Statistical approach for comparison between methods of bacterial enumeration in drinking water

A general statistical procedure based on the likelihood ratio test is presented for the purpose of comparing estimates of mean bacterial density derived from different sets of data. This approach is much more appropriate than the conventional ways of analyzing bacteriological results (e.g., analysis of variance) which usually require previous transformation of the data. An illustrative application of the method compares three distinct titration techniques for enumerating heterotrophic bacteria in drinking water at 20°C incubation temperature. It was shown that both the standard plate count (SPC) and the membrane filter (MF) procedures supplied substantially the same information, whereas the microplate technique using the most probable number (MPN) for total bacterial enumeration could yield considerably different estimates: MPN values were significantly lower in three cases and significantly higher in one case out of a total of five experiments. The results consistently indicate a strong interaction between the technique used and the sample analyzed. Three different media (nutrient agar, R-2A low nutrient agar and m-SPC agar) were then evaluated for enumerating heterotrophic bacteria, using the MF technique at 48, 72 and 96 h of incubation time at 20°C. Although the media recovered approximately the same numbers of bacteria after 96 h of incubation, statistically significant discrepancies occurred after intermediate periods of incubation, perhaps because the relative rates of bacterial growth differed among media.     

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％。     

The filtration-acclimatization method for isolation of an important fraction of the not readily cultivable bacteria

We developed a novel method, the filtration-acclimatization method (FAM), which enables the isolation and cultivation of an important fraction of the bacterial diversity, which is not cultivable by standard methods. The method consists of a filtration step, which removes most of the readily cultivable bacteria able to overgrow slowly growing bacteria, and an acclimatization procedure that provides a slow transition from the low environmental substrate concentrations to the high concentration of standard microbial media. So far, we isolated in total 65 strains from surface freshwater habitats by utilizing FAM. The isolates are affiliated with Actinobacteria, Alpha-, Betaproteobacteria, Bacteroidetes, and Spirochaeta. All isolates are pure cultures and form visible colonies on agar plates with high substrate concentrations. For further analysis, strains sharing more than a 97% 16S rRNA gene sequence similarity were grouped into one taxon. Based on sequence similarities, 88% of the obtained taxa can be considered to be undescribed species (<97% similarity to closest species). The highest similarity value of the taxa to the respective closest related species ranged from 87.7% to 99.8%, and was on average 94.5%. For comparison we isolated, by direct plating of water samples on a rich agar medium, a similar number of taxa. Amongst these taxa the percentage of taxa, which can be considered to be undescribed species, was only half of the percentage found for the taxa isolated by FAM. More importantly, it was amongst the taxa obtained by the standard method no taxon that was closer related to an uncultured bacterium than to an isolate, while 56% of the taxa isolated by FAM were closely related to uncultured bacteria.     

Comparative analysis of bacterial diversity in the rhizosphere of tomato by culture-dependent and -independent approaches

The microbiome in the rhizosphere–the region surrounding plant roots–plays a key role in plant growth and health, enhancing nutrient availability and protecting plants from biotic and abiotic stresses. To assess bacterial diversity in the tomato rhizosphere, we performed two contrasting approaches: culture-dependent and -independent. In the culture-dependent approach, two culture media (Reasoner’s 2A agar and soil extract agar) were supplemented with 12 antibiotics for isolating diverse bacteria from the tomato rhizosphere by inhibiting predominant bacteria. A total of 689 bacterial isolates were clustered into 164 operational taxonomic units (OTUs) at 97% sequence similarity, and these were found to belong to five bacterial phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes). Of these, 122 OTUs were retrieved from the antibiotic-containing media, and 80 OTUs were recovered by one specific antibiotic-containing medium. In the culture-independent approach, we conducted Illumina MiSeq amplicon sequencing of the 16S rRNA gene and obtained 19,215 high-quality sequences, which clustered into 478 OTUs belonging to 16 phyla. Among the total OTUs from the MiSeq dataset, 22% were recovered in the culture collection, whereas 41% of OTUs in the culture collection were not captured by MiSeq sequencing. These results showed that antibiotics were effective in isolating various taxa that were not readily isolated on antibiotic-free media, and that both contrasting approaches provided complementary information to characterize bacterial diversity in the tomato rhizosphere.     

利用DGGE评价不同培养基回收番茄根际细菌类群的能力

用营养肉汤、YG、根系分泌物、土壤浸渍液4种培养基从番茄根际分离培养细菌,并结合变性梯度凝胶电泳(DGGE)技术,对4种培养基回收番茄根际细菌种群的能力进行了比较研究。结果表明,不同培养基和培养温度,回收到的细菌种群有一定差异;低营养浓度的YG培养基在较低的培养温度20℃下进行较长时间的培养,比高营养浓度营养肉汤培养基产生更多、更具代表性的细菌;以根系分泌物为基础的培养基从番茄根际回收到的优势菌群最多。该研究初步建立了用DGGE技术对不同培养基回收分离细菌种群能力进行评价的方法。     

Qualitative and quantitative agar invasion test based on bacterial colony/biofilm

Invasion of the culture medium is a feature frequently studied in yeasts, in which it has been related to a greater virulence, but it is practically unknown in bacteria. Recently, it has been demonstrated that several clinically relevant bacterial species were also able of invading agar media, so it was necessary to design a microbiological assay to study the expression of this character in bacteria. Accordingly, a bacterial agar invasion test based on colony/biofilm development was designed, which allows qualitative and quantitative characterization of bacterial growth into the agar culture medium. Once the culture conditions were optimized, the test was applied to 90 strains from nine bacterial species, validating its usefulness for differentiating invasive strains (positive) from those non invasive (negative). The test also allows sorting invasive strains according to agar invasion intensity (low, moderate, high) and topographic invasion pattern (peripheral, homogeneous, mixed). Moreover, an image analysis routine to quantify the invasion was developed. Implemented method enables direct measuring of two invasion parameters (invasion area and number of invasion dots), automated calculation of three relative variables (invasion relative area, invasion dots relative density, and invasion dot average area), and the establishment of strain specific frequency histograms.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Using selective media to assess aerosolization damage and ultraviolet germicidal irradiation susceptibility of Serratia marcescens

This study determined whether selective media, McConkey agar (MC) and minimal salt glucose agar (MA) are suitable for monitoring aerosolization damage of airborne Serratia marcescens in our laboratory aerosol exposure system and assessed the relationship between bacterial culturability in these media and ultraviolet germicidal irradiation (UVGI) susceptibility of the bacteria. Two types of bacterial cultures were prepared. The first culture was taken from bacteria growing on Tryptic soy agar (TSA) as complete medium (fresh culture), which provided nearly 100% of MC/MA tolerant bacteria, while the second one was prepared from freezing the fresh culture (frozen culture), which produced 55 and 81% of MC and MA tolerant bacteria, respectively. We monitored bacterial culturability in TSA, MC and MA from these cultures in the nebulizer reservoir and bioaerosls collected on a six-stage Andersen cascade bio-impactor. The results indicated that both concentration and percentage of MC/MA tolerant bacteria maintained at a similar level during nebulization. For the bioaerosols, although the concentration recovered in the media from the fresh culture was higher than that from the frozen culture, the percentage of MC/MA tolerant bacteria was similar to that before aerosolization. We concluded that MC and MA are not suitable for monitoring aerosolization damage of the bacteria. Moreover, culturability of the bacteria in MC and MA has no effect on their survival after aerosolization. With respect to the bacterial susceptibility to UVGI, MC/MA sensitive and tolerant population as well as the fresh and frozen cultures showed the same susceptibility.     

Oligotrophic and copiotrophic marine bacteria—observations related to attachment

Abstract The initial selective adhesion of bacteria, expressing growth on solid media with low, intermediate, and high nutrient concentrations, to immersed glass surfaces in seawater was examined. Copiotrophic-type bacteria grown on high nutrient medium did not show a competitive advantage as primary colonizers. As compared to bacterial numbers in bulk water, relatively higher numbers of adhered oligotrophic-type bacteria, exhibiting growth on low-nutrient media, were found during the initial phase of adhesion. Higher numbers of copiotrophic rather than oligotrophic-type bacteria were seen in the bulk water. The majority of the adherent bacteria was irreversibly bound. Characteristics such as cell size, degree of cell surface hydrophobicity, and motility of bacterial isolates from the different nutrient concentrations did not account for the observed, possibly selective, adhesion. Although bacteria expressed nutritionally different requirements and adaptations at the time of sampling, successive reinoculations of a total of 161 isolates essentially failed to show the existence of obligacy of any given nutritional type of bacteria. The expression of different nutritional adaptations of bacteria in low-nutrient marine waters was also suggested by showing the inability of oligotrophic-like bacteria to possess starvation survival mechanisms such as those displayed by copiotrophs [3].     

Evaluation of Media for Selective Isolation of Yeasts from Oral, Rectal, and Burn Wound Specimens

Six media were evaluated to determine their ability to isolate yeasts and inhibit bacteria. The media included the following: Snyder, Snyder tellurite, Sabouraud tellurite, Littman-gentamicin, molybdate, and Mycosel (BBL). Doses of mixed intestinal gram-negative bacilli and enterococci were most effectively inhibited by Snyder tellurite agar. Klebsiella pneumoniae was the most common bacterial contaminant of the other media. All six media were comparable in isolating yeasts while preventing the growth of the oral bacterial flora. The selection of a basal fungal growth medium for tellurite incorporation to inhibit bacteria but permit growth of yeasts was affected by pH. The bacteriostatic effect of tellurite was decreased with increasing pH of media while fungistatic action was increased. The arbitrary selection of Snyder and Littman agars to isolate yeast from burn wound cultures demonstrated the need to include a selective medium for these specimens. Blood, phenylethyl alcohol blood agar, and Columbia CN blood agar were all inadequate for isolating yeasts from burns. Growth of a variety of filamentous saprophytic and pathogenic dimorphic fungi grew adequately on four of five selective media tested.     

Experimental Salmonellosis

Mice were infected with smooth or rough strains of Salmonella enteritidis and Salmonella typhimurium and viable bacterial cells found in the liver of the inoculated animals were enumerated by plating homogenates of tissues on nutrient agar plates containing 0.35 M sucrose. Some rough strains of these Salmonella were recovered in the bacteria seen on these plates and appeared able to form colonies only on the sucrose-containing medium but not on an identical medium without added sucrose. This population did not appear in the liver of animals until at least 24 hr after infection. The number of bacteria capable of forming colonies only on the hypertonic medium was found to vary with the time after infection and the strain of bacteria used for infection. From the results of morphological examination of cells of the colonies developing on the hypertonic plates, these bacterial forms were thought to result from unstable L forms in the infected tissues. Possible processes of the formation of these L forms in vivo and their significance in induction of anti-infectious immunity are discussed.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Novel method for selective isolation of actinomycetes

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Novel method for selective isolation of actinomycetes.

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

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.     

Efficacy of copper and silver ions with iodine in the inactivation of Pseudomonas cepacia

Alternatives to chlorination of water have been sought for reasons which include trihalomethane formation, possible bacterial regrowth, the high concentrations of chlorine required in certain circumstances, and the taste, odour and bodily irritation in chlorine-treated water. Electrolytically generated Cu and Ag ions at low levels, in addition to very low chlorine concentrations, have been suggested as an alternative to routine chlorination. We have examined the combination of Cu and Ag ions with low levels of iodine. Pseudomonas cepacia was grown either in rich medium or under nutrient restriction prior to disinfection. Survival of the organism and its ability to regrow after treatment as well as the effects of varying buffers, metal ion and iodine concentrations were determined. Low concentrations of metal ions (100 ppb Cu and 11 ppb Ag) and iodine (200 ppb) were more effective than either metal ions or iodine alone against Ps. cepacia grown on rich agar or in low nutrient buffer. After iodination, buffer-grown suspensions recovered to their original cell concentrations within 7 d. When Cu and Ag ions were used with or without iodine, regrowth was prevented. The results show that low concentrations of Cu and Ag in combination with iodine permit effective disinfection of bacteria after cultivation on either rich media or under nutrient restriction. These results, along with published data, suggest that the combination of these metals with halogenation may have applications in the disinfection of both recreational and potable water.     

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique

The growth patterns of macrocolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0·89), pseudomonads (r = 0·93) and staphylococci (r = 0·92) after incubation at 30°C for 3 or 6 h (staphylococci) at contamination levels of above 10³ bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.     

Method for Isolation and Purification of Cyanobacteria

A method employing nutrient saturated glass fiber filters allowed the isolation of the same numbers of cyanobacteria from freshwater as were obtained with medium solidified with agar, while providing a 2- to 15-fold reduction in the number of accompanying heterotrophic bacteria. Imipenem, a broad-spectrum β-lactam antibiotic which inhibits peptidoglycan biosynthesis, was superior to some other β-lactam antibiotics for reducing the numbers of heterotrophic bacterial contaminants associated with freshly isolated cyanobacteria to a level which facilitated the production of axenic cyanobacterial cultures.     

Efficacy of copper and silver ions with iodine in the inactivation of Pseudomonas cepacia.

Alternatives to chlorination of water have been sought for reasons which include trihalomethane formation, possible bacterial regrowth, the high concentrations of chlorine required in certain circumstances, and the taste, odour and bodily irritation in chlorine-treated water. Electrolytically generated Cu and Ag ions at low levels, in addition to very low chlorine concentrations, have been suggested as an alternative to routine chlorination. We have examined the combination of Cu and Ag ions with low levels of iodine. Pseudomonas cepacia was grown either in rich medium or under nutrient restriction prior to disinfection. Survival of the organism and its ability to regrow after treatment as well as the effects of varying buffers, metal ion and iodine concentrations were determined. Low concentrations of metal ions (100 ppb Cu and 11 ppb Ag) and iodine (200 ppb) were more effective than either metal ions or iodine alone against Ps. cepacia grown on rich agar or in low nutrient buffer. After iodination, buffer-grown suspensions recovered to their original cell concentrations within 7 d. When Cu and Ag ions were used with or without iodine, regrowth was prevented. The results show that low concentrations of Cu and Ag in combination with iodine permit effective disinfection of bacteria after cultivation on either rich media or under nutrient restriction. These results, along with published data, suggest that the combination of these metals with halogenation may have applications in the disinfection of both recreational and potable water.