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.     

Evaluation of media and techniques to enumerate heterotrophic microbes from karst and sand aquifer springs

Several media and techniques were compared for their efficiency to enumerate viable heterotrophs from both a karst and sand aquifer spring. A medium designed to enumerate bacteria from nutrient-poor waters (HCFU) as well as R2A medium proved superior to tryptic soy agar; however, the difference was always less than one order of magnitude. Membrane filtration resulted in lower counts of microbes than the spread plate, multitube turbidity, or drop plate methods from samples of both sand and karst springs. The drop plate technique yielded higher viable counts from the sand spring and basin of the karst spring, with a precision of 21% (coefficient of variation) and a maximum plating efficiency of 3.4% (viable count/direct count × 100). Subsequently, 63% of isolates from drop plates were recovered on HCFU. Microcolonies were visible by epifluorescence microscopy, acridine orange staining, and subsequent examination of excised agar sections containing drops. Correspondence to: A.T. Mikell, Jr.     

Comparison of media for enumeration of Clostridium perfringens from foods

Many media have been developed to enumerate Clostridium perfringens from foods. In this study, six media [iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin polymyxin sulfadiazine perfringens (OPSP) agar] were compared in a prestudy, of which four (IS, TSC, SCA, and DCA) were selected for an international collaborative trial. Recovery of 15 pure strains was tested in the prestudy and recovery of one strain from foodstuffs was tested in the collaborative trial. Results from the prestudy did reveal statistical difference of the media but recoveries on all media were within the microbiological limits (+/-30%) of IS, which was set as a reference medium. Recoveries on the media tested in the collaborative trial were statistically different as well, but these differences were of no microbiological-analytical relevance. Food matrices did not affect the recovery of C. perfringens in general. DCA and SCA, in particular, are labor-intensive to prepare and DCA frequently failed to produce black colonies; gray colonies were quite common. Since IS medium is nonselective, it was concluded that TSC was the most favorable medium for the enumeration of C. perfringens from foods.     

Enumeration of Klebsiella spp. in cold water by using MacConkey-inositol-potassium tellurite medium

MacConkey-inositol-potassium tellurite agar was field tested for its ability to selectively enumerate Klebsiella species from the waters of the Saint John River Basin, which include fresh and marine waters. Water temperature varied from 1 to 6 degrees C during the survey period. Results of the study indicated that 77% of the typical colonies on MacConkey-inositol-potassium tellurite medium were Klebsiella species, but the total Klebsiella population enumerated was greatly underestimated.     

Enumeration by DNA colony hybridization of virulent Yersinia enterocolitica colonies in artificially contaminated food.

A genetic probe was used to identify and enumerate virulent Yersinia enterocolitica colonies in 11 artificially contaminated foods. Efficiency of enumeration, determined by autoradiography after DNA colony hybridization, ranged from 66 to 100% (average, 86%) and was influenced by the number of indigenous bacteria. The use of nitrocellulose filters and agar medium had little effect on efficiency of enumeration.     

Enumeration of Klebsiella spp. in cold water by using MacConkey-inositol-potassium tellurite medium.

MacConkey-inositol-potassium tellurite agar was field tested for its ability to selectively enumerate Klebsiella species from the waters of the Saint John River Basin, which include fresh and marine waters. Water temperature varied from 1 to 6 degrees C during the survey period. Results of the study indicated that 77% of the typical colonies on MacConkey-inositol-potassium tellurite medium were Klebsiella species, but the total Klebsiella population enumerated was greatly underestimated.     

Enumeration by DNA colony hybridization of virulent Yersinia enterocolitica colonies in artificially contaminated food

A genetic probe was used to identify and enumerate virulent Yersinia enterocolitica colonies in 11 artificially contaminated foods. Efficiency of enumeration, determined by autoradiography after DNA colony hybridization, ranged from 66 to 100% (average, 86%) and was influenced by the number of indigenous bacteria. The use of nitrocellulose filters and agar medium had little effect on efficiency of enumeration.     

Tannin-protein complex-degrading enterobacteria isolated from the alimentary tracts of koalas and a selective medium for their enumeration.

Tannin-protein complex (T-PC)-degrading enterobacteria (T-PCDE) were isolated from the feces and from a layer of bacteria attached to the cecal wall of koalas. The T-PCDE were facultatively anaerobic, gram-negative, pleomorphic, nonmotile bacilli. The bacteria were also oxidase and catalase negative and resistant to vancomycin, reduced nitrates to nitrites, and grew on MacConkey agar. Growth on tannin-treated agar media showed a distinctive clear zone around the colony. From these observations, a selective agar plate medium (vancomycin- and tannin-treated Wilkins-Chalgren anaerobe agar) was developed to enumerate T-PCDE isolated from the feces of koalas. This medium was highly selective in the enumeration of the fecal T-PCDE and inhibited the growth of concomitant T-PC-degrading Streptococcus bovis. The T-PCDE were isolated from 10 of the 12 captive koalas studied; in 8 of these 10 koalas, the facultatively anaerobic bacterial flora was dominated (more than 60%) by T-PCDE. Viable numbers of T-PCDE were, in most of the animals, much larger (more than 100 times) than the numbers of T-PC-degrading S. bovis, suggesting that T-PCDE played a more active role in digesting T-PC in the alimentary tracts of koalas.     

Tannin-protein complex-degrading enterobacteria isolated from the alimentary tracts of koalas and a selective medium for their enumeration.

Tannin-protein complex (T-PC)-degrading enterobacteria (T-PCDE) were isolated from the feces and from a layer of bacteria attached to the cecal wall of koalas. The T-PCDE were facultatively anaerobic, gram-negative, pleomorphic, nonmotile bacilli. The bacteria were also oxidase and catalase negative and resistant to vancomycin, reduced nitrates to nitrites, and grew on MacConkey agar. Growth on tannin-treated agar media showed a distinctive clear zone around the colony. From these observations, a selective agar plate medium (vancomycin- and tannin-treated Wilkins-Chalgren anaerobe agar) was developed to enumerate T-PCDE isolated from the feces of koalas. This medium was highly selective in the enumeration of the fecal T-PCDE and inhibited the growth of concomitant T-PC-degrading Streptococcus bovis. The T-PCDE were isolated from 10 of the 12 captive koalas studied; in 8 of these 10 koalas, the facultatively anaerobic bacterial flora was dominated (more than 60%) by T-PCDE. Viable numbers of T-PCDE were, in most of the animals, much larger (more than 100 times) than the numbers of T-PC-degrading S. bovis, suggesting that T-PCDE played a more active role in digesting T-PC in the alimentary tracts of koalas.     

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.     

Growth ofThiobacillus ferrooxidans on solid media containing heterotrophic bacteria

Summary the study of the chemolithotrophic bacteriumThiobacillus ferrooxidans, an organism important in mining and metallurgical applications, has always been complicated by the inability to cultivate and enumerate many wild strains on solid agar. Here, we have described a plate-count procedure that uses an acidophilic heterotrophic bacterium to scavenge organic material from normal agar, permitting colony formation of the autotroph without visible evidence of growth of the heterotroph. This technique could enable anyT. ferroxidans isolate, including those that are agar-sensitive, to grow and develop colonies on solid medium.     

Modification of Cycloserine Cefoxitin Fructose Agar to Suppress Growth of Yeasts from Stool Specimens

Cycloserine cefoxitin fructose agar is a widely used selective isolation medium for Clostridium difficile from stool specimens. Yeasts often colonize in the intestine of C. difficile disease patients and, if colonized heavily, pure culture of C. difficile can be delayed. The aim of this study was to modify cycloserine cefoxitin fructose agar to suppress the growth of yeasts. Antimicrobial activities of three commonly available antifungal agents were tested against recent clinical isolates of Candida species. Amphotericin B was most active in inhibiting all isolates by ≤0.5 mg/L concentration. Cycloserine cefoxitin fructose agar was modified by adding 2 mg/L of amphotericin B. Serial ten-fold dilution of stool specimens from 126 suspected C. difficile -associated diarrhea patients were cultured both on cycloserine cefoxitin fructose agar plates and modified agar plates. Yeasts grew from 60 specimens on cycloserine cefoxitin fructose agar, but none grew on the modified medium. Growth of C. difficile was detected from 37 and 39 of 126 specimens on cycloserine cefoxitin fructose agar and modified medium, respectively. The number of C. difficile colonies was similar on both media. In conclusion, 2 mg/L of amphotericin B supplementation to cycloserine cefoxitin fructose agar can facilitate the isolation of C. difficile from stool specimens which are densely colonized with yeasts.     

Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

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.     

Evaluation of a Medium for the Rapid Recovery of Escherichia coli from Shellfish

A medium (A-1) which shortens the time necessary to identify and enumerate Escherichia coli found in estuarine water was evaluated for use for recovery of E. coli found in shellfish. Productivity of E. coli by this medium was comparable to that of the lengthier American Public Health Association method, and the occurrence of false positives was substantially reduced.     

Development of a selective myclobutanil agar (MBA) medium for the isolation of Fusarium species from asparagus fields

A new selective myclobutanil agar medium for the detection of Fusarium, species is proposed. Ten media formulations based on various selective agents (pentachloronitrobenzene (PCNB), Rose Bengal, malachite green, sodium hypochlorite, captan, benomyl, chlorotalonil, myclobutanil, thiram, and cupric sulfate) were compared. First, mycelium growth and colony appearance of Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Epicoccum nigrum, Fusarium sp., Fuisarium solani, Fusarium moniliforme, Fusarium oxysporum f.sp. dianthi, Penicillium sp., and Trichoderma viride isolates were compared. Second, the ability of the different media to isolate and enumerate fusaria from asparagus fields was evaluated. The myclobutanil-based medium showed the highest selectivity to Fusarium spp. growth but required a slightly longer incubation time (>5 d) than peptone-pentachloronitrobenzene-based agar (PPA) (< 5 d). PPA allowed a faster fusaria growth but also permited the growth of other moulds. The other media were less selective and did not allow to isolate fusaria or to differenciate them from other growing fungi.     

A note on the isolation of Staphylococcus aureus from raw minced meat

Conventional Baird-Parker medium (BP) and three modified BP-type media were tested for their suitability to detect and to enumerate Staphylococcus aureus in raw minced meat. BP with pig plasma gave the most rapid results without the need for extensive confirmatory tests. A method which combined a liquid modification of BP medium with BP agar supplemented with acriflavine, polymyxin and sulphamezathine, was cheaper, more sensitive, and did not necessitate additional identification. Conventional BP incubated at 37 degrees and 43 degrees C yielded less satisfactory results.     

Improved Method for Detection of Vibrio parahaemolyticus in Seafood

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

A note on the isolation of Staphylococcus aureus from raw minced meat

Conventional Baird-Parker medium (BP) and three modified BP-type media were tested for their suitability to detect and to enumerate Staphylococcus aureus in raw minced meat. BP with pig plasma gave the most rapid results without the need for extensive confirmatory tests. A method which combined a liquid modification of BP medium with BP agar supplemented with acriflavine, polymyxin and sulphamezathine, was cheaper, more sensitive, and did not necessitate additional identification. Conventional BP incubated at 37 and 43C yielded less satisfactory results.     

Differential roles of auxin efflux carrier PIN proteins in hypocotyl phototropism of etiolated Arabidopsis seedlings depend on the direction of light stimulus

In a recent study, we demonstrated that although the auxin efflux carrier PIN-FORMED (PIN) proteins, such as PIN3 and PIN7, are required for the pulse-induced first positive phototropism in etiolated Arabidopsis hypocotyls, they are not necessary for the continuous-light-induced second positive phototropism when the seedlings are grown on the surface of agar medium, which causes the hypocotyls to separate from the agar surface. Previous reports have shown that hypocotyl phototropism is slightly impaired in pin3 single mutants when they are grown along the surface of agar medium, where the hypocotyls always contact the agar, producing some friction. To clarify the possible involvement of PIN3 and PIN7 in continuous-light-induced phototropism, we investigated hypocotyl phototropism in the pin3 pin7 double mutant grown along the surface of agar medium. Intriguingly, the phototropic curvature was slightly impaired in the double mutant when the phototropic stimulus was presented on the adaxial side of the hook, but was not impaired when the phototropic stimulus was presented on the abaxial side of the hook. These results indicate that PIN proteins are required for continuous-light-induced second positive phototropism, depending on the direction of the light stimulus, when the seedlings are in contact with agar medium.