Bacteriological Analysis of Water & Chlorination of Boreholes/Wells - Practical Training

6-8 July, 2016 Gutu, Zimbabwe

12-14 July, 2016 Matobo, Zimbabwe

Oxfam

Zimbabwe National Water Authority

Ministry of Health and Child Care

Sections to Be Covered in this Training

Section 1 – Types and importance of “indicator bacteria”

Section 2 – Collection of bacteria samples in the field

Section 3 – Using DelAqua kits for bacteria analysis of water

Section 4 – Compartment Bag Tests for bacteria analysis

Section 5 – Chlorination of boreholes/wells

Section 6 – Field testing - chlorine residual, pH and turbdity

Section 7 – Links for DelAqua training videos online

Section 1

Types of and the Importance of “Indicator Bacteria”

Types of Indicator Bacteria

• Thermotolerant (Fecal) Coliform Bacteria are an indicator of contamination by feces (“fecal”) from humans and/or animals

• “Total Coliform” include both non-fecal coliform bacteria (from plants, etc.) and fecal coliform bacteria

3 Different Types of Bacteria Will be Analyzed for During This Training

Source Clackamas

The More Fecal (or E. coli) Bacteria in Drinking Water the Higher the Risk of Cholera, Typhoid, etc.

Poster at Gutu District Laboratory

Bacteria Concentration

High Risk Groups – Waterborne Disease

While all ages are at risk from cholera, certain groups are at significantly higher risk of waterborne disease/diarrhea/mortality. Higher risk groups include:

• Children less than 5 years old (especially less than 2)

• People living with HIV/AIDS

• Pregnant women

• Malnourished children

• Elderly

• People who drink water from a surface water source

Section 2

Collection of Bacteria Samples in the Field

Examples of Potential Sampling Locations

Prior to Collecting Sample Remove Any Attachments from Tap to Remove Any Deposits in Pipe/Piping

Important to Identify Sample Location, Time and Date of Collection and Sample Number on Whirl-Pak & in Notebook

Taking a Sample with a Whirl-Pak Bag (steps 1 &2)

Taking a Sample with A Whirl-Pak Bag(steps 3 & 4)

Taking a Sample with a Whirl-Pak Bag (steps 5 and 6)

(Gutu, Zimbabwe 7-7-2016)

Using Whirl-Pak Bags to Collect Bacteria Samples

Photos by T. Mahin

For Group Discussion

• Assuming a limited budget, what locations/water sources should be a priority for bacterial analysis?

• How many/how often are bacteria samples are currently being taken in your work area?

• What are the current problems in collecting and analyzing an adequate number of samples?

Section 3

Using DelAqua Kits for Bacteria Analysis of Water

Oxfam-DelAqua Water Testing Kit

• DelAqua bacteria testing kit was developed by Oxfam to be able to be used in areas without reliable electricity

• It uses a membrane filtration approach to analyze “thermotolerant” coliform bacteria which are also called “fecal coliform” bacteria and also “total coliform” bacteria the higher risk of disease (e.g. typhoid, cholera, etc.)

Incubator and On/off Switches and Indicator Lights and Fuse

Filtration Components (in DelAqua Kit)

Bacteria Analysis with the DelAqua –Making the Culture Media

You Will Need the Following items to Prepare the Culture “Medium/Media”

Mixing the Culture Medium Using Membrane Laurel Sulphate Broth (MLSB)

Mixing the MLSB (continued)

Pouring into Small Bottles and Sterilising the MLSB

Making MLSB Medium (Gutu Training)

Photo by T. Mahin

Sterliising Step

Sterilising Petri Dishes and the Media

Possible methods to use to strerilise petri dishes and media:

1. Autoclave

2. Pressure cooker

Sterilization of Filtration Equipment Step

Dry the Sample Cup

Pour a Few Drops of Methanol into the Sample Cup

Carefully Ignite the Methanol in the Sample Cup Using the Cigarette Lighter

Methanol Dispenser

After the Methanol Burns for Several Seconds, Place the Filtration Head ono the Cup

Keep the Filtration Apparatus Sealed for at Least 15 minutes Before Use

Methanol Warning

• Methanol must be stored safely away from children (in a lab or office). It can be deadly if it is drinken. It should be used in well ventilated areas.

• For general lab cleanup ethanol is less toxic and would be better.

• It should not be used to wash hands.

Absorbent Pads Step

Using the Absorbent Pad Dispenser, Place One Pad Into Each Petri Dish

Petri-dish

Absorbent pad dispenser

Tip - If absorbent pad dispenser is broken, tweezers (sterilised with lighter) can be used

Pour the Media onto the Absorbent Pad

Pad

Petri Dish Cover

Using Pipette to Pour Media onto Absorbent Pad

Handling Petri Dishes

Filtering the Sample Through The Membrane Filter

Assemble the Filtration Apparatus

Remove Membrane Filter from Packet with Sterilised Tweezers

To Sterilise Tweezers, Flame the Tip with a Lighter

Place the Membrane Filter Onto the Bronze Disc

Fit the Filter Funnel and Plastic Collar

Filter Funnel

Plastic Collar

The 3 Plastic Collar Adjustment Positions

Pour the Sample into the Filter Funnel

100 ml Sample

Attach the Vacuum Pump and Create a Vacuum to Draw the Sample Through the Membrane (DelAqua)

From www.delaqua.org

Creating a Vacuum (Wagtech Potatest)

From Wagtech Potatest Users Manual

After Filtering the Sample, Remove the Membrane Filter

Lower The Membrane Filter onto an Absorbent Pad (that already has had medium poured in it)

Starting at the edge of the petri dish, use a rolling motion to place the membrane filter on top of the absorbent pad (with medium). This will prevent air being trapped between the pad and the membrane filter.

Place the Lid and Mark with non-Permanent Pen

Incubation of Samples

Incubation of Samples

• All 16 petri dishes must be placed in the Incubator regardless of how many have samples

• To ensure proper and even heating of the samples

• Samples must be in the incubator for 16 to 18 hours for DelAqua and 18 hours for Wagtech Potatest

• Thermotolerant (faecal) coliform samples set at 44 degrees C and Total Coliform at 37 degrees C

Reading and Interpreting the Samples

Yellow colonies (but not red dots) are Thermotolerant (faecal) bacteria

From Wagtech PotatestUsers Manual

From Wagtech Potatest Users Manual

Sample on the right = 0 CFU/100 but how many thermotolerantcoliform bacteria you think are on the sample on the left?

Sample on Right is “Diluted” 10 to 1 by Only Filtering 10 mL of Sample Instead of 100 mL (Why?)

Results on right must be multiplied by 10 to get bacteria/ 100 mL since only 10 mL was used for test?

For discussion:How many bacteria/100 mL (dots) would you report for the sample on the right?

After Samples are Read, Petri Dishes Ban be Sterilised in a Pot of Boiling Water

For Group Discussion

•What does it mean to dilute the sample for bacteria analysis?

•When do you want to dilute (use a smaller volume of water) samples?

For Group Discussion

• What steps should be taken if the fecal (“thermotolerant”) coliform bacteria in the drinking water sample tested are greater than 5 and/or greater than 10 CFU/100 ml?

• Who should take the steps?

Section 4

Compartment Bag Test for Bacteria (E. coli) Analysis

Compartment Bag Test for E. coli Bacteria Testing

CBT Results (from Nepal after the earthquake)

Photo by T Mahin

How many E. coli/100 ml are there in this sample?

Section 5

Chlorination of boreholes/wells

Chlorinating Wells after Rehabilitation

Ground Water

Source for above -https://microbewiki.kenyon.edu

Dewatering Boreholes/Wells after Chlorinating

• For boreholes, the volume of water to be dewatered is not that large so can be dewater by pumping to waste using the hand pump (test chlorine residual to know if enough water was wasted)

• For “deep wells” (dug wells) the volume of water is much larger so hand pumping to waste is not adequate. Generally a submersible pump and generator would be needed.

The Chlorine Dosage for Disinfecting a Well (from Oxfam Guidelines)

Use Proper Protection When Handling High Strength Chlorine (HTH)

Photo by T. Mahin

Turbidity

High turbidity requires more chlorine to oxidise organic matter and iron

Alternate Approach to Measure Turbidity

High Turbidity in Wells

• High turbidity after a rehabilitated well has stabilized can be a sign of poor protection at the top of the well allowing surface water contamination (usually during the wet season)

• During the dry season it is usually caused by inadequate cleaning of the well as part of rehabilitation/installation

• High turbidity (> 20 NTU) can interfere with chlorination of wells

Sealing the Top of a Well

Use of Turbidity Tube from DelAqua Kit

For discussion: Why and when should we measure turbidity?

Gutu, Zimbabwe (7-7-2016) Photos by T. Mahin

Use of Turbidity Tubes

Use of Turbidity Tubes

Use of Turbidity Tubes

From Wagtech Potatest Users Manual

Section 6

Chlorine Residual Testing in the Field

Examples of Chlorine Residual Field Testing Equipment

• Comparator (“pool tester”)

• Chlorine test strips

For Discussion: Why Is it important to check the chlorine residual before doing bacteria analysis after rehabilitating boreholes/wells?

•What will be the impact on bacteria analysis if the water being tested has chlorine in it?

•What could the source of chlorine be for boreholes or deep wells?

•How can sample be collected to prevent having chlorine in the sample?

To Check Chlorine Residual Fill the Comparator Cells with Sample Water

• For a tap sample let the water run for at least 1 minute

• For a sample from a hand pump, pump some water to bucket or waste prior to collecting sample

Drop One DPD Tablet Into the Right Hand Cell (C12)

Invert the Comparator Until the Tablet is Dissolved

Photo by T. Mahin

For Discussion: What is the Chlorine Level of This Sample?

Chlorine Test Strips

A simple easy to read way to test chlorine

Photo by T. Mahin

Measuring pH

Photo by T. Mahin

Measuring pH at a School in Gutu, Zimbabwe

For Discussion: What is the pH of this Sample?

DelAqua Training Videos Online (web sites checked on 7-3-2016)

Media preparation training video (4 min, 1 sec.):

https://youtu.be/ls1pA7f_6ho

Sterelising petri dishes and media training video:

https://youtu.be/9agCi4IfZlU

Sterelising the filtration apparatus training video:

https://youtu.be/0eILlwRpsjY

• Counting colonies training video:

https://youtu.be/5vgrJ2UYzM0

User Manuals

• For the DelAqua Manualhttp://www.oxfam.org.uk/equipment/catalogue/resources-included-available/water-and-sanitation/water-treatment-and-testing/Delagua%20english_manual_2000-1.pdf

• For the Wagtech Potatest Manualhttp://www.palintest.com/application/files/9514/5546/6923/Potatest_User_Manual_EN_LR.pdf

Information Source Credit

• Many of the pictures of the Oxfam-DelAqua equipment and instructions on its use has been copied from either the Oxfam-DelAqua users manual and/or the DelAqua training videos.

Photo by T. Mahin

MoHCC Presentation at Matobo Training

Thank you for your participation!

For questions about this presentation you can email Tom Mahin at: tom.mahin@gmail.com