 deep brown not as dark as those of E. The colonies developing on endo or eosin methylene blue agar may be typical (unnucleated. Neighbouring colonies show little Colonies are slightly raised. Colonies are considerably raised and marked by convex. Incubate the secondary broth tubes and agar slants at 35 ± 0. with or without metallic sheen) atypical (opaque.5°C for 24 ± 2 hrs or 48 ± 3 hrs and if gas is not produced in 24 hrs gram stained preparation from these agar slant cultures are made. Confluence Elevation Appearance by transmitted light Appearance by reflected light Gram Staining Reagents 1. aerogenes on eosin or EMB agar can be done by referring the following table. internal structure of central dark portion is difficult to discern. Lugol’s solution. From each of these plates fish out one or two colonies and transfer to lauryl tryptose broth fermentation tubes and to nutrient agar slants. Striated internal structure is often observed in young colonies. 8. 10. are larger than those of E. unnucleated. the test may be considered as positive completed test and this demonstrates the presence of coliform organisms. mucoid. coli and A. The gas formation in the secondary lauryl tryptose broth tubes and the demonstration of gram-negative non-spore forming rod shaped bacteria in agar culture may be considered a satisfactory positive completed test. 9. Neighbouring colonies run together tendency to run together. metallic sheen is not observed except in a depressed center.coli and A. 5. Much lighter than E. button like. Incubate the Petri dishes (inverted) at 35 ± 0. coli. aerogenes on eosin or EMB agar Characteristic Size Escherichia coli Well isolated colonies are 2–3 mm in diameter. surface is flat or slightly concave.5°C for 24 ± 2 hrs. 6. Aerobactor aerogenes Well isolated colonies. quickly. often concentrically ringed with a greenish metallic sheen. Dark.Test for Coliforms in Water 87 4. 7. almost black centre that extend across more than three fourths of the diameter of the colony. Differentiation of E. rarely convex.
1 and 0. may be estimated by Thomas’ simple formula: MPN/100 mL= No. 8. Prepare a light emulsion of the bacterial growth on an agar slant in a drop of distilled water on a glass slide. 3.1mL combination. Air-dry or fix by passing the slide through a flame and stain for 1minute with ammonium oxalate-crystal violet solution. 10. The MPN for combination not appearing on the table or for other combinations of tubes and dilutions. If on the other hand a combination corresponding to 1. Cells that do not decolorise but retain the crystal violet stain (deep blue) are defined as gram positive. Cells that decolorise and accept the safranin stain are pink and are defined as gram negative. 6. 2. These values are prepared for 10. Acetone alcohol. and 0. Counter-stain with safranin for 15 seconds and then rinse with tap water. 0. Step Procedure Gram + ve lnitial stain Mordent Decolonisation Counter stain Crystal violet for 30 sec. lodine for 30 sec. 95% ethanol for 10-20 sec. The values are at the 95% confidence limit for each of the MPN determined. Steps in the gram staining is shown in the following table. If the combination is 100. 4.1. The MPN for the variety of planting series are presented in table in Appendix III. the MPN is 0. Blot dry with blotting paper and view through the microscope.88 A Comprehensive Laboratory Manual for Environmental Science and Engineering 3. Counter stain 4. Rinse the stained slide in tap water. 5. 7. 1mL.01 mL is planted. of positive tubes × 100 mL sample in negative tubes × mL sample in all tubes. Stains purple Remains purple Remains purple Remains purple Results Gram – ve Stains purple Remains purple Becomes colourless Stains pink Computation of MPN The number of positive finding of coliform group organisms resulting from the multiple portion decimal dilution planting should be computed as the combination of positives and recorded in terms of the Most Probable Number (MPN). Rinse the slide in tap water and then apply Lugol’s solution for 1minute. Decolorise with acetone alcohol till the stain is just removed. Procedure 1. record MPN as 10 times the value shown in the table. Safranin for 20-30 sec.1 times the value in the table.
......... 1 mL and 0.. of positive tubes × 100 =... Case (ii) For other combinations and dilutions MPN/100 mL= No.. or time of description observation Date and time of incubation Results after incubation for various volumes of samples inoculated (mL) + ve or – ve 10 10 10 1 1 1 0....1 0..1 Presumptive test 24 hrs Presumptive test 48 hrs Confirmed test 48 hrs Completed test 24 hrs Completed test 48 hrs Number of + ve tubes mL of sample in – ve tubes Test case Calculation Case (i) For three each of 10 mL..Test for Coliforms in Water 89 Observation Sample Date and no..1 mL sample concentration combinations MPN from the MPN table (Appendix-III) =.1 0....... mL sample in negative tubes × mL sample in all tubes Result MPN/100 mL =.
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Discussion
Questions 1. What are E.coli? Are they harmful to human beings? Why is their presence tested in the waters to be supplied for domestic consumption? 2. What is coliform index? 3. Define MPN.
Test for Coliforms in Water
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Experiment No. Nessler’s reagent 2. Stock ammonium solution 1. colour and substances precipitated by hydroxyl ion interfere with the determination.8 M conc.). Zinc sulphate solution 3. The yellow colour or reddish brown colour typical of ammonia N can be measured in a spectrophotometer in the wavelength of 400–500 nm with a light path of 1cm. Apparatus 1. which have low ammonia concentration. ___________________ Date ___________________ AMMONIA NITROGEN Aim To determine the ammonia nitrogen of the given sample of water. natural water and highly purified effluents. EDTA reagent as stabiliser 4. The sample containing ammonia must be analysed immediately after collection.00 mg. Spectrophotometer. Direct Nesslerisation Direct Nesslerisation is used only for purified water. pH meter Reagents 1. if not 0. whose intensity of colour is directly proportional to the amount of NH3 present. Principle Colorimetric method.00 mL = 1. Turbidity. H2SO4/L should be added to the sample stored at 4°C. It combines with NH3 in alkaline solution to form a yellowish brown colloidal dispersion. using Nessler’s reagent is sensitive to 20mg/L of ammonia N and may be used up to 5mg/L of ammonia N. or Nessler tube tall form (50 mL or 100 mL capacity) 2.
2. 10.0. 0. 5. 4. A blank using distilled ammonia free water is treated with Nessler’s reagent as above.0. 1. 6. 2.7.4. From the absorbance of a solution of unknown concentration. 100 mL ZnSO4 solution is added to 100 mL sample and to it is added 0.5. 3. This is mixed thoroughly. A graph with mg of NH3 along x-axis and absorbance along y-axis is plotted and a straight-line graph is drawn. 5. Then the sample is put in 1cm standard tubes of spectrophotometer and the absorbance noted at 400– 500 nm wavelengths. 9. Residual chlorine is removed by means of a dechlorinating agent (one or two drops sodium thiosulphate solution) 2. 3. 7. EDTA reagent is added to 50 mL of sample. 0. A calibration curve is prepared as follows: With 0.0 mL of standard NH4Cl solution in 50 mL distilled water standard diluted samples are prepared. 4. 1. To this is added 2 mL of Nessler’s reagent (proportional amount to be added (if the sample volume is less). Calculation ammonia N in mg/L = where. 8.0.5. Each sample is Nesslerised as indicated earlier and the absorbance is noted down.4. The floc formed is allowed to settle and the clear supernatent is taken for Nesslerisation. 11. 1.5 mL of NaOH solution to obtain a pH of 10. The absorbance is fixed as zero. Observation The observation is presented in Tables A and B respectively. 0. If the sample contain Ca or Mg.7. Table A: Observation for calibration Stock ammonia solution in mL Ammonia Absorbance A mL of sample A = µg N found colorimetrically.94 A Comprehensive Laboratory Manual for Environmental Science and Engineering Procedure 1.0. 2. the µg of NH3 present can be read from the calibration curve.
Ammonia Nitrogen 95 Table B Sample no. or description Ammonia nitrogen in mg/L Discussion. Absorbance Ammonia nitrogen in µ g from graph Ammonia nitrogen in mg Results Sample no.
Discuss the significance of ammonia nitrogen in water. What is the source of ammonia nitrogen in water?. 2.96 A Comprehensive Laboratory Manual for Environmental Science and Engineering Questions 1.
Ammonia Nitrogen 97.
98 A Comprehensive Laboratory Manual for Environmental Science and Engineering.
Place the stand in a cool water bath and add 2 mL