The microbiological quality of water: the nature of the problem

Improvements in methods for the detection and enumeration of microbes in water, particularly the application of techniques of molecular biology, have highlighted shortcomings in the ”standard methods” for assessing water quality. Higher expectations from the consumer and increased publicity associated with pollution incidents can lead to an uncoupling of the cycle which links methodological development with standard-setting and legislation. The new methodology has also highlighted problems within the water cycle, related to the introduction, growth and metabolism of microbes. A greater understanding of the true diversity of the microbial community and the ability to transmit genetic information within aquatic systems ensures that the subject of this symposium and volume provides an ideal forum to discuss the problems encountered by both researcher and practitioner.

A survey of the microbiological quality of commercial frozen prawn products

The paper presents the results of a bacteriological survey carried out on 2,917 samples of frozen prawn, 55 samples of raw material, 35 samples of water, 4 samples of ice and 42 samples of various equipment used for processing. The survey covered a period of three years (1960-63) and comprised of samples collected from five of the leading processing factories in Cochin. Frozen products tested consisted of headless (marine and fresh water), peeled and deveined and cooked frozen samples. Statistical analysis of the data shows that there is no significant variation between samples and between factories with respect to product quality. The standard plate count varied between 1.0x10(4 superscript) and 1.0x10(6 superscript) per gram for headless and between 1.0x10(4 superscript) and 1.0x10(7 superscript) for peeled and deveined and cooked frozen samples. Majority of the samples had bacterial load well within the limits prescribed for such products.

Microbiological quality of some iced fishes of Imphal market, Manipur

Imphal is the main marketing centre of fish in Manipur. As fish production of the state is not sufficient to meet the demands, about 120 metric tons of iced fishes are annually brought from other states and sold in this market. Microbiological quality of iced Wallago attu, Labeo rohita, L. gonius and Aorichthy aor in respect of total fungal count (TFC), total plate count of bacteria (TPC), Most Probable Number (MPN) of coliforms, Streptococci, Staphylococcus, Salmonella and Escherichia coli in four tissues (skin, muscle, gill and intestine) were analysed. In all cases, the counts were highest in the gills and lowest in the muscles. The values of TFC, TPC, coliforms, Streptococci and Staphylococci were 0-10³/g 10(sup)6-10⁸/g, 2-α/g, 10-10⁵/g, 10-10⁵/g respectively. E. coli and Salmonella were not detected in any of the samples while the ice used in the preservation contained 10⁵-10⁷ of TPC per gram. The microbiological qualities of the iced fishes of Imphal market were adjudged poor. The extremely high counts of bacteria might be due to (1) poor quality and left over fishes being packed, (2) contact with contaminated ice and (3) repeated thawing and freezing during the process of marketing and transportation.

Organoleptic and microbiological quality changes of catla (Catla catla) in immediate and delayed ice storage and at ambient temperature

An investigation was carried out on the quality changes of Catla (Catla calla) stored immediately (0 h) in ice, after 6 hours in ice and at ambient temperature. The samples were examined for organoleptic and microbiological parameters in summer. Organoleptically, the acceptability of fish varied between 16-20 days in both the iced storage conditions and 12-13.5 hours at ambient temperature (28°C). When fish were organoleptically just acceptable on the 16th day of storage, bacterial load were 6.23 and 6.17 log10 cfu/g, respectively for 0 hour and after 6 hours iced fish. But on the 20th day of storage, when fish were just unacceptable SPC were 6.51 and 6.62 log10 cfu/g. In case of ambient temperature storage condition standard plate count was 8.36 log10 cfu/g on 13.5 hours, when fish were organoleptically just unacceptable. At the time of rejection for fish stored in ice (0 hour and after 6 hours) on 20th day, gram negative and gram positive values were 55.45%, 44.55% and 44.52%, 55.48% respectively. While fish were rejected after 13.5 hours at ambient temperature gram negative and gram positive bacteria were found as 43.02% and 56.98%. The differences in SPC, gram positive and gram negative bacteria between the storage times were statistically significant (p<0.05).

Microbiological quality study of Macrobrachium rosenbergii (de Man 1879) during storage at -20°C temperature

The shelf life of fresh water prawn Macrobrachium rosenbergii by applying low temperature was investigated. M. rosenbergii preserved at -20°C was subjected for quality assessment before storage and at 15, 30, 45, and 90 days of storage period. The quality assessments as done microbiological viz. total bacterial count (TBC), total mould count (TMC), total yeast count (TYC), total coliform count (TCC) and salmonella count. All the samples were acceptable during 90 days because the upper limit of all spoilage indicator was not exceeding within the experimental time period.

The impact of molecular biology on assessment of water quality: advantages and limitations of current techniques

The advent of molecular biology has had a dramatic impact on all aspects of biology, not least applied microbial ecology. Microbiological testing of water has traditionally depended largely on culture techniques. Growing understanding that only a small proportion of microbial species are culturable, and that many microorganisms may attain a viable but non-culturable state, has promoted the development of novel approaches to monitoring pathogens in the environment. This has been paralleled by an increased awareness of the surprising genetic diversity of natural microbial populations. By targeting gene sequences that are specific for particular microorganisms, for example genes that encode diagnostic enzymes, or species-specific domains of conserved genes such as 16S ribosomal RNA coding sequences (rrn genes), the problems of culture can be avoided. Technical developments, notably in the area of in vitro amplification of DNA using the polymerase chain reaction (PCR), now permit routine detection and identification of specific microorganisms, even when present in very low numbers. Although the techniques of molecular biology have provided some very powerful tools for environmental microbiology, it should not be forgotten that these have their own drawbacks and biases in sampling. For example, molecular techniques are dependent on efficient lysis and recovery of nucleic acids from both vegetative forms and spores of microbial species that may differ radically when growing in the laboratory compared with the natural environment. Furthermore, PCR amplification can introduce its own bias depending on the nature of the oligonucleotide primers utilised. However, despite these potential caveats, it seems likely that a molecular biological approach, particularly with its potential for automation, will provide the mainstay of diagnostic technology for the foreseeable future.

Survey on the quality of Jaadi available [in] Sri Lankan market

A quality of survey was conducted at the fish curing yards in a northwest coast and the southern coast in Sri Lanka. A total of 40 samples different varieties of fishes were collected from the market and jaadi curing yards and all were evaluated for the quality, fungal and insect infestation. Samples were analyzed for proximate composition chemical, microbiological and sensory quality. Thirty percent of the total analyzed samples of fish were found to be unfit for consumption. Samples collected from Negombo were found to the infected with maggots. Only 42% samples had dry matter above 50%. All the samples showed a protein content above 20%. The highest protein content was 27.92% in hurulla. Over 90% of the samples had TVN at acceptable quality limits (>40). The TBC for 33% of the samples were in the range 104-105/g range, while 48% were in the range of 107-108/g due to contamination of maggots and fungi. The Survey showed jaadi had a high level of protein in its composition. But defects of curing process such on imperfect cleaning inadequate salting resulted in low (Chemical and microbiological) quality of the product.

Cultural methods of detection for microorganisms: recent advances and successes

Most microbiological methods require culture to allow organisms to recover or to selectively increase, and target organisms are identified by growth on specific agar media. Many cultural methods take several days to complete and even then the results require confirmation. Alternative techniques include the use of chromogenic and fluorogenic substances to identify bacteria as they are growing, selective capture using antibodies after short periods of growth, molecular techniques, and direct staining with or without flow cytometry for enumeration and identification. Future microbiologists may not use culture but depend on the use of specific probes and sophisticated detection systems.

Detection of specific bacteria in water: implications of survival strategy

It is widely recognised that conventional culture techniques may underestimate true viable bacterial numbers by several orders of magnitude. The basis of this discrepancy is that a culture in or on media of high nutrient concentration is highly selective (either through ”nutrient shock” or failure to provide vital co-factors) and decreases apparent diversity; thus it is unrepresentative of the natural community. In addition, the non-culturable but viable state (NCBV) is a strategy adopted by some bacteria as a response to environmental stress. The basis for the non-culturable state is that cells placed in conditions present in the environment cannot be recultured but can be shown to maintain their viability. Consequently, these cells would not be detected by standard water quality techniques that are based on culture. In the case of pathogens, it may explain outbreaks of disease in populations that have not come into contact with the pathogen. However, the NCBV state is difficult to attribute, due to the failure to distinguish between NCBV and non-viable cells. This article will describe experiences with the fish pathogen Aeromonas salmonicida subsp. salmonicida and the application of molecular techniques for its detection and physiological analysis.

The health significance of heterotrophic bacteria in drinking water

Tap water is not sterile; it contains organisms which grow in water distribution systems or inside taps and their fittings. The absence of known pathogenic bacteria is assured by the absence of the indicator organisms but concerns have been raised in the past few years that drinking water fulfilling the standards laid down in the EC Directive ECC 80/778 may still cause disease. These concerns have arisen from several sources: the fact that a cause has been identified in only half of all suspected waterborne outbreaks of disease; reports have suggested that heterotrophic bacteria possessing single pathogenic mechanisms such as haemolysin may cause disease; reports of heterotrophic organisms causing water contact diseases in hospitals. These concerns led to a reappraisal of the pathogenic potential of heteretrophic bacteria, by carrying out an extensive literature search and review commissioned by the UK Water Research Company. This research identified many papers showing an association between drinking water and heterotrophic bacteria but only very few reports of suspected waterborne disease associated with the heterotrophs. The organisms demonstrating potential to cause disease were species of Aeromonas and Yersinia, but typing of organisms identified in patients and isolated from the water revealed very few similarities. The potential of Aeromonas and Yersinia to cause waterborne disease is thought to be very low and the Communicable Disease Surveillance Centre database of laboratory infections due to these two genera of organisms was analysed to produce population-related incidences for each health region in England and Wales. Additionally a laboratory questionnaire revealed different levels of ascertainment of these two organisms in different laboratories of the Public Health Laboratory Service.

Viruses and drinking water

There is no evidence to indicate that there is a risk of acquiring a virus infection through the consumption of properly treated drinking water, provided the integrity of the distribution system is maintained and there is no post-treatment contamination. The consumption of inadequately treated, untreated or post-treatment contaminated water is, however, associated with a risk of hepatitis A, hepatitis E and viral gastroenteritis. The use of the standard bacterial indicators for water monitoring provides an adequate safeguard against viral contamination.

Production of positive controls for calcivirus-specific PCR using recombinant baculiovirus technology

Recent advances in our knowledge of the genetic structure of human caliciviruses (HuCVs) and small round-structured viruses (SRSVs) have led to the development of polymerase chain reaction (PCR)-based molecular tests specific for these viruses. These methods have been developed to detect a number of human pathogenic viruses in environmental samples including water, sewage and shellfish. HuCVs and SRSVs are not culturable, and no animal model is currently available. Therefore there is no convenient method of preparing viruses for study or for reagent production. One problem facing those attempting to use PCR-based methods for the detection of HuCVs and SRSVs is the lack of a suitable positive control substrate. This is particularly important when screening complex samples in which the levels of inhibitors present may significantly interfere with amplificiation. Regions within the RNA polymerase regions of two genetically distinct human caliciviruses have been amplified and used to produce recombinant baculoviruses which express RNA corresponding to the calicivirus polymerase. This RNA is being investigated as a positive control substrate for PCR testing, using current diagnostic primer sets. Recombinant baculovirus technology will enable efficient and cost-effective production of large quantities of positive control RNA with a specific known genotype. We consider the development of these systems as essential for successful screening and monitoring applications.

Persistence of viable but non-culturable bacteria during the production and distribution of drinking water

The direct measurement of in situ respiring bacteria using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) shows that, especially for Gram-negative bacteria, large numbers of viable but non-culturable (VBNC) bacteria are present in finished water from a conventional water treatment plant, and the regrowth of bacteria along distribution networks can be seen rapidly by using this very sensitive technique. The level of bacterial inactivation with chlorine is much less important than has been previously supposed (based on experiments with non-injured laboratory strains of bacteria and classical culture techniques). Threshold values of VBNC bacteria leaving water treatment plants or regrowing along distribution systems have to be determined for better control of coliform regrowth and health- risks associated with the consumption of drinking water.

Biodiversity in drinking water distribution systems:a brief review

In drinking water distribution systems, three groups of living organisms are usually found in the biofilm and circulating water: heterotrophic bacteria, free-living protozoa, and macro-invertebrates. Indirect evidence suggests that protozoa grazing in distribution systems can partially eliminate biomass production and accidental microbiological pollution. This paper examines the biodiversit in drinking water distribution systems.

Tastes and odours in potable water:perception versus reality

Tastes and odours are amongst the few water quality standards immediately apparent to a consumer and, as a result, account for most consumer complaints about water quality. Although taste and odour problems can arise from a great many sources, from an operational point of view they are either ”predictable” or ”unpredictable”. The former - which include problems related to actinomycete and algal growth - have a tendency to occur in certain types of water under certain combinations of conditions, whereas the latter - typically chemical spills - can occur anywhere. Long-term control is one option for predictable problems, although biomanipulation on a large scale has had utile success. Detection and avoidance is a more practicable option for both predictable and unpredictable problems, particularly if the distribution network can be serviced from other sources. Where these are not feasible, then water treatment, typically using activated carbon, is possible. In general there is a reasonable understanding of what compounds cause taste and odour problems, and how to treat these. An efficient taste and odour control programme therefore relies ultimately on good management of existing resources. However, a number of problems lie outside the remit of water supply companies and will require more fundamental regulation of activities in the catchment.