m-Endo Media Research Articles

Evaluation of the growth, lipid, and hydrocarbon accumulation abilities in five strains of Botryococcus braunii (races A, B, and L) (Trebouxiophyceae, Chlorophyta)

The green alga Botryococcus braunii can photosynthetically fix CO2 and accumulate abundant hydrocarbons, which has driven interest in this species as a sustainable biofuel. In this study, five strains of B. braunii (SAG 807-1, SAG 2532, SAG 30.81, SC-1, and SC-2) were identified based on the nucleotide sequence of 18S rDNA, of which SC-1 and SC-2 were isolated from the water bodies of southern China by our laboratory. Then, the effects of different media and initial nitrogen concentrations on the growth, total lipid contents, fatty acid profiles, and hydrocarbon compositions in these five strains were compared. Molecular identification revealed that B. braunii SC-1 and SC-2 belong to races B and L, respectively, while SAG 807-1, SAG 2532, and SAG 30.81 belong to race A. Compared with mBBM and mEndo media, SAG 807-1, SAG 2532, SC-1, and SC-2 grew faster in mBG-11 media. The highest total lipid content of SAG 807–1, SAG 30.81, and SC-1 reached >45 % of cell dry weight (DW), with the maximum total lipid content of SC-2 reaching 66.52 % of DW. The major fatty acid components of the five strains of B. braunii were palmitic acid (C16:0), oleic acid (C18:1), and erucic acid (C22:1). The maximum hydrocarbon content was 22.13 % DW in B. braunii SC-1, of which 1.4 % DW was squalene. Squalene is widely used as an antioxidant and as an adjuvant in vaccines.

Suitability of total coliform â-d-galactosidase activity and CFU counts in monitoring faecal contamination of environmental water samples

Total coliforms are a group of bacteria found in high numbers in  mammalian intestines; hence their presence in water indicates the possible contamination with faecal material. Total and faecal coliform counts were monitored over a period of 18 months using mFC, m-Endo and CM1046 media together with enzymatic assays on 215 environmental water samples obtained from the Eastern Cape Province of South Africa. A positive correlation, with an R2 value of 0.9393 was observed between faecal and total coliform colony units employing mFc and m-Endo media, and 0.8818 using CM1046 media. Also,a positive correlation was observed between Escherichia coli colony-forming units and â-d-galactosidase (B-GAL) activity (R2=0.8542). Overall, this study indicated that faecal contamination of environmental water samples could be monitored by measuring total coliform â-galactosidase activity and total coliform colony-forming units.

Suitability of total coliform â-d-galactosidase activity and CFU counts in monitoring faecal contamination of environmental water samples

Total coliforms are a group of bacteria found in high numbers in  mammalian intestines; hence their presence in water indicates the possible contamination with faecal material. Total and faecal coliform counts were monitored over a period of 18 months using mFC, m-Endo and CM1046 media together with enzymatic assays on 215 environmental water samples obtained from the Eastern Cape Province of South Africa. A positive correlation, with an R2 value of 0.9393 was observed between faecal and total coliform colony units employing mFc and m-Endo media, and 0.8818 using CM1046 media. Also,a positive correlation was observed between Escherichia coli colony-forming units and â-d-galactosidase (B-GAL) activity (R2=0.8542). Overall, this study indicated that faecal contamination of environmental water samples could be monitored by measuring total coliform â-galactosidase activity and total coliform colony-forming units.

Evaluation of media for simultaneous enumeration of total coliform and Escherichia coli in drinking water supplies by membrane filtration techniques

This study evaluated three different dehydrated media for simultaneous detection and enumeration of total coliform (TC) and Escherichia coli in drinking water samples with a standard membrane filtration procedure. The experiment indicated that the differential coliform agar (DCA) medium was the most effective among the tested media in enumerating TC and E. coli, without the need for extensive accompanying confirmation tests. The results for DCA medium were highly reproducible for both TC and E. coli with standard deviation of 6.0 and 6.1, respectively. A high agreement (82%) was found between DCA and m-Endo media on 152 drinking water samples in terms of TC positive. The DCA medium also reduced concealment of background bacteria.

THE AC/TC BACTERIAL RATIO: A TOOL FOR WATERSHED QUALITY MANAGEMENT

An inexpensive tool for indicating microbial quality in watersheds is presented that utilizes the conventional total coliform test, comparing the relative concentrations of different colonies that form on a membrane filter fed by m-Endo media. These bacterial colonies can be classified into 3 types; typical (TC), atypical (AC), and background. The ratio of the concentrations of AC colonies to those of TC is related to water quality, fecal loadings, and fecal age. The AC/TC ratio relies upon shifts in populations between indigenous and introduced bacteria, with the indigenous bacteria providing a baseline against which the concentrations of the introduced are evaluated. When the AC/TC ratio is low ( 20) and can be related to healthier water quality conditions. Different types of runoff have different AC/TC values with human sewage at the lowest end of the spectrum with a value of 1.5 under normal conditions.

The utility of the AC/TC ratio for watershed management: a case study

A human-impacted watershed was monitored during the dry summer seasons in 2002 and 2003 to investigate the impact of providing access to sewer mains to local village residences. Faecal coliform concentrations were monitored at select sites along the 30-mile stretch of creek, together with faecal streptococci, enterococci and total coliforms. Analysis of the results found that levels of faecal coliforms were inadequate at identifying significant known influxes of human and animal sewage established by sanitary survey. However, the bacterial ratio of atypical colonies to total coliform colonies (AC/TC), obtained from the total coliform membrane filter assay on m-Endo media, correctly indexed human faecal impact of inadequately sewered villages located along the creek. In addition, the AC/TC ratio correctly classified the predominant source of faecal runoff in the creek headwaters as agricultural, and indicated when aged agricultural faecal material was introduced by tributaries. An approach for watershed management that uses the AC/TC ratio in addition to levels of bacteria is proposed.

Artificial neural network modelling: a summary of successful applications relative to microbial water quality

Artificial neural networks (ANN) are modelling tools that can be of great utility in studies of microbial water quality. The ability of ANNs to work with complex, inter-related multiparameter databases and provide superior predictive power in non-linear relationships suits their application to microbial water quality studies. To date ANNs have been successfully applied (a) for the prediction of peak microbial concentrations, (b) to sort land use associated faecal pollution sources and relative ages of runoff and (c) towards the selection and study of surrogate parameters. Predictions of peak microbial contamination or faecal pollution sources have been greater than 90% accurate. The importance of a subgroup of organisms that are isolated by the total coliform membrane filter test on m-Endo media in defining faecal sources was revealed through parameter selection exercises. The result is the definition of a new bacterial ratio that can be directly related to the age of faecal contamination in animal impacted runoff.

Analysis of Bottled Water for Escherichia coli and Total Coliforms

U.S. Food and Drug Administration regulations governing bottled water include microbiological quality guidelines based on coliform counts. Recently, a new MF medium for simultaneous detection of total coliforms and Escherichia coli was developed. This medium, m-ColiBlue24 (m-CB) was compared to m-Endo medium and an International Organization for Standardization standard coliform medium, lactose agar with Tergitol 7. Coliform analysis was conducted on 104 brands of bottled water from 10 countries. Some samples were additionally analyzed for heterotrophic plate count and Pseudomonas sp. populations, including P. aeruginosa. Presumptive coliform colonies were found in 5.8% of the samples with m-CB, 1.9% with m-Endo and 11.5% with lactose agar with Tergitol 7. None of the presumptive coliforms from any of the three media were verified as true coliforms in subsequent analysis. Consequently, the presumptive recovery rates actually represented false-positive error (FPE) rates. The FPE for m-CB and m-Endo were not statistically different (P < 0.05) but the FPE for lactose agar with Tergitol 7 was significantly larger.

Recovery of coliform bacteria from rural groundwater supplies using reduced oxygen concentrations during incubation.

The effect of decreased oxygen concentration during incubation of M-Endo medium on detection of coliforms from rural groundwater supplies was examined. Incubation oxygen concentrations of 0¿(anaerobic GasPak), 4, 8, and 21% (atmospheric) were examined. Our findings point to several advantages of using anaerobic incubation for the isolation of coliforms: (i) higher verification rates with concomitant decreases in occurrence of false-positive coliforms; (ii) overall reduction in growth of nonsheen colonies; and (iii) reduction in colony size for nonsheen organisms, thereby minimizing crowding effects and facilitating enumeration of coliform colonies. However, these advantages were not sufficient to permit increased recovery of total coliforms as compared with aerobic incubation. In addition, the increased frequency of detecting false-negative coliforms during anaerobic incubation is a disadvantage to this method. While detection of total coliforms was reduced under conditions of anaerobiosis,the detection of fecal coliforms and (or) E. coli was not impeded.

Sheen formation and growth response of groundwater bacteria to reduced oxygen concentrations during incubation of M-Endo medium.

In vitro pure-culture studies were conducted to assess growth and sheen formation of groundwater bacteria on M-Endo medium incubated under reduced oxygen concentrations (0, 4, 8, 12, and 16%). Coliform and noncoliform bacteria were isolated from 17 untreated, rural groundwater supplies on M-Endo medium. All 16 coliform isolates tested were capable of sheen formation at oxygen concentrations of 4% or greater, yet some of these same isolates (Enterobacter aerogenes, Enterobacter cloacae, and Hafnia alvei) were either unable to grow or failed to produce a metallic sheen when incubated under strict anaerobiosis. Approximately 70% of the 21 noncoliform isolates examined exhibited growth inhibition at oxygen concentrations of 8% or less. The growth of a false-positive coliform isolate of Serratia fonticola was inhibited when incubated under reduced oxygen concentrations of 16% or less. Our findings suggest that the selectivity of M-Endo medium, and resultant inhibition of noncoliforms and false-positive coliforms, is enhanced by incubation in the absence of oxygen. However, the failure of strict anaerobiosis to permit detection of total coliforms such as Hafnia and Enterobacter spp. may compromise the reliability of this technique for evaluating the sanitary quality of some waters. On the other hand, oxygen concentrations of 4, 8, 12, and 16% permitted adequate sheen development of all coliforms tested while inhibiting some noncoliforms.

Improved membrane filtration method incorporating catalase and sodium pyruvate for detection of chlorine-stressed coliform bacteria

In vitro pure culture studies were conducted on three different strains of Escherichia coli (K-12, EPA 00244, and SWEI) to determine the effect of chlorination on catalase activity. In each case, stationary-phase cells exhibited significant (P less than 0.001) reductions in enzyme activity following exposure to chlorine. Mean differences in activity between control and chlorine-stressed cells ranged from 8.8 to 20.3 U/mg of protein for E. coli SWEI and EPA 00244, respectively. Following initial enzyme studies, resuscitation experiments utilizing the membrane filtration technique were conducted on chlorinated sewage effluent. Five different amendments, including catalase (1,000 U per plate), heat-inactivated catalase (1,000-U per plate), sodium pyruvate (0.05%), a catalase-sodium pyruvate combination (1,500 U/0.01%), and acetic acid (0.05%), were tested for the ability to enhance detection of chlorine-stressed cells on M-fecal coliform (M-FC), mT7, M-Endo, and tryptone-glucose-yeast extract (TGY) media. Significant (P less than 0.001) increases in recovery of fecal coliforms on M-FC, total coliforms on mT7 and M-Endo, and total heterotrophs on TGY were obtained on plates containing catalase, pyruvate, or the combination of these compounds. Supplementation with heat-inactivated catalase and acetic acid did not improve recovery of chlorine-stressed cells compared with recovery on nonamended media. Subsequent analysis of colonies from plates containing compounds which enhanced recovery indicated coliform verification percentages of greater than 80% on M-FC, greater than 90% on mT7, and greater than 94% on M-Endo media. These data suggest that the addition of peroxide-degrading compounds to various standard recovery media may improve detection of both coliform and heterotrophic bacteria in chlorinated waters.

Improved detection of acid mine water stressed coliform bacteria on media containing catalase and sodium pyruvate

Pure culture suspensions of two strains of exponential and stationary phase Escherichia coli exhibited significant reductions in catalase activity following exposure to acid mine water (AMW). The exogenous addition of catalase (500-2000 U) or sodium pyruvate (0.05-5%) to a nonselective recovery medium resulted in enhanced detection (12- to 465-fold) of AMW-stressed E. coli as compared with recovery on the medium lacking these supplements, whereas addition of 3,3'-thiodipropionic acid failed to improve recovery. Additional in vitro experiments utilizing selective M-FC, mT7, and M-Endo media containing 1000 U catalase or 1.0% pyruvate similarly resulted in improved detection of AMW-stressed cells, with the exception of M-Endo containing pyruvate. Appropriately modified media were then used to analyze an AMW-impacted stream by the membrane filtration technique. Addition of catalase, pyruvate, or a combination of both significantly improved recovery of fecal and total coliforms without promoting growth of noncoliforms. Supplementation of plate count agar with pyruvate and (or) catalase enhanced detection of total heterotrophs. These findings suggest that addition of catalase or pyruvate to standard recovery media may improve detection of coliform and total heterotrophic bacteria in AMW-impacted waters.

Detection of Acinetobacter spp. in rural drinking water supplies

A bacteriological survey was conducted of untreated, individual groundwater supplies in Preston County, W.Va. Nearly 60% of the water supplies contained total coliforms in excess of the U.S. Environmental Protection Agency maximum contaminant level of 1 CFU/100 ml. Approximately one-third of the water systems contained fecal coliforms and/or fecal streptococci. Acinetobacter spp. were detected in 38% of the groundwater supplies at an arithmetic mean density of 8 CFU/100 ml and were present in 16% of the water supplies in the absence of total coliforms, posing some concern about the usefulness of total coliforms as indicators of the presence of this opportunistic pathogen. Slime production, a virulence factor for A. calcoaceticus, was not significantly different between well water isolates and clinical strains, suggesting some degree of pathogenic potential for strains isolated from groundwater. In addition, several Acinetobacter isolates were able to interfere with sheen production by some coliform bacteria on M-Endo medium, adding further to the possible significance of Acinetobacter spp. in groundwater supplies.

Synergistic False-Positive Coliform Reaction on M-Endo MF Medium

The reported occurrence of synergistic false-positive coliform reactions on M-Endo MF medium was investigated. The objectives of the study were (i) to isolate populations of bacteria which produced such false-positive reactions, (ii) to determine whether false-positive sheen reactions are a result of synergistic interaction, and (iii) to determine the metabolic intermediates involved in false-positive sheen production. Samples of river water were subjected to coliform analysis by the membrane filter technique with M-Endo MF medium. Suspect sheen-forming colonies were analyzed to determine purity and identity of cultures. Mixed cultures were separated, and individual isolates were examined for sheen production. The isolates not producing a sheen were recombined and tested further for sheen production. Those mixtures reproducing the sheen were characterized biochemically and tested to determine the metabolic intermediates involved. Chromatographic analysis of the metabolites showed that individual isolates produced an assortment of neutral organic compounds including 2-butanone, 2,3-butanedione, formaldehyde, and butyraldehyde, whereas acetaldehyde or propionaldehyde was produced only by the mixed cultures. Tests showed that both propionaldehyde and acetaldehyde could react to produce a sheen on M-Endo medium. The conclusion was reached that synergistic false-positive coliform reactions do indeed occur on M-Endo MF medium.

Synergistic False-Positive Coliform Reaction on M-Endo MF Medium

The reported occurrence of synergistic false-positive coliform reactions on M-Endo MF medium was investigated. The objectives of the study were (i) to isolate populations of bacteria which produced such false-positive reactions, (ii) to determine whether false-positive sheen reactions are a result of synergistic interaction, and (iii) to determine the metabolic intermediates involved in false-positive sheen production. Samples of river water were subjected to coliform analysis by the membrane filter technique with M-Endo MF medium. Suspect sheen-forming colonies were analyzed to determine purity and identity of cultures. Mixed cultures were separated, and individual isolates were examined for sheen production. The isolates not producing a sheen were recombined and tested further for sheen production. Those mixtures reproducing the sheen were characterized biochemically and tested to determine the metabolic intermediates involved. Chromatographic analysis of the metabolites showed that individual isolates produced an assortment of neutral organic compounds including 2-butanone, 2,3-butanedione, formaldehyde, and butyraldehyde, whereas acetaldehyde or propionaldehyde was produced only by the mixed cultures. Tests showed that both propionaldehyde and acetaldehyde could react to produce a sheen on M-Endo medium. The conclusion was reached that synergistic false-positive coliform reactions do indeed occur on M-Endo MF medium.