because of the recovery of greater numbers of Streptococcus bovis and Streptococcus equinus strains. Percentages of confirmed fecal streptococci obtained on GTC, KF, PSE, and TA agars were 70, 95, 80, and 74, respectively. Differences between these percentages were not statistically significant, but they indicated that selectivity of GTC agar could be improved. Advantages of using GTC agar to isolate fecal streptococci from foods include a short incubation time (16 to 18 h) and large, distinct colonies that facilitate rapid enumeration and subsequent confirmation.     

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.     

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.     

Evaluation of Pfizer selective enterococcus and KF media for recovery of fecal streptococci from water by membrane filtration.

Pfizer selective enterococcus (PSE) and KF agars were compared for their recovery of fecal streptococci from sewage effluent on membrane filters. The results showed that PSE agar is highly selective for the enterococci. The tan color resulting from esculin hydrolysis, which was not always visible on the surfaces of the colonies, is not considered a necessary differential characteristic on PSE agar since more than 90% of all colonies recovered on membrane filters were confirmed as fecal streptococci and 86% were confirmed as enterococci. The detection of esculin hydrolysis on membrane filters was not improved by using the new Millipore type HC filter. KF agar recovered significantly greater numbers of organisms but was not as selective, with 83% of the typical colonies being confirmed as fecal streptococci and 54% as enterococci. An attempt to improve the selectivity of KF agar while retaining its inclusiveness by incubation at 45 C was not successful.     

Pfizer Selective Enterococcus Agar Overlay Method for the Enumeration of Fecal Streptococci by Membrane Filtration

The use of Pfizer selective enterococcus (PSE) agar with the membrane filter technique for the enumeration of fecal streptococci is limited due to the inability of the characteristic black precipitate, indicative of esculin hydrolysis, to diffuse from the medium through the membrane. A modification of the membrane filter technique that consisted of placing the membrane on PSE agar and overlaying it with tempered PSE agar was evaluated by comparing recovery, selectivity, and other parameters with M-enterococcus and KF-streptococcus agars, two selective media routinely used with the membrane filter technique for the enumeration of fecal streptococci in water and wastewater. No statistically significant differences could be demonstrated in the recovery capabilities of the three media. Inasmuch as the PSE overlay technique requires only 24 h of incubation as opposed to 48 h for the other two media, this modification may have some merit in water pollution monitoring programs.     

Comparison of fluorescent gentamicin-thallous-carbonate and KF streptococcal agars to enumerate enterococci and fecal streptococci in meats.

Two selective and differential media were compared for their abilities to enumerate enterococci and fecal streptococci in pork, beef, and poultry products. Counts obtained on KF streptococcal (KF) agar were compared with counts obtained on fluorescent gentamicin-thallous-carbonate (fGTC) agar. Reactions of 13 known enterococcal species were also observed. All 13 species of enterococci as well as Streptococcus bovis and Streptococcus equinus grew equally well on fGTC agar. KF streptococcal medium allowed growth of most species of enterococci but not S. bovis and S. equinus. Quantitative comparisons between the two media inoculated with pure cultures of known species of enterococci revealed equivalent plate counts following incubation. However, when meat samples were plated, counts on fGTC agar were consistently and significantly higher than counts on KF agar for all sample sources.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Comparison of Selective Media for Isolation of Presumptive Group D Streptococci from Human Feces

Pfizer Selective Enterococcus (PSE) agar, a medium containing bile, sodium azide, and esculin, was evaluated for its sensitivity and selectivity for detection and enumeration of presumptive group D streptococci in human feces. SF broth and SF broth plus agar (1.5%), representing selective media in common use, were studied simultaneously. Presumptive group D streptococci were recovered on PSE agar from the feces of all 25 subjects. No growth was observed in 8% of specimens in SF broth. No gram-negative organisms were recovered in any medium. PSE agar has the advantages of selecting out Streptococcus bovis, earlier appearance of distinctive reactions, and lack of requirement for special incubation temperature.     

Evaluation of media for monitoring fecal streptococci in seawater

The selectivity of KF streptococcus agar (KF) for monitoring fecal streptococci (FS) in seawater was examined in 234 samples of Mediterranean water and compared with the selectivity of M-Enterococcus agar (M-Ent) for 124 samples and with bile-esculin-azide agar (BEA) for 17 samples. KF was found to be unsuitable for marine water because Vibrio alginolyticus and other gram-negative bacilli indigenous to this environment grew well on it and produced red colonies identical to those of FS. In 26% of samples, some with high counts of red colonies on the membrane filters (MF), there were no streptococci, only gram-negative bacilli and staphylococci, and in an additional 23.1% the streptococci constituted less than 50% of the "typical" red colonies on the MF. V. alginolyticus also produced FS-like colonies on MF incubated on BEA but was not isolated from MF incubated on M-Ent. Although staphylococci grew and produced FS-like colonies on all three media, M-Ent was the most selective since no gram-negative bacilli were isolated from MF incubated on it.     

Evaluation of media for monitoring fecal streptococci in seawater.

The selectivity of KF streptococcus agar (KF) for monitoring fecal streptococci (FS) in seawater was examined in 234 samples of Mediterranean water and compared with the selectivity of M-Enterococcus agar (M-Ent) for 124 samples and with bile-esculin-azide agar (BEA) for 17 samples. KF was found to be unsuitable for marine water because Vibrio alginolyticus and other gram-negative bacilli indigenous to this environment grew well on it and produced red colonies identical to those of FS. In 26% of samples, some with high counts of red colonies on the membrane filters (MF), there were no streptococci, only gram-negative bacilli and staphylococci, and in an additional 23.1% the streptococci constituted less than 50% of the "typical" red colonies on the MF. V. alginolyticus also produced FS-like colonies on MF incubated on BEA but was not isolated from MF incubated on M-Ent. Although staphylococci grew and produced FS-like colonies on all three media, M-Ent was the most selective since no gram-negative bacilli were isolated from MF incubated on it.     

Enumeration of Food-Borne Clostridium perfringens in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar

The SFP (Shahidi-Ferguson perfringens), TSC (tryptose-sulfite-cycloserine), EY (egg yolk)-free TSC, and OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agars have been tested for their suitability to enumerate Clostridium perfringens in naturally contaminated foods. Complete recoveries of C. perfringens were obtained in each of the four media, but only the TSC and EY-free TSC agars were sufficiently selective to ensure subsequent confirmatory tests without interference from facultative anaerobes. Because of some disadvantages associated with the use of egg yolk, EY-free TSC agar is recommended for enumeration of C. perfringens in foods. Several conditions for convenient shipment of foods and C. perfringens isolates with minimum loss of viability have been tested. The highest viable counts were preserved when foods were mixed 1:1 (wt/vol) with 20% glycerol and kept in a container with dry ice. Isolated C. perfringens strains remained viable for at least 2 weeks at ambient temperatures on blood agar slopes with a 2% agar overlay in screw-cap culture tubes.     

Further Studies on the Suitability of Various Media Containing Antibacterial Antibiotics for the Enumeration of Moulds in Food and Food Environments

Oxytetracycline–glucose–yeast extract agar (OGYA), gentamicin–glucose–yeast extract agar (GGYA) with the antibiotic added separately or sterilized with the medium, chlortetracycline–Rose Bengal agar (CRA), chloramphenicol-streptomycin agar (PYA) and to a lesser extent, oxytetracycline–gentamicin–glucose–yeast extract agar (OGGYA) with or without Rose Bengal added, have been compared for the selective enumeration of moulds in foods. The results obtained from dried cereal products show that the media are almost equally productive and selective when applied to such foods, but Rose Bengal limits the size of mould colonies. When examining fresh proteinaceous foods, such as minced meat and chicken, CR agars and to a certain extent gentamicin-containing agars show the distinct advantage of being much more inhibitory towards the psychrotrophic Gram negative rods that predominate in the associated flora of such foods. Oxytetracycline–glucose–yeast extract agar lost its bacteriostatic properties when heavily challenged with proteinaceous substrates and/or incubated for longer periods at 37° or even at 25°. For such applications chloramphenicol was found to be the antibiotic of choice.     

Evaluation of recovery methods to detect faecal streptococci in polluted waters

AIMS: This paper compares the faecal streptococci count on 25 samples of polluted waters obtained with three techniques: most probable number (MPN), membrane filtration (MF) and pour plate (PP) methods. Although the PP method is a simple technique, familiar to water bacteriologists, it is not recommended in the international methods. METHODS AND RESULTS: For the MPN method, azide dextrose broth and ethyl violet azide broth were employed. For the MF technique, Millipore filters were placed onto azide maltose agar (KF agar), while for the PP method, 1 ml of a decimal water dilution was added to (Kennel Faecal) KF medium. Regression analysis and Friedman's ANOVA were performed to determine the relationship between faecal streptococci counts obtained with the three techniques. Statistical analysis of the results showed that the MPN, MF and PP techniques were equally valid with respect to faecal streptococci enumeration in polluted waters. CONCLUSION: Since the PP method was found to be as good as the other techniques, it may be preferred in polluted waters. It is more economical in terms of both time and materials than the MPN count, and it is as accurate as the MF count. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that the PP method, although not recommended internationally, is a reliable alternative to MF and MPN.     

Selective agars for the isolation of Streptococcus iniae from Japanese flounder, Paralichthys olivaceus, and its cultural environment

Two kinds of selective agar were developed for the isolation of Streptococcus iniae, the causal agent of streptococcosis, from Japanese flounder (Paralichthys olivaceus) and from culture tanks in flounder farms. The selective agars were heart infusion agar with added thallium acetate and oxlinic acid (TAOA), and colistin sulphate and oxolinic acid (CSOA). For samples containing various bacterial flora, selective agars were supplemented with defibrinated horse blood in order to distinguish beta-haemolytic colonies of Strep. iniae. Streptococcus iniae was quantitatively isolated from the brain and kidney of diseased flounders in pure culture. Two-thirds of isolates picked up from selective blood agars inoculated with intestinal samples were identified as Strep. iniae. The bacterial colony numbers of deposits and water from culture tanks on selective blood agars were about 10-10(5) times smaller than those on control heart infusion agar; Strep. iniae was isolated from few deposit and water samples.     

Incidence and relationship of group D streptococci with other indicator organisms in meats.

Raw and processed meats were analyzed for presumptive group D streptococci using KF streptococcus agar. Counts were compared with coliform, presumptive Escherichia coli, and Enterobacteriaceae counts but no meaningful relationships were observed. Results indicated that group D streptococci and E. coli type I were principally contaminants from the packing plant, rather than at retail level. The predominating group D streptococcus in both beef and pork cuts was Streptococcus faecalis, while in processed meat (bologna), the predominating group D streptococci were Streptococcus faecium var. durans and Streptococcus faecium. Streptococcus bovis was not detected among the isolates from any meat samples. Marked differences were noted in numbers of group D streptococci in processed meat from different manufacturers. The results did not support the use of group D streptococci as alternative indicator organisms for meats. However, the association of group D streptococci with packing plant contamination may prove to be of value.     

Modification of kanamycin-esculin-azide agar to improve selectivity in the enumeration of fecal streptococci from water samples.

Kanamycin-esculin-azide agar was modified by increasing the concentration of sodium azide to 0.4 g liter-1 and replacing kanamycin sulfate with 5 mg of oxolinic acid liter-1. The modification, named oxolinic acid-esculin-azide (OAA) agar, was compared with Slanetz-Bartley and KF agars by using drinking water and seawater samples. The OAA agar showed higher specificity, selectivity, and recovery efficiencies than those obtained by using the other media. In addition, no confirmation of typical colonies was needed when OAA agar was used, which significantly shortens the time of sample processing and increases the accuracy of the method.     

Use of fecal streptococci as indicators of pollution in soil.

The survival, recovery, and identification of Streptococcus isolates from soil was investigated by (i) examination of survival in soil under different moisture and temperature conditions, (ii) evaluation of media combinations for recovering fecal streptococci from soil, and (iii) partial identification of isolates from diverse habitats. Cool, moist conditions prolonged the survival of Streptococcus faecalis in soil for at least 12 weeks, whereas freezing was lethal, with the populations being reduced up to 95% when several freeze-thaw treatments occurred. Media evaluations indicated that both the efficiency of recovery and enumeration of the fecal streptococci from soil can be influenced by the combination of media used. Taxonomic data revealed a need to develop procedures to differentiate between isolates of fecal origin and plant-derived streptococci that possess many of the cultural reactions of S. faecalis. It was found that recent fecal isolates exhibited a much greater incidence of multiple antibiotic resistance than soil or vegetation isolates, and this characteristic, coupled with the use of enterococci as indicators of fecal contamination in soil systems, is discussed.     

Use of fecal streptococci as indicators of pollution in soil.

The survival, recovery, and identification of Streptococcus isolates from soil was investigated by (i) examination of survival in soil under different moisture and temperature conditions, (ii) evaluation of media combinations for recovering fecal streptococci from soil, and (iii) partial identification of isolates from diverse habitats. Cool, moist conditions prolonged the survival of Streptococcus faecalis in soil for at least 12 weeks, whereas freezing was lethal, with the populations being reduced up to 95% when several freeze-thaw treatments occurred. Media evaluations indicated that both the efficiency of recovery and enumeration of the fecal streptococci from soil can be influenced by the combination of media used. Taxonomic data revealed a need to develop procedures to differentiate between isolates of fecal origin and plant-derived streptococci that possess many of the cultural reactions of S. faecalis. It was found that recent fecal isolates exhibited a much greater incidence of multiple antibiotic resistance than soil or vegetation isolates, and this characteristic, coupled with the use of enterococci as indicators of fecal contamination in soil systems, is discussed.