How to measure coliforms - CWC

1 World Bank & Government of The Netherlands fundedTraining module # WQ - 23 How to measure coliformsNew Delhi, June 1999 CSMRS Building, 4th Floor, Olof Palme Marg, Hauz Khas,New Delhi 11 00 16 IndiaTel: 68 61 681 / 84 Fax: (+ 91 11) 68 61 685E-Mail: Consultants BV & DELFT HYDRAULICS withHALCROW, TAHAL, CES, ORG & JPSH ydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 1 Table of contentsPage1 Module context22 Module profile33 Session plan44 Overhead/flipchart master65 Evaluation sheets136 Handout157 Additional handout188 Main text20 Hydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 21. Module contextThis module describes a laboratory exercise on measurement of coliform bacteria. Modulesin which prior training is required to complete this module successfully and other relatedmodules in this category are listed designing a training course, the relationship between this module and the others,would be maintained by keeping them close together in the syllabus and place them in alogical sequence.

2 The actual selection of the topics and the depth of training would, ofcourse, depend on the training needs of the participants, their knowledge level and skillsperformance upon the start of the titleCodeObjectives1 Basic water qualityconceptsaWQ - 01 Become familiar with common waterquality parameters Appreciate important water qualityissues2 Basic chemistry conceptsaWQ - 02 Convert units from one to another Understand the basic concepts ofquantitative chemistry Report analytical results with thecorrect number of significant digits3 How to prepare standardsolutionsWQ - 04 Recognise different types ofglassware Use an analytical balance andmaintain it Prepare standard solutions4 Introduction to microbiologyaWQ - 20 classify different types ofmicroorganisms identify certain water borne diseases5 Microbiological laboratorytechniquesaWQ - 21 Explain

3 Methods of bacteriaidentification Discuss methods of bacteriaenumeration Follow methods of good laboratorypractice6 coliforms as indicators offaecal pollutionaWQ - 22 Identify the main water qualityproblems caused by microorganisms Explain why coliform bacteria aregood indicators Explain the principles of the coliformanalysis methoda- prerequisiteHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 32. Module profileTitle:How to measure coliformsTarget group:HIS function(s): Q2, Q3, Q5, Q6 Duration:1 session of 160 min and 2 sessions of 45 min eachObjectives:After the training the participants will be able to measure total and faecal coliforms in water samplesKey concepts: Culture media preparation Serial dilution Inoculation Reading MPN tableTraining methods:Lecture, laboratory exercisesTraining toolsrequired:Board, flipchart, OHS, laboratoryHandouts:As provided in this moduleFurther readingand references: Standard Methods: for the Examination of Water andWastewater, APHA, AWWA, WEF/1995.

4 APHA PublicationHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 43. Session planNoActivitiesTimeTools1 Preparations Collect/prepare the following samples Sample A: Surface water Sample B: Contaminated tap water (1 mL sewage/10 Ltap water) Sample C:Boiled sample B Estimate the requirement of dilution and culture tubes,andpipettes for the presumptive test Prepare dilution water, culture medium accordingly anddispense in tubes. Culture tubes should contain an inverteddurham tube. Put caps or cotton plugs. Sterilise glassware, culture and dilution tubes2 Introduction: Ask participants to name a few water borne diseases Recapitulate use of coliforms as indicators of faecal pollution10 min3 Bacteriological testing Discuss importance of advance preparations How to correctly estimate requirements What steps are required in preparations and testing Take the group around the laboratory and show theincubators and the sterilisers30 minOHS4 ExerciseSession I Briefly describe the exercise and ask the participants to readthe main text and SAP for total coliforms Demonstrate serial dilution and inoculation of tubes usingaseptic technique.

5 Ask participants to practice undersupervision. Divide the participants in groups of 3. Ask them to preparedilutions and inoculate lauryl tryptose tubes for presumptivetest. The inoculated tubes should be immediately put in theincubator. Simultaneously the groups should prepare BGBL and ECbroths and dispense them in tubes, sterilise and store for usein subsequent sessions20 min20 min40 min40 minhandoutHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 5 NoActivitiesTimeTools4 Exercise (contd.)Session II Ask participants to record the positive tubes after incubationfor 24 h. Explain that in order to complete the exercise, thenegative tubes will not be incubated for additional 24 h asrequired in the standard procedure The participants will start the confirmed test for total coliformsand test for faecal coliforms by transferring inocula from thepositive tubes to the respective culture media Ascertain that the incubators are set at required temperature45 min5 Exercise (contd.)

6 Session IIIAsk participants to record the number of positive tubes anddetermine MPN of total coliforms and faecal coliforms15 min6 ConclusionAsk participants to write report and discuss results30 minHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 64. Overhead/flipchart masterOHS format guidelinesType of textStyleSettingHeadings:OHS-TitleArial 30-36, with bottom border line (not:underline)Text:OHS-lev1 OHS-lev2 Arial 24-26, maximum two levelsCase:Sentence case. Avoid full text in :Use occasionally and in a consistent wayListings:OHS-lev1 OHS-lev1-NumberedBig for definite series of steps. Avoidroman numbers and :None, as these get lost in photocopying andsome colours do not reproduce at of a table will ease horizontal alignmentover more lines (columns)Use equation editor for advanced formattingonlyHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 7 How to measure coliforms1.

7 Preparations2. Sample inoculation3. Total coliform bacteria4. Faecal coliform bacteria5. Reading MPNH ydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 8 Preparations Test once started cannot be delayed Preparations according to number & source of samples culture medium and culture tubes dilution water and dilution tubes pipettes, transfer loop sterilisation, autoclave, hot air oven, flame incubators at required temperatureHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 9 Sample inoculation Drinking water: no dilution, 10 mL in 10 tubes Surface waters: 10,1, mL in 5 tubes each Polluted waters: 1, , mL in 5 tubes each Grossly polluted water: smaller inocula Good practice to have more than 3 inoculaHydrology Project Training Module File.

8 23 How to measure Version 05/11/02 Page 10 Total coliform bacteria Presumptive test lauryl tryptose broth, enrichment medium for lactose fermenters incubate at 35 oC check for gas & turbidity, 24 2 h, 48 3 h Confirmed test positive presumptive tubes brilliant green lactose bile broth, selective medium for coliforms incubate at 35 oC check for gas & turbidity, 24 2 h, 48 3 hHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 11 Faecal coliform bacteria Positive presumptive tubes EC medium Incubate at oC Elevated temperature is selective for faecal coliforms Check for gas & turbidity, 24 2 h, 48 3 hHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 12 MPN /100 mL Bacteria are randomly distributed in the sample Multiple tubes inoculated with various sample sizes Smaller the sample, greater the chance of negative reaction The density is statistically estimatedHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 135.

9 Evaluation sheetsHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 14 Hydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 156. HandoutHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 16 How to measure coliforms1. Preparations2. Sample inoculation3. Total coliform bacteria4. Faecal coliform bacteria5. Reading MPNP reparations Test once started cannot be delayed Preparations according to number & source of samples culture medium and culture tubes dilution water and dilution tubes pipettes, transfer loop sterilisation, autoclave, hot air oven, flame incubators at required temperatureSample inoculation Drinking water: no dilution, 10 mL in 10 tubes Surface waters: 10,1, mL in 5 tubes each Polluted waters: 1, , mL in 5 tubes each Grossly polluted water.

10 Smaller inocula Good practice to have more than 3 inoculaTotal coliform bacteria Presumptive test lauryl tryptose broth, enrichment medium for lactose fermenters incubate at 35 oC check for gas & turbidity, 24 2 h, 48 3 h Confirmed test positive presumptive tubes brilliant green lactose bile broth, selective medium for coliforms incubate at 35 oC check for gas & turbidity, 24 2 h, 48 3 hFaecal coliform bacteria Positive presumptive tubes EC medium Incubate at oC Elevated temperature is selective for faecal coliforms Check for gas & turbidity, 24 2 h, 48 3 hMPN /100 mL Bacteria are randomly distributed in the sample Multiple tubes inoculated with various sample sizes Smaller the sample, greater the chance of negative reaction The density is statistically estimatedHydrology Project Training Module File: 23 How to measure Version 05/11/02 Page 17 Add copy of Main text in chapter 8, for all Project Training Module File: 23 How to measure Version 05/11/02 Page 187.