Microflora of Water
Microorganisms inhabit the water of all basins – from seas and oceans to lakes, rivers, streams, or bogs. They are spread everywhere and can be found even on the bottoms of ocean trenchs at depths up to 4000-9000 m.
The flora of rivers and lakes depends on water pollution and therefore from the quality of wastewater treatment and purification.
The representatives of many bacterial genera – Pseudomonas (e.g., P. fluorescens), Aeromonas, Plesiomonas, Micrococcus (M. roseus), Nitrosomonas, Nitrobacter and others – can be determined in water as the common aquatic microorganisms. Anaerobic bacteria are infrequently found in water, correlating with its pollution.
The degree of water contamination by various microorganisms is designated as saprobity. It generally comprises the total amount of all the living matter present in water including animal and plant decay remnants.
There are three zones of saprobity depending on the degree of water pollution.
Polysaprobic zone is highly contaminated water, with a mass of organic substances and a few oxygen contents. The total count of microorganisms in 1 ml exceeds 1,000,000. Coliform bacteria and anaerobic bacteria dominate there.
Mesosaprobic zone is characterized by a moderate pollution of water that is followed by the mineralization of organic matter by active oxidation and nitrification. The total microbial count in 1 ml of water is in the range about 104-105 microbial cells. The number of coliform bacteria is greatly reduced.
Oligosaprobic zone corresponds to pure water. The total number of microorganisms is generally low, about 10 to 1000 microbial cells in 1 ml of water. The representatives of coliform bacteria are not determined.
The water is an appropriate medium for transmission of the diseases predominantly by fecal-oral route (waterborne diseases). The most common infections transmitted by water include the broad group of bacterial and viral enteric infections (salmonellosis, shigellosis, colienteritis, cholera, campylobacteriosis, hepatitis A and E) as well as leptospirosis, tularemia, amoebic dysentery, fungal infections.
Many pathogenic bacteria remain alive in water for a long time. For instance, shigellae survive in water for 7-9 days, salmonellas – about 3 months, Vibrio cholera and El Tor – for many months, Francisella tularensis – for about of 3 months, leptospirae – from several weeks up to 4-5 month.
There are two main parameters (indices) indicating water sanitary quality.
Primary one is total plate count (or total viable count) in 1 ml of water.
Another one is the quantity of fecal indicator microorganisms. They have to be equal or less than their numbers established by regulation acts.
Total plate count of water is the quantity of mesophilic chemoorganotrophic bacteria in 1 ml of water capable of producing colonies after incubation at 37oC for 24 h. It should be less than 50 colony-forming units (CFU) per 1 ml (cm3) for tap water. In that case tap water is considered as clean satisfying sanitary regulations.
In the well water and in open reservoirs the amount of microbes in 1 ml should not exceed 1000.
The test for total microbial count determination in tap water is performed as follows. Tap is flamed, and then the water is opened and flows for 5 minutes. Then 1 ml of water is taken, poured into sterile Petri dish and mixed with 6-8 ml of melted and cooled up to 45oC meat peptone agar. After pour plating agar is hardened and the probe is incubated in thermostat at 37oC for 24 h. Then the total quantity of colonies is counted.
Indicator microorganisms of water are evaluated by determination of E. coli and its variants (so-called coliform bacteria). They reflect the possibility of fecal pollution of water. The coliform bacteria comprise the members of Enterobacteriaceae family from Escherichia, Citrobacter, Enterobacter, Klebsiella genera. They are gram-negative rods without spores, oxydase negative, fermenting lactose and mannitol to acid and gas products at 37oC in 24 h. These bacteria are discharged to the environment with feces from humans or animals.
Among total coliform bacteria there are thermotolerant bacteria, fermenting carbohydrates at 44oC for 24 h. These bacteria indicate fresh fecal environmental pollution.
Standards for tap water include the count of total coliform bacteria and thermotolerant bacteria in 100 ml of water. They should be absent in 300 ml of examined water probe.
Due to epidemiological situation some additional parameters of water quality (the quantity of coli phages, enteroviruses, С. perfringens spores) are estimated. These agents must also be absent.
Taking into account the enormous epidemiological role of water in relation to enteric infections, the tests for the assessment of indicator bacteria in water must be rapid, not laborious and highly reliable.
There are two basic testing methods for determination of quantity of fecal indicator bacteria in water.
First one is the membrane filtration method that is performed in several steps.
Three 100 ml portions of water are filtered through 3 separate nylon filters placed in sterile conditions into the funnel manifold (apparatus for the membrane filtration). The filters are removed and put onto Endo agar or similar medium. After incubation at 37oC for 24 h the quantity of red lactose-positive colonies is evaluated. If the growth of lactose-positive colonies is absent, the test means negative and the quality of water corresponds to normality. In the opposite case the investigation is continued. After counting lactose-positive colonies the gram-stained slides are prepared and examined (for colibacteria gram-negative rods should be revealed). Oxydase test is performed that is negative for coli-bacteria. Then the colony sample is inoculated into semi-solid lactose-peptone media for incubation at 37oC within 24 h. Gas and acid production is detected and the conclusion about quantity of total coliform bacteria is made. It indicates the fecal water pollution regardless of its terms.
For identification of fresh fecal water pollution the quantity of thermotolerant bacteria is assessed. Additional examination includes inoculation of culture into semi-solid lactose-peptone media for incubation at 44oC within 24 h. If gas and acid production due to lactose fermentation is revealed, the conclusion about thermotolerant coli-bacteria (E. coli) presence is made, indicating fresh fecal water pollution.
Titration method is used for water testing in case of membrane filtration method inaccessibility or in case of opaque water with many suspended particles. It is based on lactose-peptone medium fermentation similar with previous method.