II. Biochemical tests for Differentiation of Faecal (Escherichia coli) and Non-Faecal (Enterobacter sp.) coliform present in water samples: IMViC Test:Since, E. coli and E aerogenes bear a close resemblance to each other in their morphological and cultural characteristics, four biochemical tests are performed to differentiate them. These tests are collectively known as the IMViC tests. Each letter of IMViC stands for a reaction/property or a product, which can be used for both to characterize E. coli and to differentiate it from E. aerogenes (I - indole production, M - methyl red test, Vi - Voges-Proskauer reaction, and C - citrate utilization). Colonies from the nutrient agar slant of the completed test, described above, are used to inoculate Hi Media IMViC test kit with 50 microlitre/loopful of culture and the kit is incubated at 370C for 24-48 hours.1. Indole test (I): E. coli produce/synthesize an enzyme, tryptophanase, which forms indole, pyruvic acid and ammonia from tryptophan, whereas E. aerogenes cannot catabolize tryptophan and do not produce indole.2. Methyl red test (M): Methyl red is an acid-base indicator that turns red in a slightly acid medium. Both the organisms produce acid from glucose, E. coli produce large amount of acids thus a low pH, which turn the indicator (methyl red) to red colour whereas E. aerogenes cultures produce only small amounts of organic acids and consequently do not produce the colour change.3. Voges-Proskauer (Vi) test: It detects the presence of acetyl methyl carbinol. E. coli do not produce acetyl-methyl-carbinol in glucose peptone medium but E. aerogenes do.4. Citrate utilization test (C): E. aerogenes is capable of utilizing sodium citrate as its sole source of carbon, i.e. it will grow in a chemically defined medium in which sodium citrate is the only source of carbon. E. coli does not grow under these circumstances.(ii) Membrane Filter Technique:This technique has become common and is preferred. It involves the use of a sterile filter disk having pores fine enough to retain bacteria (</= 0.22μ). The filter disk is placed in filtration unit, and a measured volume of water is filtered through it. The bacteria retained on the surface of the filter disk are removed and placed upon the surface of Eosin Methylene Blue agar medium or on a sterile absorbent pad that has been previously saturated with an appropriate broth culture mediumin Petri dish and incubated. The colonies develop on the surface of the membrane wherever bacteria are entrapped. Eosin Methylene Blue culture medium, which is both selective and differential medium for coliforms is used. The dark colour of colonies is characteristics of coliform, which are counted and then from this value the total number of coliforms in original water sample can be determinedBiochemical Characterization of Microbial Flora of Water and WastewaterDrinking water should be aesthetically acceptable, being clear, odourless, without disagreeable taste, free from chemical toxins and pathogenic micro-organisms. Diseases like typhoid, cholera, diarrhea, poliomyelitis and viral hepatitis A and B are water borne. Natural water sources usually contain some saprophytic bacteria like Pseudomonas, Serratia, Flarobacterium, Chromobacterium, Acinetobacter and Alcaligenes specis. Aerobie spore former bacilli, Enterobacter sps may also be washed into natural waterbodies. These are harmless. Only pathogens introduced into water by excremental or sewage pollution pose a risk to human health.The primary test employed as an indicator of fecal pollution of water is the presence of coliform bacteria because they are present in feces of human beings and other warm blooded animals in

large numbers and can be detected in water, even in high dilutions. Presence of thermotolerant E. coli provides definite proof of fecal pollution.Objective: To characterize water and wastewater microflora biochemicallyPrinciple/Theory:The challenge of waste water treatment is to remove (1) Compounds with high O2 demand (2) pathogenic organisms and viruses and (3) a multitude of human made chemicals. Biochemical tests are one of the easiest and cheaper means for identification. Here two different types of readymade biochemical test kits from Hi Media labs will be used for the biochemical characterization. Besides this, given below is identification of different groups of bacteria on the basis of media’s and biochemical methods using conventional techniquesConventional way to characterize water microbes:Collection of SamplesThe sample containers should be clean or sterilized. Sodium thiosulfate should be added to samples of chlorinated water to inactivate residual chlorine which may power bacterial counts. Samples should be immediately analyzed for microbiological testing.Differential Coliform testEijkman test is usually employed to find out whether the coli forms of bacteria detected in presumptive test are E.coli. After usual presumptive test, subcultures are made from all the bottles showing acid and gas production to fresh tubes of single strength MacConkey broth. They are incubated at 440C strictly. Thermotolerant E. coli give definite proof of fecal pollution. Those showing gas in Durham’s tubes, contain E.coli. Confirmation of E.coli can be done by testing for indole production and citrate utilization.IMViC Tests to differentiate enteric bacteriaIndole production: Tryptophan is an essential amino acid that can be metabolized by tryptophanase produced by some bacteria. Ability to hydrolyze tryptophan with the production of indole, a nitrogen compound is not a characteristic of all microorganisms and therefore serves as a biochemical marker. Indole can be detected chemically. Tryptone (digested protein) is used as a substrate in this test.Tryptophan ____________________ Indole + Pyruvic acid + AmmoniaTryptophanaseMethod: Inoculate two 1.0% tryptone broth tubes with test culture. Along with one control; incubate tubes at 370C for 24-48 h. To about 6.0 ml of culture, add 0.3ml Kovac’s reagent (p-dimethylaminobezaldehyde,5g, amyl alcohol, 75 ml and conc. HCl ,25 ml). Mix well. Reddening of upper layer of broth within few min. indicates a positive indole test.Methyl Red (MR) Test: Sugars (hexose monosaccharide) are oxidized by all enteric organisms for energy production but end products vary with the organisms in use. Methyl red (a pH indicator) detects the presence of large concentrations of acids. This test differentiates between E.coli and Enterobacter aerogens particularly. Both organisms initially produce organic acids but at later stages E.coli maintains acidic condition while Enterobacter aerogens converts these acids to non acid products such as 2,3 butanediol and acetoin resulting in a rise in pH.Methyl red at 4.0 ph turns red, indicating a positive test. At 6.0 ph indicator remains yellow.

Glucose +H2O______Lactic, acetic and formic acids + CO2 + H2 (pH4.0) __Red color

MRVP broth contains glucose, 0.5, proteose peptone 0.5, K2HPO4 in 100 ml water. Do not adjust pH.Method: Inoculate 5 ml MRVP broth with bacterial culture and incubate for 48 h at 37 0C. Appearance of a distinct red colour on adding alc. methyl red solution shows positive test.Voges-Proskauer (VP) test: This test measures the production of neutral end products such as acetylmethyl carbinol from organic acids from glucose by some bacteria (Enterobacter aerogens)Glucose + O2_________Acetic acid__________2, 3 dibutanediol, acetyl methylcarbinol +CO2 +H2 (pH 6.0)In the presence of Barritts reagent, acetyl methyl carbinol is oxidized to a diacetyl compound, imparting red colour to the medium.Nonacidic compounds produced from glucose fermentation by E.aerogens are detected .Barritts reagent consists of a mixture of alcoholic alpha nephthol and 40 % KOH.Citrate utilization: In the absence of fermentable sugars, some bacteria can utilize citrate as a carbon source which depends on the presence of enzyme citrate permease (positive test by Protease sps.)Citric acid__________ Oxalacetic acid +acetic acid _________ Pyruvic acidCitraseSimmon citrate agar contains citrate as its only carbon and energy source. Colour change from green to blue is a positive test of citrate utilization

I) IMViC Test Kit (HiMedia)Principle: Each HiMViC kit is a standardized colorimetric identification system utilizing four conventional biochemical tests and eight carbohydrate utilization tests. The tests are based on the principle of pH change and substrate utilization. On incubation, organisms undergo metabolic changes which are indicated by colour change in the media that can be either interpreted visually or after addition of the reagent(s).Requirements: HiIMViC Biochemical Test Kit (KB 001)1. Each kit contains sufficient material to perform 10 test2. Kovac’s reagent (R008) for indole test3. Methyl Red reagent (I007) for Methyl Red test4. Baritt reagent A (R029) for Voges-Proskauer’s test5. Baritt reagent B (R030) for Voges- Proskauer’s test

Procedure:1) Preparation of inoculum* KB001 cannot be used directly on clinical specimens. The organisms to be identified have to be first isolated and purified. Only pure cultures should be used.* Isolate the organism to be identified on a common medium like Nutrient Agar (M001/M1274) or Brain Heart Infusion Agar (M211). Pick up a single well isolated colony and inoculate in 5 ml Brain Heart Infusion broth and incubate at 37oC for 4-6 hours until the inoculum turbidity is ≥ 0.1 0D at 620 nm or 0.5 Mcfarland standard. Alternatively, a homogeneous suspensionmade in 2-3 ml sterile saline can be used for inoculation. The density of the suspension should be adjusted to 0.1 0D at 620 nm or 0.5 Mcfarland standard.

Note

* Erroneous false negative results may be obtained if the inoculum turbidity is less than 0.1 O* Results are more prominent when enriched culture instead of suspension

2) Inoculation of the strip* Open the kit aseptically. Peel off the sealing tape* Inoculate each well with 50 μl of the above inoculum by surface inoculation method* Alternatively the strip can be inoculated by stabbing each individual well with a loopful of inoculum3) Incubation : Temperature of incubation : 35-37oC, Duration of incubation :

18-24 hours.Observations & Result*Interpret results as per the standard given in the Result Interpretation Chart. Addition of reagents in well nos 1, 2 and 3 should be done at the end of incubation period that is after 18-24 hrs. Following reagents to be added to the respective wells.

Indole Test : Well No. 1* Add 1-2 drops of Kovac’s reagent (R008). Development of reddish pink colour with in 10 seconds indicates a positive reaction.* Reagent remains pale coloured if the test is negative.

Methyl Red Test : Well No. 2* Add 1-2 drops of Methyl Red reagent (I007).* Reagent remains red in colour if the test is positive.* Reagent decolourises and turns yellow if the test is negative.

Voges Proskaeur’s Test : Well No. 3* Add 2-3 drops of Baritt reagent A (R029) and 1-2 drops of Baritt reagent B (R030).* Pinkish red colour development within 5-10 minutes indicates a positive test.* No change in colour or a slight copper colour (due to reaction of Baritt reagent A with Baritt reagent B) denotes a negative reaction.

Important points to be taken into consideration while interpreting the result1) Allow the reagents to come to room temperature after removal from the refrigerator.2) In case of carbohydrate fermentation tests, some microorganisms show weak reaction. In this case record the reaction as ± and incubate further upto 48 hours. Orange colour after 48 hours of incubation should be interpreted as a negative reaction.3) In case of Lysine and Ornithine decarboxylation reaction, incubation upto 48 hours may be required.4) At times organisms give contradictory result because of mutation or the media used for isolation, cultivation and maintenance.5) The identification index has been compiled from standard references and results of tests have been obtained in the laboratory.

Precautions

* Clinical samples and microbial cultures should be considered potentially pathogenic and handled accordingly.

* Aseptic conditions should be maintained during inoculation and handling of the strips.* Reagents should not come in contact with skin, eyes or clothing.

Disposal of used materialAfter use, strips the instruments used for isolation and inoculation (pipettes, loops etc.) must be disinfected using a suitable disinfectant and then discarded by incineration or autoclaving in a disposable bag.RESULT INTERPRETATION CHART No.

Test Reagents to be added after incubation

Principle Original colour of the medium

Positive reaction

Negative reaction

1 Indole 1-2 drops of Kovac’s reagent

Detects deamination of tryptophan

Colourless Reddish pink

Colourless

2 Methyl red 1-2 drops of Methyl reagent

Detects acid production

Colourless Red Yellow

3 Voges Proskauer’s

1-2 drops of Barrit (reagent A and 1-2 drops of Baritt reagent B

Detects acetoin production

Colourless Pinkish red Colourless/ slight copper

TITAN MEDIATitan Medium or Dehydrated Culture Medium is a combination of complex nutrient substrates formulated for the cultivation of microorganisms. The components of a Dehydrated Culture Medium must satisfy the nutritional requirements of microorganisms, which in order to live and replicate require sources of nitrogen, carbon and trace elements. The trend has been towards using more defined media components with extracts and peptones taking the place of infusions and more recently, with chromogenic / fluorogenic compounds taking the place of carbohydrates and pH indicators.

There are various uses of Dehydrated Culture Media, which can be summarized as follows:Maintenance of microorganisms in culture and subcultures.Isolation and enumeration of microorganisms in foods & beverages, water, dairy, cosmetics, brewery, pharmaceutical, agriculture and veterinary etc.Isolation of pathogenic microorganisms that cause infections.Identification of microorganisms for therapeutic, epidemiological and taxonomic purposes.Determination of the sensitivity of microorganisms to antimicrobial agents.Study of the biochemical and physiological characteristics of microorganisms and their ability to adapt to different environments.

Mass cultivation of microorganisms for the production of antibiotics, enzymes, toxins, antisera and vaccines etc.Evaluation of the biological activity of pharmaceutical preparations (antibiotics, vitamins, amino acids and disinfectants) and the specific activity of drugs.

There are various types of culture media ingredients like peptones, carbohydrates, indicators, minerals, selective agents, solidifying agent i.e., agar, enrichments and enzymatic substrates.PEPTONES AND EXTRACTS : The peptones and extracts are nutrients obtained from meat, casein, soybean, yeast cells, liver, malt, gelatin, potato and groundnut etc. Peptones are produced by enzymatic hydrolysis of proteins. Enzymatic hydrolysis of proteins break the protein molecule at specific points and tends to preserve the amino acid and vitamin content of the original raw material while acidic hydrolysis breaks all the peptide bonds and produces free amino acids losing some important amino acids. The meat infusions and extracts obtained by protein coagulation through heating are very similar to peptones.CARBOHYDRATES :Carbohydrates like glucose, lactose, mannitol and sucrose etc. are added to a Culture Medium as a source of energy to increase the rate of growth of microorganisms. They are present as fermentable substrates in combination with pH indicators for microbial differentiation

INDICATOR SUBSTANCESIndicator Substances can be classified into there types: pH indicators, oxidation-reduction indicators, and hydrogen sulphide indicators. Among the pH indicators, phenol red, neutral red, bromothymol blue and bromocresol purple have replaced the substances most used in the past such as litmus and Andrade’s indicator. The pH indicator reveals the formation of acids from carbohydrates and the formation of bases (Ammonium ions) from peptones, single amino acids or amines.Among the above-mentioned indicators, phenol red is the most sensitive since it reveals even very small variations in pH in the Dehydrated Culture Media and is most widely used.The oxidation-reduction indicators used in Culture Media are methylene blue and resazurin, which take on particular colourless to blue, and resazurin from colourless to pink.The hydrogen sulphide indicator substances are usually ferrous salts (ferric citrate, ferric sulphate, ferric ammonium sulphate, ferric ammonium citrate). The hydrogen sulphide produced by bacteria from sodium thiosulphate, reacts with the ferrous producing ferrous sulphide, which precipitates in the centre of the colony (characteristic colonies with black centres).

MINERALS :The salts (Mg, Mn, Fe, Ca, Zn, Cu) present in Dehydrated Culture Media provide necessary minerals for microbial growth. Potassium and sodium phosphates acts as buffering agents in the medium while sodium chloride maintains the osmolarity of the Culture Medium

SELECTIVE AGENTS:              The selective agents are chosen and added to Culture Media to suppress the growth of unwanted organisms favouring the growth of desired ones. The first selective agents used in Microbiology were dyes and they are still present for this purpose in some formulations. Crystal

violet is used in MacConkey agar to inhibit the Gram-positive bacteria, brilliant green is used in combination with bile salts to inhibit Gram-positive bacteria and stimulate the growth of Salmonella. Among the substances of biological origin, bile salts are the most widely used selective agents. They are present both in mixtures (Bile Salts, Bile Salt No. 3) and as pure substances (Sodium deoxycholate) to inhibit the growth of Gram-positive bacteria in media for isolation of intestinal microflora.

A new class of selective agents, which is useful both in Clinical and Industrial Microbiology, are antibiotics. These substances, alone or in mixtures, already in the media or supplied separately as supplements, have the advantage of selecting the bacterial species to be isolated with greater specificity. Organic & inorganic salts make another class of selective agents. Sodium chloride at high concentration inhibits both the Gram negative and Gram-positive organisms, with the exception of Staphylococci Sodium azide in Dehydrated Culture Media is used for the selective isolation of Streptococci and Enterococci. Sodium Selenite in a buffered medium stimulates the growth of Salmonella and inhibits the Gram-positive bacteria. Sodium Citrate, Potassium tellurite, Sodium Tertrathionate and Sodium lauryl Sulphate also belong to this class of ingredients.SOLIDIFYING AGENTS :The main solidifying agent in Dehydrated Culture Media is Agar. Robert Koch perfected the use of agar in culture substances and gave great impetus to the technique of isolation of microorganisms in pure culture. Agar is extracted from agarophyte seaweeds mainly Gellidium, Gracilaria, Plerocladia and Euchuma. Agar with different properties can be obtained based on the place of cultivation and techniques of extraction. The agar obtained from algae cultivated on the Altantic has a stronger solidifying character than agar obtained from the alage cultivated on the coasts of the Pacific. The agar in Culture Media has the unique role of a solidifying agent and has no nutritive properties as regard to microorganisms.Titan's Agar Quality is tested and certified under ideal conditions.Note: Repeated heating of agar medium decreases its Gel strength

ENRICHMENTS :To improve the fertility properties of culture media for the cultivation of fastidious microorganisms (Neisseria, Haemophillus etc.) various enrichments are added normally after autoclaving and cooling the base medium to 50°C. Blood and animal serum are the most commonly used enrichments. According to the type of microbial research to be carried out heamoglobin, albumin, egg yolk, whole eggs, chemically defined enrichment solutions are used

ENZYMATIC SUBSTANCE (CHROMOGENIC & FLUOROGENIC MEDIA) :Incorporating synthetic or natural substances, which can be cleaved in Dehydrated Culture Media by specific microbial enzymes, a big improvement has been obtained in the identification of some microbial species and genera. Depending on the substrate, cleavage of these products by microbial enzymes can lead either to a diffuse colouration or the formation of a coloured precipitate in the center of the colonies or, else to the formation of an opaque or clarification halo around the colonies for some media, identification occurs at the species level and for others at the genus or group level. The specificity of the detection is correlated to the specificity of the defected enzymatic activity and to the Culture Media formation. To increase the specificity and the sensitivity of the microbial detection in Chromogenic Culture Media, the enzymatic

substrates are to be used in combination with optimized reagents, nutrient compounds and inhibitory substances.

PRECAUTIONS WHILE USING DEHYDRATED CULTURE MEDIARead instructions carefully given over the labels and note the best before date of each lot before use.Confirm the physical characteristics of the Dehydrated Culture Media, which should be free flowing and not clumped.Since the Culture Media are highly hygroscopic, store them in cool (preferably below 25°C, unless and otherwise specified) and dry place. Protect them from direct sunlight and humid place.Ensure proper capping of the box after use.Efficiency of the autoclave should be ascertained from time to time using various physical measurements or chemicals indicators.Before using agar based media, the plates should be sterilized by keeping washed and dried plates in oven at 160° C for 2 hours.Never store the prepared media at 0°C.Bring the stored media to room temperature before use or as per instructions for use.Please place order for TITAN MEDIA along with their supplements if required.

13.2.4 BacteriophagesViruses which infect bacteria, known as bacteriophages or simply as phages,were first described from the intestinal tract of man in the early 1900s(D’Herelle 1926; Pelczar et al. 1988). The use of phages as models forindicating the likely presence of pathogenic enteric bacteria first appeared in the1930s, and direct correlations between the presence of certain bacteriophagesand the intensity of faecal contamination were reported (several references citedby Scarpino 1978).The evolving role for phages to coliforms, known as coliphages (Box 13.1;Table 13.3) however, has been to model human enteric viruses. The DNAcontainingtailed coliphages (T type) and RNA-containing phages that infect viathe F-pili (sex factor) (F-RNA coliphages) have been the most used.

III. Examination of Bottled WaterConsumption of bottled water is increasing rapidly worldwide. In the U.S. alone, over 3.6 billion gallons of bottled water were consumed in 1998 (International Bottled Water Association, Alexandria, VA). Unlike potable water, which is regulated by the U.S. EPA, bottled water is legally classified as food in the U.S. and regulated by the FDA (Federal Register. 1995. 21 CFR Part 103 et al. beverages: bottled water; final rule. 60(218) 57076-57130). FDA defines bottled water as "water that is intended for human consumption and that is sealed in bottles or other containers with no added ingredients except that it may contain safe and suitable antimicrobial agents" and, within limitations, some added fluoride. Bottled water may be used as a beverage by itself or as an ingredient in other beverages. These regulations do not apply to soft drinks or similar beverages. In addition to "bottled water" or "drinking water", in 21 CFR Part 103 FDA also defines various types of bottled water that meet certain criteria. These identities include "artesian or artesian well water", "ground water", mineral water", "purified or demineralized

water", "sparkling bottled water", "spring water" and "well water". Additionally "sterile water" is defined as water that meets the requirements under the "Sterility Test" in the United States Pharmacopeia

Coliform organisms are not necessarily pathogens and are rarely found in bottled water, however, they serve as an indicator of insanitation or possible contamination. Surveys have shown that coliforms are useful indicators of bottled water quality, but some countries also monitor additional microbial populations as indicators of bottle water quality (10, 33). Under the current bottled water quality standard, FDA has established a microbiological quality requirement that is based on coliform detection levels. These levels may be obtained by membrane filtration (MF) or by 10-tube MPN analysis of ten 10-mL analytical units.

Bacteriological water analysisBacteriological water analysis is a method of analysing water to estimate the numbers of bacteria present and, if needed, to find out what sort of bacteria they are. It is a microbiological 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 recreational waters are safe to use.The interpretation and the action trigger levels for different waters vary depending on the use made of the water. Very stringent levels applying to drinking water whilst more relaxed levels apply to marine bathing waters where much lower volumes of water are expected to be ingested by users

ApproachThe common feature of all these routine screening procedures is that the primary analysis is for indicator organisms rather than the pathogens that might cause concern. Indicator organisms are bacteria such as non-specific coliforms, Escherichia coli and Pseudomonas aeruginosa that are very commonly found in the human or animal gut and which, if detected, may suggest the presence of sewage. 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

MethodologiesBecause 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 10ml) is diluted with 100ml of sterile growth medium and an aliquot of 10ml is then decanted into each of ten tubes. The remaining 10ml 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 in each sample tube. This inverted tube catches any gas produced. The production of gas at 37 Deg Celsius is a strong indication of the presence of Escherichia coli

ATP TestingAn ATP test is the process of rapidly measuring active microorganisms in water through detection of a molecule called Adenosine Triphosphate, or 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 firefly enzyme Luciferase using a Luminometer. The amount of light produced is directly proportional to the amount of biological energy present in the sample2nd Generation ATP tests are specifically designed for water, wastewater and industrial applications where, for the most part, samples contain a variety of components that can interfere with the ATP assay

Plate countThe 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 10 and 100 colonies of the target bacterium are grown. Fewer than 10 colonies makes the interpretation statistically unsound whilst greater than 100 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.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 agar in a dish that is sealed and incubated. Typical media include Plate count agar for a general count or MacConkey agar to count gram-negative bacteria such as E. coli. Typically one set of plates is incubated at 22ºC and for 24 hours and a second set at 37ºC for 24 hours. The composition of the nutrient usually includes reagents 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 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 filters 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 count a much greater number of colonies under a binocular microscope

Pour platesWhen 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 (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.

2.4.7 BacteriophagesBacteriophages (phages) are viruses that infect and replicate in specific bacteria. The ability toidentify phages (coliphages) of E.coli, also detects fecal contamination. This is because thepresence of coliphages also indicates the presence of E.coli. The significance of coliphages asindicators of sewage contamination, and their greater persistence compared to bacterialindicators make them useful as additional indicators of treatment efficiency. A current methodof coliphage detection is through the culture of E.coli in a Tryptic Soy Agar (TSA) mediumBacteria can be useful to humans in many ways. Bacteria decompose many types of organic substances and are currently being investigated as a means of decomposing unwanted synthetic chemicals (e.g., pesticides, dyes, and petroleum) that are released into the environment.Table A: Human pathogenic bacteria

Bacteria Disease

Bacillus anthracis anthrax

Bordetella pertussis whooping cough

Corynebacterium diphtheriae diphtheria

Mycobacterium tuberculosis tuberculosis

Salmonella sp. salmonellosis, typhoid fever

Shigella sp. bacillary dysentery

Streptococcus pyogenes scarlet fever

Vibrio cholerae cholera

Yersinia pestis bubonic plague

Some of the better known waterborne diseases, caused by bacteria, are: cholera, bacillary dysentery, shigellosis, and typhoid fever.Bacteria and water:Coliform bacteria have been used to evaluate the general quality of water.  Testing for coliform bacteria is faster and cheaper than testing for specific organisms and pathogens.  U.S. Public Health Service established a standard in 1914.  Coliforms include all aerobic and facultatively anaerobic, gram-negative, non-sporeforming bacilli that, when incubated at 35 C, can ferment lactose and produce gas (CO2) within 48 hrs. Fecal coliforms are the coliform bacteria that originate specifically from the intestinal tract of warm-blooded animals (e.g., humans, beavers, racoons, etc.). They are cultured by increasing the incubation temperature to 44.5 C and using somewhat different growth media.

Two other groups of bacteria that are present in feces are: fecal streptococci and Clostridium. Clostridia spores can survive a long time during adverse conditions. This genera occurs naturally in soils and polluted waters; it is not used for monitoring purposes. Fecal streptococci and enterococci are terms that have been used interchangeably; however, there are some differences between the two groups (Table B). Fecal streptococci indicate the presence of fecal contamination by warm-blooded animals. Unlike coliforms, fecal streptococcal bacteria are not known to multiply in the environment. Also, they tend to die-off more quickly than coliforms. 

The ratio of fecal coliforms to fecal streptococci (FC/FS) can provide information on the source of contamination (Table C); however, several precautions are in order when using these ratios: (1) Bacterial concentrations can be greatly variable if the pH is outside of the 4.0 to 9.0 range, (2) The faster die-off rate of fecal streptococci will alter the ratio as time from contamination increases, (3) Pollution from several sources can alter the ratio and confuse the issue, (4) FC/FS ratios have been of limited value in identifying pollution sources in irrigation returns, bays, estuaries, and marine waters, and (5) Ratios should not be used when fecal streptococcal counts are less than 100/100 mL.

Table B: Fecal streptococci

Entercoccus Group

|-- S.faecalis --|| S.faecalis subsp. liquefaciens || S.faecalis subsp. zymogenes ||-- S.faecium | Group D

S.bovis |S.equinus --|S.avium Group Q

Table C: FC/FS ratios 

Source Ratio

Man 4.4

Duck 0.6

Sheep 0.4

Chicken 0.4

Pig 0.4

Cow 0.2

Turkey 0.1

Water quality criteria, guidelines, and standards: A health effects recreational water quality criterion is defined as a measurable relationship between the quantity of the indicator in the water and the potential risk to human health associated with using the water for recreational purposes. A water quality guideline, obtained from the criterion, is a suggested upper limit on the quantity of the indicator in the water that is associated with an unacceptable level of health risk. A water quality standard, obtained from the criterion, is a guideline set by law.The first federal water quality criteria recommendations were proposed, in 1968, by the National Technical Advisory Committee (NTAC). The criterion for bathing waters was based on studies, conducted during the late 1940's and early 1950's by the Public Health Service, of total coliform concentrations. The criterion was converted to fecal coliform concentrations using Ohio River data collected, during the original study, in 1949. The NTAC recommended that:"Fecal coliforms should be used as the indicator organism for evaluating the microbiological suitability of recreation waters. As determined by multiple-tube fermentation or membrane filter procedures and based on a minimum of not less than five samples taken over not more than a 30-day period, the fecal coliform content of primary contact recreation waters shall not exceed a log mean of 200/100 mL, nor shall more than 10% of total samples during any 30-day period exceed 400/100 mL." (NTAC, 1968)The above criterion was again recommended, in 1976, by the USEPA, even though several aspects of it had been criticized by a number of researchers (USEPA, 1986). Since then, researchers have conducted several studies in an effort to determine acceptable bacteria concentrations in recreational waters. Evaluations of such studies are presented in: Health Effects Criteria for Fresh Recreational Waters (Dufour, 1984) and Health Effects Criteria for Marine Recreational Waters (Cabelli, 1983). The Ambient Water Quality Criteria for Bacteria - 1986 (USEPA, 1986) had the following recommendations for recreational bathing waters:

Based on a statistically sufficient number of samples (generally not less than five samples equally spaced over a 30-day period), the geometric mean of the indicated bacterial concentrations should not exceed one

or the other of the following:Freshwater: E. coli: 126 per 100 mL, or enterococci 33 per 100 mLMarine Water: enterococci: 35 per 100 mLIn shellfish harvesting areas, the geometric mean fecal coliform concentration must not exceed 14 bacteria per 100 mL, with not more than 10% of the samples exceeding 43 bacteria per 100 mL (USEPA, 1987; Mueller et al., 1987). Total coliform bacteria should not exceed 70 per 100 mL, with not more than 10% of the samples taken during any 30-day period exceeding 230 colonies per 100 mL (Mueller et al., 1987). Diseases such as paratyphoid and infectious hepatitis

may be spread through the consumption of bacteria-contaminated shellfish. For more information on shellfish, please refer to the Shellfish section.The standards actually utilized by the various states differ (USEPA, 1988a; USEPA, 1988b). The original criterion, based on fecal coliform concentrations, is still in use by many states. In many cases, water quality classifications have been developed that change the concentration of fecal coliforms required to meet the intended use of the water (e.g., primary contact, secondary contact, shellfish, etc.). A more recent report (Francy, Myers, and Metzker, 1993), conducted using water samples collected from several areas in Ohio, concluded that: "The difference between the use of E. coli and fecal coliform bacteria is that E. coli can be used to establish guidelines and standards on the basis of an acceptable level of risk as determined by a regulatory agency and the public. The relation between fecal coliform bacteria and E. coli concentrations can vary, whereas the epidemiological literature shows that the relation between E. coli and swimming- associated illness is strong and consistent over geographic boundaries."Drinking water requirements: The EPA has set the following maximum contaminant levels (MCLs) on treated drinking water:For systems that analyze at least 40 samples per month, no more than 5% of the samples may be total coliform positive.For systems analyzing fewer than 40 samples per month, no more than 1% of the samples may be total coliform positive (AWWA, 1990b).The number of samples collected each month is based on population (Table 4). Table 4: Drinking Water Sampling (abbreviated table adapted from Bordner and Winter, 1978)

PopulationMinimum number of samples per month

Population

Minimum number of samples per month

25 to 1,000 1 96,001 to 111,000 100

4,101 to 4,900 5250,001 to 290,000

160

10,301 to 11,100 12410,001 to 450,000

200

15,501 to 16,300 18550,001 to 600,000

230

18,901 to 19,800 22720,001 to 780,000

260

24,001 to 24,900 28970,001 to 1,050,000

300

28,001 to 33,000 351,520,001 to 1,630,000

360

50,001 to 54,000 602,060,001 to 2,270,000

410

64,001 to 70,000 753,020,001 to 3,320,000

450

76,001 to 83,000 85 4,690,001 or 500

more

Irrigation effects: Human diseases can occur from the consumption of crops that have been irrigated with polluted water. Crops that are eaten raw (e.g., celery, lettuce, tomatoes, peppers) are especially dangerous for the transmission of disease-causing organisms. Because some bacteria will dessicate (or dry-out) and die from prolonged exposure to air, the risk for illness can be decreased by delaying the harvest and consumption of crops (IHD-WHO, 1978).Recreation effects: Immersion in bacteria-contaminated water can result in infections of the eyes, ears, nose, and throat (Mueller et al., 1987). Epidemiological studies that associated the occurrence of gastrointestinal illness with coliform concentrations were used to develop the criteria for fresh and marine recreational waters that were previously discussed. From bacteriological data, it was estimated that fecal coliform concentrations of 200 per 100 mL would cause 8 illnesses per 1,000 swimmers at fresh water beaches and 19 illnesses per 1,000 swimmers at marine beaches (USEPA, 1986).Bacteria sources: Bacteria can enter water via either point or nonpoint sources of contamination. Point sources are those that are readily identifiable and typically discharge water through a system of pipes. Sewered communities may not have enough capacity to treat the extremely large volume of water sometimes experienced after heavy rainfalls. At such times, treatment facilities may need to bypass some of the wastewater. During bypass or other overflow events, bacteria- laden water is discharged directly into the surface water as either sanitary sewer overflow (SSO) or as combined stormwater overflow (CSO). Power outages and flooding can also contribute to the discharge of untreated wastewater. Treated wastewater and treatment plant residuals can also have adverse impacts on sensitive areas. Estuaries may be particularly susceptible to contamination from offshore sewage sludge dumping and offshore sewage pipe outfalls (Kennish, 1992).Nonpoint sources are those that originate over a more widespread area and can be more difficult to trace back to a definite starting point. Typical nonpoint sources include agricultural, residential and urban areas. Agricultural sources include livestock excrement from barnyards, pastures, rangelands, feedlots, and uncontrolled manure storage areas. Land application of manure and sewage sludge can also result in water contamination, which is why states require permits, waste utilization plans, or other forms of regulatory compliance.Failed on-site wastewater disposal systems (septic systems) in residential or rural areas can contribute large numbers of coliforms and other bacteria to surface water and groundwater. Stormwater runoff from residential, rural, and urban areas can transport waste material from domestic pets and wildlife into surface waters.Bacteria transport: Bacteria-laden water can either leach into groundwater and seep, via subsurface flow, into surface waters or rise to the surface and be transported by overland flow. Bacteria in overland flow can be transported freely or within organic particles. Overland flow is the most direct route for bacteria transport to surface waters. Underground transport is less direct, because the movement of water and bacteria is impeded by soil porosity and permeability constraints.Analytical methods: There are a variety of methods and media available for the detection and enumeration of indicator organisms. Two manuals that go into great detail describing these techniques are Standard Methods for the Examination of Water and Wastewater (AWWA, APHA, and WEF, 1992) and Microbiological Methods for Monitoring the Environment: Water and Wastes (Bordner and Winter, 1978). A brief listing of the method types is presented below:

Total and fecal coliforms: Total coliforms are incubated at 35±0.5 C, whereas fecal coliforms are further distinguished by incubation at 44.5±0.2 C.a. Multiple-tube most probable number (MPN) fermentation technique: This method is applicable for treated and untreated water; however, untreated water will likely require a greater dilution range. Statistical tables (MPN tables) are utilized to determine the number of bacteria present and the range in the 95% confidence interval based on the number of positive culture tubes. The procedure has three steps, presumptive, confirmed, and completed, that can take seven days to complete. b. Membrane filter procedure: This method will result in discrete bacterial colonies that may be further identified. Highly turbid water and noncoliform bacteria can interfere with the test. This test can require the processing of several sample dilutions in order to obtain filter plates with the appropriate range of colonies for valid enumeration. c. Presence-absence test: A qualitative test, rather than quantitative, that can be used on routine water distribution samples. Postive drinking water samples would be further analyzed.d. Defined-substrate: Media to which special, selective components have been added. Such additives help identify the target organism through color changes or other responses. The responses are brought about through metabolic or enzymatic processes specific to the target organism.Fecal streptococci: As with fecal coliforms, these organisms undergo incubation steps at both 35±0.5 C and 44.5±0.2 C.a. Fecal streptococci most probable number procedure: This method is similar to that for coliforms; however, different media are utilized.b. Fecal streptococci membrane filter procedure: Again, this test is similar to that used for coliforms, except for the type of media used.Methods are continually being revised, streamlined, and updated in an effort to: reduce analysis time, reduce costs, and increase accuracy.BIOCHEMICAL TESTS OFTEN USED FOR IDENTIFICATION OF BACTERIA

1. Sugar Fermentation

- Phenol Red Glucose, Phenol Red Lactose and Phenol Red Sucrose broths (PRG) (PRL) (PRS)

- testing to determine whether bacteria can ferment these sugars- phenol red pH indicator dye is red at neutral pH and yellow in acid pH- fermentation of sugars results in the production of acids (A) and/or gases (G)- gas detection is detected using the small inverted tube called a Durham tube- test needs to be read within 48 hours

NEGATIVE (-) POSITIVE (+)

2. Citrate Utilization

- Simmon’s Citrate agar is used to determine if an organism can use citrate as its only carbon source using the enzyme citrase (also contains ammonia as the only nitrogen source)

- citrate utilization is an aerobic process and a slant tube is used to increase exposure of bacterial growth to the air; inoculate the slant

- pH indicator is Bromthymol blue, which is green at neutral pH but turns Prussian blue at pH levels above 7.63. Urease Test

- filter sterilized broth tubes containing urea and phenol red pH indicator- detects the presence of urease which converts urea into the highly alkaline product

ammonia- when pH of media becomes alkaline, phenol red turns bright pink- Proteus species causing UTI’s cause rapid color changes within 24 hours

NEGATIVE (-) POSITIVE (+)4. Indole Production

- media containing the amino acid tryptophan is inoculated (TSB in our lab)- the test is trying to determine whether bacteria possess the enzyme tryptophanase which

will produce the byproduct indole from the catabolism of tryptophan- Kovac’s reagent is used after inoculation and incubation to read the results

- the alcohol in Kovac’s reagent floats on the media and another chemical in the reagent reacts with indole to form a red color in the alcohol layer

- SIM media (sufide-indole-motility media) can also test for the production of tryptophanase

NEGATIVE (-) POSITIVE (+)

NEGATIVE (-) POSITIVE (+)5. Lysine Decarboxylase Production

- media containing the amino acids lysine, ornithine or arginine are typically used to detect the presence of decarboxyase enzymes which remove a carboxyl group (COOH) from the amino acid, we are using lysine in our lab.

- fermentation of glucose in the broth must occur to trigger the activity of the decarboxylase enzymes, therefore a sterile mineral oil layer is pipetted onto the media after inoculation to reduce the amount of oxygen exposure.

- in our lab we tested for the production of lysine decarboxylase enzymes which remove a carboxyl group from lysine to produce the alkaline end product cadaverine.

- the pH indicator in the broth is Bromcresol purple which is yellow in acid pH and purple at neutral pH and above.

- if the microbe we are testing for does not ferment glucose, the test is of no value.- if the microbe only ferments glucose but does not have the enzyme lysine decarboxylase,

the media will turn yellow.- if the microbe ferments glucose, the media will initially turn yellow and then as the lysine

decarboxylase is activated the media will become purple again.

NEGATIVE (-) POSITIVE (+)6. Nitrate reduction

- used to detect the ability of microbes to reduce nitrate (NO3) to nitrite (NO2) or beyond to N2 gas or NH4 (ammonia) using the enzyme nitrate reductase

- tryptic nitrate media is used in our lab for this test- if the microbe has nitrate reductase enzymes, it can reduce the nitrate to nitrite and this

can be detected by the addition of 2 reagents: nitrate reagent A (sulfanilic acid) and nitrate reagent B (alpha naphthylamine). The production of nitrite will react with the two reagents and produce a red color change in the broth.

- If no color change occurs after the addition of nitrate reagents A and B, it can mean that nitrate is still present in the broth or that the nitrate was reduced to a compound other than nitrite, such as N2 gas or ammonia. To tell the difference, add zinc powder to the broth. Zinc powder reduces nitrate to nitrite and will create a red color which is a negative test, since the nitrate wasn’t reduced by the microbe but was instead reduced by the zinc. If the zinc powder produces no color change then the test is positive and the nitrate was reduced beyond nitrite to N2 gas or ammonia which cause no color change with the nitrate reagents A and B.

NEGATIVE (-) POSITIVE (+)7. Methyl Red/Voges Proskauer Broth

A. Voges-Proskauer Test- some microbes do not produce stable acids from glucose fermentation but instead

produce 2,3 butanediol from glucose breakdown and in the process an intermediate chemical acetoin is produced.

- 2 reagents: Barritt’s A (alpha-naphthol) and Barritt’s B (KOH) are added to a 48 hour culture of the MRVP broth. Take a pipette to aliquot out a small amount of the broth to do the VP test and return the broth for further incubation for the MR test if necessary

- a wine red color change with the addition of Barritt’s reagents A and B is a positive test detecting the presence of acetoin and therefore 2,3 butanediol; a brown or copper color is negative

NEGATIVE (-) POSITIVE (+)

B. Methyl Red Test - some microbes produce mixed acids (lactic, succinic, formic, acetic) with the

fermentation of glucose, if these acids are stable and can persist in the presence of the buffer in the media, they will reduce the pH of the media to below 5. The test needs 5 days of incubation to ensure the presence of stable acids.

- after the proper incubation period, a few drops of methyl red reagent is added to the broth; methyl red is red at pH less than 5 and yellow at pH greater than 6

NEGATIVE (-) POSITIVE (+)

8. Catalase production

- Many microbes use oxygen to metabolize glucose in the electron transport system and as a result produce toxic hydrogen peroxide (H2O2) intracellularly. To remove this toxic byproduct many cells contain catalase which breaks hydrogen peroxide down into water and O2 gas. Growth can be removed from a TSA slant and put on a glass slide to which a few drops of H2O2

are added. Bubbling is a positive test (weak bubbling can be seen under low power of your microscope).

NEGATIVE (-) POSITIVE (+)

9. Oxidase production

- Organisms that use the electron transport system to metabolize glucose utilize an enzyme called cytochrome oxidase. To detect the presence of this enzyme we add oxidase reagent to organisms streaked on a TSA plate or to bacteria placed on filter paper. A color change to pink then eventually blue-black is a positive test for the presence of the enzyme10. Gelatinase Test

- this test utilizes nutrient gelatin (NG) deeps to determine whether a microbe can digest and liquefy gelatin using hydrolytic gelatinases

- this media is inoculated using an inoculating needle and needs to be incubated for up to 7 days

- a positive test results in the gelatin being hydrolyzed into amino acids, which means the media will become a liquid; in a negative test the media stays solid

- to determine whether the microbes liquefied the media or whether the media just melted in the incubator (gelatin melts at 28° C), put the media in a refrigerator for 5-10 minutes and see if it is still liquid

- Staph aureus (gelatinase +) and Staph epidermidis (gelatinase - ) can be distinguished using nutrient gelatin

NEGATIVE (-) POSITIVE (+)

11. Triple Sugar Iron Agar

- TSI slant tubes are used- stab the butt and streak the slant- A multitest medium that contains glucose (1/10th the amount of the other sugars), sucrose

and lactose; thiosulfate (H2S indicator) and phenol red pH indicator- 4 reactions are possible:a) slant color: red (K) or yellow (A)b) butt color: red (K) or yellow (A)c) gas bubbles: + or –d) H2S precipitate: black ppt. (+) or no black ppt (-)

- a yellow butt indicates glucose fermentation- a yellow slant indicates the fermentation of sucrose and lactose

NEGATIVE (-) POSITIVE (+)

VARIOUS REACTIONS POSSIBLE

12. Phenylalanine Deaminase Test

- this test looks for the production of phenylalanine deaminase, which removes an amine group from the amino acid phenylalanine to produce phenylpyruvic acid and ammonia.

- phenylalanine agar slants are inoculated - 10% ferric chloride reagent is needed to read the test; the appearance of a green color

when the reagent is added is positive, a yellow color is negative

NEGATIVE (-) POSITIVE (+)13. Sulfide-Indole-Motility Media

- this multitest media can test for the production of H2S production (as does the TSI media), can test for indole production from tryptophan, and can test for motility (bacteria that possess flagella)

- to detect motility, the media is inoculated with an inoculating needle and after 24 hours we look for cloudiness radiating out from the stab line

- to test for indole production add Kovac’s reagent to the media- the media contains cysteine (an amino acid) which when catabolized by cysteine

desulfurase produces H2S as a byproduct. The H2S reacts with ferrous sulfate in the tube to produce a black precipitate; bacteria not producing H2S have no black precipitate produced

NEGATIVE (-) + INDOLE + MOTILITY + H2S gas

14. Litmus Milk

- litmus milk contains the pH indicator litmus, which is pink in acid pH and blue in alkaline pH

- to look to see whether a bacterium can use lactose (milk sugar) as a carbon source and break down lactose to lactic acid, using the enzyme β-galactosidase, we can look for the media to turn pink

- to test for the ability of organisms to ferment lactose and oxidize litmus (acts as a H acceptor), we can look to see if the media turns white

- curd (clot) formation can be detected by bacteria that produce lactic acid or rennin, both of which cause the milk protein casein to clot

- proteolysis (peptonization) occurs when microbes are unable to use the lactose as an energy source and must use the milk proteins instead for this purpose. As the milk proteins are broken down into their amino acid building blocks, ammonia turns the litmus a deep purple in the upper part of the tube while the lower part becomes a clear brown liquid.

Sometimes the litmus will detect the partial breakdown of casein and the production of alkaline end product by a blue appearance in the media.

15. OPTIONAL- Adonitol, Arabinose and Sorbitol fermentation - Coagulase

ENRICHED, SELECTIVE and DIFFERENTIAL MEDIA

1. MANNITOL SALT AGAR (MSA)- 7.5% salt (selective) - phenol red pH indicator (differential)- Staph. aureus can ferment mannitol to produce acid end products;

Staph. epidermidis cannot

2. EOSIN METHYLENE BLUE AGAR (EMB)- lactose and the dyes eosin and methylene blue permit the differentiation between

lactose fermenting enterics (like E. coli) and non-fermenters- E. coli colonies have a green metallic sheen appearance- lactose fermenting enterics (coliforms) other than E. coli produce pink colonies- non-lacotse fermenting enterics ( like Salmonella and Shigella) produce

colorless colonies- the dyes are therefore differential, but they are also selective since they are

inhibitory to gram positive bacteria and to nonenterics- E. coli is an indicator species for fecal contamination of water in the

coliform test

3. MacCONKEY AGAR- crystal violet in the media inhibits gram positive organisms- lactose, bile salts and the pH indicator neutral red allows the differentiation of

enteric bacteria based on their ability to ferment lactosea) coliforms:

- ferment lactose producing acid- bacterial colonies are red- E. coli produce greater amounts of acid causing the media around

the colonies to also turn red-pink

b) non-coliforms

- typhoid fever and bacillary dysentery are due to pathogens in this group

- do not ferment lactose, don’t make acid- colonies are uncolored and sometimes transparent

4. BLOOD AGAR (BA)- differential media, used to determine the hemolytic ability of bacteria- sheep blood is used as an enrichment ingredient to grow fastidious bacteria such

as Streptococcus spp.- chocolate agar is made by adding sheep blood to the agar before the medium

cools, causing the blood to cook in the media turning it brown- there are four (4) possible outcomes when reading blood agar:

a) no growthb) gamma hemolysis: no change in the media surrounding the colonies

c) alpha hemolysis: incomplete RBC lysis- hemoglobin methemoglobin- greenish-brown (“drab”) halo in media surrounding colonies

d) beta hemolysis: complete lysis of RBC’s in media results in a “zone of clearing” in the media surrounding the colonies

5. PHENYLETHYL ALCOHOL AGAR (PEA)- used to isolate gram positive cocci - the alcohol makes the media selective, inhibiting gram negative bacteria by

dissolving their LPS layer6. STARCH AGAR

- when looking to see whether microbes produce amylase- the reagent iodine reacts with starch to produce a blue black color- a zone of clearing around the microbes is a positive test

7. SPIRIT BLUE AGAR or TRIBUTYRIN AGAR- when looking to see whether bacteria produce lipase which hydrolyze

triglycerides into their monomers- SBA contains olive oil and spirit blue dye- Tributyrin is a triglyceride (simple fat)- clear halo around growth is positive

8. SKIM MILK AGAR- when looking to see whether bacteria produce caseinase- a zone of clearing is positive

9. DNase AGAR- methyl green dye in media detects the presence of DNA- a zone of clearing is positive

10. CRYSTAL VIOLET AGAR- selective media used to grow gram negative bacteria- crystal violet is an inhibitory dye against gram + bacteria

Useful pH range and colour change of some indicator dyes

Indicator dye Useful pH range Colour change acid to alkaline

Cresol red (pK1) 02.-1.8 Red-Yellow

Thymol blue (pK1) 1.2-.2.8 Red-yellow

Bromophenol blue 3.0-4.6 Yellow-Blue

Bromocresol green 3.8-5.4 Yellow-Blue

Methyl red 4.4-6.0 Red-Yellow

Chlorophenol red 4.8-6.4 Yellow-Red

Bromocresol purple 5.2-6.8 Yellow-Purple

Bromothymol blue 6.0-7.6 Yellow-Blue

Phenol red 6.8-8.4 Yellow-Red

Gresol red (pK2) 7.2-8.8 Yellow-Red

Thymol blue (pK2) 8.0-9.6 Yellow-Blue

Phenolphthalein 8.3-10.0 Yellow-Red

Tolyl red 10.0-11.6 Red-yellow

Parazo orange 11.0-12.6 Yellow-Orange

Acyl blue 12.0-13.6 Red-Bule

Cresol Red

chemical structure of cresol red

Cresol red is a pH indicator and molecular weight marker for agarose gels. Unlike bromophenol blue and xylene cyanol, cresol red does not inhibit Taq polymerase in PCRs.

advantage: much less shadow on EtBr pictures unlike xylene cyanol and bromophenol blue

molecular weight 404 g/mol

in 1% agarose at ~1000 bp (between xylene cyanol 5000bp and bromophenol blue 500bp)

in 3% agarose at >100bp (xylene cyanol 300bp)

pH indicator

amber - acidic pH

yellow - neutral

red – alkaline

BThe relationship between the activity of hydrogen ions [exactly hydronium ions, H(H2O)n+] and

concentration of a solution is fundamentally important to determine the extent of a chemical reaction, as it affects the equilibria and kinetics of a wide variety of chemical and biochemical reactions. The hydrogen-ion activity refers to the effective concentration of unassociated hydrogen ions, the form that directly affects physicochemical reaction rates and equilibria. The symbol, pH, numerically relates the hydrogen ions concentration or activity. The pH is approximately equal to the negative logarithm of H+ concentration expressed in molarity. pH 7 is neutral; above it alkalinity increases and below it acidity increases. pH indicators are usually weak acidic or basic organic tautomers which exist in more than one structural form of which at least one form is characteristically colored in relation to different electronic configuration of the bound. Indicators should not change color exactly at one pH value, but within a wide pH range. The transition point of an indicator is defined as the point at which the acid and alkaline forms of the indicator exist in equal concentrations.

Bromcresol Purple (CAS #: 115-40-2): from pH 5.2 (yellow) to pH 6.8 (purple)

Bromothymol blue (CAS #: 76-59-5): from pH 6.0 (yellow) to pH 7.6 s

Cresol Red (CAS #: 1733-12-6): from pH 7.0 (orange) to pH 8.8 (purple)

Phenol Red Sodium Salt (CAS #: 34487-61-1): from pH 6.8 (yellow) to pH 8.2 (red)

pH measurements - indicators

pH indicators are usually weak acids or weak bases that change their color depending on their dissociation (protonation) state. Sometimes both forms are colored, sometimes only one. In most cases you may assume that to completely change color of bicolored indicator pH must change by 2 units. However, human eye is more sensitive to some colors than to others, thus some color changes can be perceived over wider pH range.

pH indicators can be used to check pH of the solution, although they are rarely added directly. Only in acid-base titrations indicator should be added to the solution. To check pH it is much more convenient to use pH strips. It is worth of noting here that pH strips are nothing else but pieces of paper impregnated with indicator or a mixture of indicators.

pH indicators - colors and color change pH range

indicator namepHcolor

pHcolor

Cresol red0.2red

1.8yellow

Bromocresol purple5.2yellow

6.8purple

Bromothymol blue6.0yellow

7.6blue

Phenol red6.4yellow

8.2red/violet

Neutral red6.8blue/red

8.0orange/yellow