bacteriological water analysis
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Bacteriological water analysis is a method of analysing water to estimate the numbers of
bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
present and, if needed, to find out what sort of bacteria they are. It represents one aspect of
water quality Water quality refers to the chemical, physical, and biological characteristics of water based on the standards of its usage. It is most frequently used by reference to a set of standards against which compliance, generally achieved through tr ...
. It is a
microbiological Microbiology () is the scientific study of microorganisms, those being unicellular (single cell), multicellular (cell colony), or acellular (lacking cells). Microbiology encompasses numerous sub-disciplines including virology, bacteriology, pro ...
analytical procedure which uses samples of water and from these samples determines the concentration of bacteria. It is then possible to draw inferences about the suitability of the water for use from these concentrations. This process is used, for example, to routinely confirm that water is safe for human consumption or that bathing and
recreation Recreation is an activity of leisure, leisure being discretionary time. The "need to do something for recreation" is an essential element of human biology and psychology. Recreational activities are often done for enjoyment, amusement, or pleasur ...
al waters are safe to use. The interpretation and the action trigger levels for different waters vary depending on the use made of the water. Whilst very stringent levels apply to
drinking water Drinking water is water that is used in drink or food preparation; potable water is water that is safe to be used as drinking water. The amount of drinking water required to maintain good health varies, and depends on physical activity level, a ...
, more relaxed levels apply to marine bathing waters, where much lower volumes of water are expected to be ingested by users.


Approach

The common feature of all these routine screening procedures is that the primary analysis is for indicator organisms rather than the
pathogen In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ ...
s that might cause concern. Indicator organisms are bacteria such as non-specific
coliform Coliform bacteria are defined as either motile or non-motile Gram-negative non- spore forming Bacilli that possess β-galactosidase to produce acids and gases under their optimal growth temperature of 35-37°C. They can be aerobes or facultativ ...
s, ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' and ''
Pseudomonas aeruginosa ''Pseudomonas aeruginosa'' is a common encapsulated, gram-negative, aerobic–facultatively anaerobic, rod-shaped bacterium that can cause disease in plants and animals, including humans. A species of considerable medical importance, ''P. aerugi ...
'' that are very commonly found in the human or animal gut and which, if detected, may suggest the presence of
sewage Sewage (or domestic sewage, domestic wastewater, municipal wastewater) is a type of wastewater that is produced by a community of people. It is typically transported through a sewer system. Sewage consists of wastewater discharged from residenc ...
. Indicator organisms are used because even when a person is infected with a more pathogenic bacteria, they will still be excreting many millions times more indicator organisms than pathogens. It is therefore reasonable to surmise that if indicator organism levels are low, then pathogen levels will be very much lower or absent. Judgements as to suitability of water for use are based on very extensive precedents and relate to the probability of any sample population of bacteria being able to be infective at a reasonable statistical level of confidence. Analysis is usually performed using culture, biochemical and sometimes optical methods. When indicator organisms levels exceed pre-set triggers, specific analysis for pathogens may then be undertaken and these can be quickly detected (where suspected) using specific culture methods or
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
.


Methodologies

The most reliable methods are direct plate count method and membrane filtration method. mEndo Agar is used in the membrane filtration while VRBA Agar is used in the direct plate count method. VRBA stands for violet red bile agar. A media that contains bile salts which promotes the growth of gram negative and has inhibitory characteristic to gram positive although not complete inhibitory. These media contain lactose which is usually fermented by lactose fermenting bacteria producing colonies that can be identified and characterised. Lactose fermenting produce colored colonies while non lactose fermenting produce colorless ones. Because the analysis is always based on a very small sample taken from a very large volume of water, all methods rely on statistical principles.}


Multiple tube method

One of the oldest methods is called the multiple tube method. In this method a measured sub-sample (perhaps 10 ml) is diluted with 100 ml of sterile growth medium and an
aliquot Aliquot ( la, a few, some, not many) may refer to: Mathematics *Aliquot part, a proper divisor of an integer *Aliquot sum, the sum of the aliquot parts of an integer *Aliquot sequence, a sequence of integers in which each number is the aliquot sum ...
of 10 ml is then decanted into each of ten tubes. The remaining 10 ml is then diluted again and the process repeated. At the end of 5 dilutions this produces 50 tubes covering the dilution range of 1:10 through to 1:10000. The tubes are then incubated at a pre-set temperature for a specified time and at the end of the process the number of tubes with growth in is counted for each dilution. Statistical tables are then used to derive the concentration of organisms in the original sample. This method can be enhanced by using indicator medium which changes colour when acid forming species are present and by including a tiny inverted tube called a
Durham tube Durham tubes are used in microbiology to detect production of gas by microorganisms. They are simply smaller test tubes inserted upside down in another test tube so they are freely movable. The culture media to be tested is then added to the la ...
in each sample tube. The Durham inverted tube catches any gas produced. The production of gas at 37 degrees Celsius is a strong indication of the presence of ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
''.


ATP testing

An ATP test is the process of rapidly measuring active microorganisms in water through detection
adenosine triphosphate Adenosine triphosphate (ATP) is an organic compound that provides energy to drive many processes in living cells, such as muscle contraction, nerve impulse propagation, condensate dissolution, and chemical synthesis. Found in all known forms of ...
(ATP). ATP is a molecule found only in and around living cells, and as such it gives a direct measure of biological concentration and health. ATP is quantified by measuring the light produced through its reaction with the naturally occurring enzyme
firefly luciferase Firefly luciferase is the luciferase, light-emitting enzyme responsible for the bioluminescence of fireflies and click beetles. The enzyme catalyses the oxidation of firefly luciferin, requiring oxygen and Adenosine triphosphate, ATP. Because of ...
using a luminometer. The amount of light produced is directly proportional to the amount of biological energy present in the sample. Second generation ATP tests are specifically designed for water,
wastewater Wastewater is water generated after the use of freshwater, raw water, drinking water or saline water in a variety of deliberate applications or processes. Another definition of wastewater is "Used water from any combination of domestic, industr ...
and industrial applications where, for the most part, samples contain a variety of components that can interfere with the ATP assay.


Plate count

The plate count method relies on bacteria growing a colony on a nutrient medium so that the colony becomes visible to the naked eye and the number of colonies on a plate can be counted. To be effective, the dilution of the original sample must be arranged so that on average between 30 and 300 colonies of the target bacterium are grown. Fewer than 30 colonies makes the interpretation statistically unsound whilst greater than 300 colonies often results in overlapping colonies and imprecision in the count. To ensure that an appropriate number of colonies will be generated several dilutions are normally cultured. This approach is widely utilised for the evaluation of the effectiveness of water treatment by the inactivation of representative microbial contaminants such as ''E. coli'' following ASTM D5465. The laboratory procedure involves making serial dilutions of the sample (1:10, 1:100, 1:1000, etc.) in sterile water and cultivating these on
nutrient A nutrient is a substance used by an organism to survive, grow, and reproduce. The requirement for dietary nutrient intake applies to animals, plants, fungi, and protists. Nutrients can be incorporated into cells for metabolic purposes or excret ...
agar in a dish that is sealed and incubated. Typical media include
plate count agar Plate Count Agar (PCA), also called Standard Methods Agar (SMA), is a microbiological growth medium commonly used to assess or to monitor "total" or viable bacterial growth of a sample. PCA is not a selective medium. The total number of living a ...
for a general count or
MacConkey agar MacConkey agar is a selective and differential culture medium for bacteria. It is designed to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacteria and differentiate them based on lactose fermentation. ...
to count
Gram-negative bacteria Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...
such as ''E. coli''. Typically one set of plates is incubated at 22 °C and for 24 hours and a second set at 37 °C for 24 hours. The composition of the nutrient usually includes
reagent In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...
s that resist the growth of non-target organisms and make the target organism easily identified, often by a colour change in the medium. Some recent methods include a fluorescent agent so that counting of the colonies can be automated. At the end of the incubation period the colonies are counted by eye, a procedure that takes a few moments and does not require a
microscope A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisibl ...
as the colonies are typically a few millimetres across.


Membrane filtration

Most modern laboratories use a refinement of total plate count in which serial dilutions of the sample are vacuum filtered through purpose made
membrane filter Membrane technology encompasses the scientific processes used in the construction and application of membranes. Membranes are used to facilitate the transport or rejection of substances between mediums, and the mechanical separation of gas and li ...
s and these filters are themselves laid on nutrient medium within sealed plates. The methodology is otherwise similar to conventional total plate counts. Membranes have a printed millimetre grid printed on and can be reliably used to count the number of colonies under a binocular microscope.


Pour plate method

When the analysis is looking for bacterial species that grow poorly in air, the initial analysis is done by mixing serial dilutions of the sample in liquid nutrient agar which is then poured into bottles which are then sealed and laid on their sides to produce a sloping agar surface. Colonies that develop in the body of the medium can be counted by eye after incubation. The total number of colonies is referred to as the
total viable count Total viable count (TVC), gives a quantitative estimate of the concentration of microorganisms such as bacteria, yeast or mould spores in a sample. The count represents the number of colony forming units (cfu) per g (or per ml) of the sample. A TV ...
(TVC). The unit of measurement is cfu/ml (or colony forming units per millilitre) and relates to the original sample. Calculation of this is a multiple of the counted number of colonies multiplied by the dilution used.


Pathogen analysis

When samples show elevated levels of indicator bacteria, further analysis is often undertaken to look for specific pathogenic bacteria. Species commonly investigated in the temperate zone include ''
Salmonella typhi ''Salmonella enterica'' subsp. ''enterica'' is a subspecies of ''Salmonella enterica'', the rod-shaped, flagellated, aerobic, Gram-negative bacterium. Many of the pathogenic serovars of the ''S. enterica'' species are in this subspecies, includin ...
'' and ''
Salmonella Typhimurium ''Salmonella enterica'' subsp. ''enterica'' is a subspecies of ''Salmonella enterica'', the rod-shaped, flagellated, aerobic, Gram-negative bacterium. Many of the pathogenic serovars of the ''S. enterica'' species are in this subspecies, includi ...
''. Depending on the likely source of contamination investigation may also extend to organisms such as ''
Cryptosporidium ''Cryptosporidium'', sometimes informally called crypto, is a genus of apicomplexan parasitic alveolates that can cause a respiratory and gastrointestinal illness (cryptosporidiosis) that primarily involves watery diarrhea (intestinal cryptosp ...
spp''. In tropical areas analysis of ''
Vibrio cholerae ''Vibrio cholerae'' is a species of Gram-negative, facultative anaerobe and comma-shaped bacteria. The bacteria naturally live in brackish or saltwater where they attach themselves easily to the chitin-containing shells of crabs, shrimps, and oth ...
'' is also routinely undertaken.


Types of nutrient media used in analysis

MacConkey agar MacConkey agar is a selective and differential culture medium for bacteria. It is designed to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacteria and differentiate them based on lactose fermentation. ...
is culture medium designed to grow Gram-negative bacteria and stain them for lactose fermentation. It contains bile salts (to inhibit most Gram-positive bacteria), crystal violet dye (which also inhibits certain Gram-positive bacteria), neutral red dye (which stains microbes fermenting lactose),
lactose Lactose is a disaccharide sugar synthesized by galactose and glucose subunits and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from ' (gen. '), the Latin word for milk, plus the suffix '' - ...
and
peptone Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. A p ...
. Alfred Theodore MacConkey developed it while working as a bacteriologist for the Royal Commission on Sewage Disposal in the
United Kingdom The United Kingdom of Great Britain and Northern Ireland, commonly known as the United Kingdom (UK) or Britain, is a country in Europe, off the north-western coast of the continental mainland. It comprises England, Scotland, Wales and North ...
. Endo agar contains peptone, lactose,
dipotassium phosphate Dipotassium phosphate (K2HPO4) (also dipotassium hydrogen orthophosphate; potassium phosphate dibasic) is the inorganic compound with the formula K2HPO4.(H2O)x (x = 0, 3, 6). Together with monopotassium phosphate (KH2PO4.(H2O)x), it is often used ...
, agar, sodium sulfite, basic
fuchsin Fuchsine (sometimes spelled fuchsin) or rosaniline hydrochloride is a magenta dye with chemical formula C20H19N3·HCl.
and was originally developed for the isolation of ''Salmonella typhi'', but is now commonly used in water analysis. As in MacConkey agar, coliform organisms ferment the lactose, and the colonies become red. Non-lactose-fermenting organisms produce clear, colourless colonies against the faint pink background of the medium. ''mFC medium'' is used in membrane filtration and contains selective and differential agents. These include rosolic acid to inhibit bacterial growth in general, except for faecal coliforms,
bile Bile (from Latin ''bilis''), or gall, is a dark-green-to-yellowish-brown fluid produced by the liver of most vertebrates that aids the digestion of lipids in the small intestine. In humans, bile is produced continuously by the liver (liver bile ...
salts inhibit non-enteric bacteria and aniline blue indicates the ability of faecal coliforms to ferment lactose to acid that causes a pH change in the medium. U.S. Geological Survey. Ohio Water Microbiology Laboratory, Columbus, OH. (January 2007)
"mFC agar method for fecal coliforms."
''Analytical Methods.''
''TYEA medium'' contains
tryptone Tryptone is the assortment of peptides formed by the digestion of casein by the protease trypsin. Tryptone is commonly used in microbiology to produce lysogeny broth (LB) for the growth of '' E. coli'' and other microorganisms. It provides a so ...
,
yeast extract Yeast extracts consist of the cell contents of yeast without the cell walls; they are used as food additives or flavorings, or as nutrients for bacterial culture media. They are often used to create savory flavors and umami taste sensations, a ...
, common salt and
L-arabinose Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group. For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, or structural ...
per liter of glass distilled water and is a non selective medium usually cultivated at two temperatures (22 and 36 °C) to determine a general level of contamination (a.k.a. colony count).


See also

*
Water testing Water (chemical formula ) is an inorganic, transparent, tasteless, odorless, and nearly colorless chemical substance, which is the main constituent of Earth's hydrosphere and the fluids of all known living organisms (in which it acts as a ...
*
Water quality Water quality refers to the chemical, physical, and biological characteristics of water based on the standards of its usage. It is most frequently used by reference to a set of standards against which compliance, generally achieved through tr ...


References

{{DEFAULTSORT:Bacteriological Water Analysis Aquatic ecology Water Microbiology techniques Water quality indicators