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.     

Interaction of deoxycholate with the sodium channel of squid axon membranes

Deoxycholate can react with sodium channels with a high potency. The apparent dissociation constant for the saturable binding reaction is 2 microM at 8 degrees C, and the heat of reaction is approximately -7 kcal/mol. Four independent test with Na-free media, K-free media, tetrodotoxin, and pancuronium unequivocally indicate that it is the sodium channel that is affected by deoxycholate. Upon depolarization of the membrane, the drug modified channel exhibits a slowly activating and noninactivating sodium conductance. The kinetic pattern of the modified channel was studied by increasing deoxycholate concentration, lowering the temperature, chemical elimination of sodium inactivation, or conditioning depolarization. The slow activation of the modified channel can be represented by a single exponential function with the time constant of 1--5 ms. The modified channel is inactivated only partially with a time constant of 1 S. The reversal potential is unchanged by the drug. Observations in tail currents and the voltage dependence of activation suggest that the activation gate is actually unaffected. The apparently slow activation may reflect an interaction betweem deoxycholate and the sodium channel in resting state.     

Bacteriological analysis of water by potentiometric measurement of lipoic acid reduction: preliminary assays for selective detection of indicator organisms

The practical task of adapting an original potentiometric technique to the bacteriological analysis of water is discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculated one by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redox potential of the cultures was followed during incubation by combined gold versus reference electrodes. When the incubation temperature was regulated at 36 degrees C, most organisms were able to grow and to reduce the coenzyme, generating changes in the redox potential of the culture. However, very few organisms developed significant reductive activity when the temperature was increased to 41 degrees C and when the broth was provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli and Klebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometric responses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signals as compared with K. pneumoniae. The time required for 100-mV shift of potential to be detected was related to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth. Experiments on natural surface water samples showed the the potentiometric method, associated with the selective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmental water. The calibration curve relating the time required for a 100-mV shift of potential to be detected to the number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistent with the composite standard curve of detection times obtained with six different laboratory strains of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriological analysis of water by potentiometric measurement of lipoic acid reduction: preliminary assays for selective detection of indicator organisms.

The practical task of adapting an original potentiometric technique to the bacteriological analysis of water is discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculated one by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redox potential of the cultures was followed during incubation by combined gold versus reference electrodes. When the incubation temperature was regulated at 36 degrees C, most organisms were able to grow and to reduce the coenzyme, generating changes in the redox potential of the culture. However, very few organisms developed significant reductive activity when the temperature was increased to 41 degrees C and when the broth was provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli and Klebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometric responses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signals as compared with K. pneumoniae. The time required for 100-mV shift of potential to be detected was related to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth. Experiments on natural surface water samples showed the the potentiometric method, associated with the selective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmental water. The calibration curve relating the time required for a 100-mV shift of potential to be detected to the number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistent with the composite standard curve of detection times obtained with six different laboratory strains of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600

1. The technique of DNA-RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25-45 degrees C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate-salts or glucose-salts media the ratio was 0.046+/-0.005 and in enriched broth it was 0.087+/-0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate-salts or glucose-salts media, the messenger RNA fraction constituted 2.2+/-0.3% of the total cellular RNA. In enriched broth 3.6+/-0.3% of the total RNA was messenger.     

The Inhibitory Effect of Sodium Deoxycholate on Escherichia coli

SUMMARY: Sodium deoxycholate (0.2%) inhibited growth of Escherichia coli in semi-digested meat and milk, but had very little effect in media in which the source of nitrogen was amino acids or ammonia. The addition of more digested forms of protein (peptone and casein hydrolysate) to a less digested form (beef infusion) overcame the inhibitory effect. The significance which this phenomenon may have in controlling the population of E. coli in the small intestine is discussed.     

Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli

Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe. It is vitally important for sodium export and pH homeostasis in this organism. Recently, the sod2 gene has been cloned and sequenced. However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed. In the present work we examined physiological consequences of expression of sod2 in E. coli. To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E. coli strain MH1 which has impaired sodium exchange. The effect of sod2 expression on E. coli varied depending on the E. coli genotype. When sod2 was expressed in BL21 cells which have normal N a⁺/H⁺ antiporters, the result was a Li⁺ sensitive phenotype. LiCl completely arrested or prevented growth of BL21 E. coli transformed with the sod2 gene. The effect on growth was pronounced in media of low external pH. Sod2 was then expressed in E. coli MH1 which is devoid of endogenous Na⁺/H⁺ antiporters. These cells became more resistant to external LiCl, but only in Na⁺ containing media. In the absence of external Na⁺, the presence of sod2 reduced growth. The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na⁺/H⁺ antiporter in conjunction with different housekeeping systems of E. coli host cells.     

Use of Sodium Polyanethol Sulfonate in the Preparation of 5% Sheep Blood Agar Plates

An alternative method to defibrinating sheep blood for use in bacteriological media is described. The new procedure incorporates sodium polyanethol sulfonate in a concentration of 0.05% (vol/vol). In testing 117 bacterial and fungal isolates, no significant differences were found with respect to adequate growth, pigment production, hemolytic reactions, and other physical attributes. Further tests demonstrate that the sodium polyanethol sulfonate in sheep blood agar plates does not cause any aberrations in zone sizes around disks used in antibiotic susceptibility tests. Consequently, the method represents a suitable alternative to the use of defibrinated sheep blood in the preparation of bacteriological media.     

Effect of anionic detergents on wild-type and envelope mutants of Escherichia coli and Pseudomonas aeruginosa

Escherichia coli UB1005 (DCO), an envelope mutant (DC2), Pseudomonas aeruginosa 799 and an envelope mutant (799/61) were exposed to sodium deoxycholate (DOC), sarkosyl and sodium lauryl sulphate (SLS). DOC was the least effective lytic agent, but the two Ps. aeruginosa strains, especially 799/61, were more susceptible to DOC and sarkosyl than the E. coli ones. SLS was an efficient lysing agent, although Ps. aeruginosa 799 was the least susceptible of the four strains. DC2 was lysed more rapidly and to a greater extent than UB1005 by all three agents. The mutant strains, especially DC2, were more sensitive to selective media than the wild-type ones.     

The Recovery of Indicator Bacteria on Selective Media

S ummary . The recovery of Pseudomonas aeruginosa, Escherichia coli , and Streptococcus faecalis from aqueous suspending media has been studied with a rich plating medium (trypticase-soy agar) and selective media. Tap water was highly toxic to all strains investigated. Recovery of Ps. aeruginosa was most successful when phosphate buffer was the diluent. Phosphate buffer did not improve the recovery of E. coli. Streptococcus faecalis remained viable when suspended in double distilled water, deionized distilled water or phosphate buffer. Following a lag period all strains grew in 0.1% peptone water or stream water. Injury preventing recovery of viable cells on selective media occurred during suspension in all aqueous media tested, including those which supported growth. These observations suggest difficulties inherent in the interpretation of bacteriological results obtained during surveys of water sources and a need to reduce the selectivity of recovery media against injured cells.     

Solubilization of the Semliki Forest virus membrane with sodium deoxycholate.

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at 0.9 +/- 0.1 mM free equilibrium concentration when 2.2 +/- 0.2 - 103 mol of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at 1.5 +/- 0.1 mM sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.     

Influence of enteric bacteria conditioned media on recovery of Escherichia coli O157:H7 exposed to starvation and sodium hypochlorite

AIMS: To examine the effect that starvation and sodium hypochlorite stress have on virulence of Escherichia coli O157:H7 and the influence of conditioned media on recovery of stressed cells. METHODS AND RESULTS: Escherichia coli O157:H7 was starved for 5 days then exposed to 1 microg ml(-1) sodium hypochlorite, suspended in defined media Dulbecco's Modified Eagle's Medium supplemented with conditioned media, sampled over a 12-h period; and assayed for growth, production of Shiga toxin (Stx), and attachment to HCT-8 cells. During recovery, stressed and control cells grown in conditioned media exhibited greater attachment efficiencies to HCT-8 cells then cells in DMEM alone. Production of Stx by treated cells mimicked Stx production by control cells suggesting that components of conditioned media assist in recovery. Results showed that levels of autoinducer-2 fluctuate during recovery and growth suggesting involvement of a quorum sensing mechanism during the recovery of stressed E. coli O157:H7. CONCLUSIONS: The recovery of stressed E. coli O157:H7 exposed to starvation conditions and HOCl is positively affected by the presence of autoinducer-2 thereby influencing virulence factor production. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens in a stressed state prior to ingestion can rapidly recover in the presence of bacterial by-products; exhibiting virulence characteristics and presenting a microbial food safety hazard.     

Transport and utilization of the biosynthetic intermediate shikimic acid in Escherichia coli.

Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poor growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced. Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport. Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% cotransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not cotransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that both classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (Km) for shikimate transport.     

Specificity for field enumeration of Escherichia coli in tropical surface waters

In remote rural areas in developing countries, bacteriological monitoring often depends on the use of commercial field media. This paper evaluates a commercial field medium used for the enumeration of Escherichia coli in different surface waters under primitive field conditions in rural Pakistan. In order to verify the field kit, 117 presumptive E. coli isolates have been tested, finding a specificity of only 40%. By excluding some strains based on colony colours, the calculated specificity could be increased to 65%. Thus, it is suggested that prior to use in a tropical environment, the specificity of any commercial medium used should be tested with representative tropical isolates, in order to increase the specificity.     

Quantitative determination of Escherichia coli from coliforms and faecal coliforms in sea water.

Escherichia coli concentration in sea water was determined by the MUG test after primary growth on membrane filters used to determine total coliforms or faecal coliforms. A good correlation (r = 0.86) was found between E. coli obtained from coliforms versus those from faecal coliforms. Verification procedures showed that all the MUG-positive colonies obtained on both media were E. coli. Evaluation of this data and the literature indicated that this technique for estimation of E. coli in sea water is a useful addition to laboratory procedures without generally increasing the time and the expense of the analysis of recreational water.     

Influence of prior growth conditions on low nutrient response of Escherichia coli in seawater

By use of experimental microcosms, it was demonstrated that the survival of Escherichia coli in nutrient-free seawater depended on the age of cells and on some physicochemical conditions during their prior growth. Cells grown in a bacteriological medium, with an acid or an alkaline pH, at high temperature (44 degrees C), or in the absence of oxygen were more sensitive to exposure to seawater of low nutrient content. In contrast, some complex media allowed production of cells adapting more rapidly to seawater. Cells grown in urine were far more sensitive than those grown in all bacteriological media tested. The sensitivity of all cells was highest when they were harvested during the early exponential phase of growth.     

Solubilization of the semliki forest virus membrane with sodium deoxycholate

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest virus have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at $\text{0.9 ± 0.1}\text{mM}$ free equilibrium concentration when $\text{2.2 ± 0.2 ß 10}{}^3\text{mol}$ of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at $\text{1.5 ± 0.1}\text{mM}$ sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.     

Inhibitory action of a non-metabolizable fatty acid on the growth of Escherichia coli: role of metabolism and outer membrane integrity.

The inhibitory action of decanoic acid on both Escherichia coli K-12/154 (normal lipopolysaccharide) and E. coli RC59 (defective lipopolysaccharide) was studied. A correlation was found between the doubling time of E. coli 154 growing in different media and the lethal effect of 0.4% decanoic acid on this bacterium. Decanoic acid (0.4%) exerted a lytic action on glucose-starved and NaN3-inhibited cells of E. coli 154 and RC59. Exponentially growing cultures of both strains were not affected by the addition of 0.4% methyldecanoate, but cells of E. coli RC59 reaching the stationary phase were attacked by that compound. A bactericidal action of 0.4% methyldecanoate on exponential E. coli 154 and RC59 was observed when sodium azide was also present in the media. Concentrations lower than 0.01% methyldecanoate had a lytic effect on spheroplasts from E. coli 154 and RC59. These results indicate that the inhibitory action of a non-metabolizable fatty acid on E. coli depends on the cellular metabolic activity and the outer membrane integrity.     

Isolation and Some Properties of Cell Envelope Altered Mutants of Escherichia coli

Mutants of Escherichia coli which have a defect in their permeability barrier were selected. The technique used was to employ a strain of E. coli having a deletion in the gene for lactose permease and to select for mutants which can grow on lactose at 40 C. Twenty such mutants were isolated and six of these were found to be more sensitive to actinomycin D, sodium deoxycholate, and sodium dodecyl sulfate than was the parental strain. They were also more sensitive to the antibiotics vancomycin and bacitracin, which inhibit peptidoglycan biosynthesis. These mutants were no more sensitive to several different colicins or phages than was the wild-type strain. One of the mutants selected by this technique has an abnormal morphology when grown on certain carbon sources in minimal medium, and this mutant is more extensively studied in the accompanying paper.