1. 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

2. 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

3. Section 1
Types of and the Importance of
“Indicator Bacteria”

4. 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

5. 3 Different Types of Bacteria Will be Analyzed for
During This Training

6. Source Clackamas

7. The More Fecal (or E. coli) Bacteria in
Drinking Water the Higher the Risk
of Cholera, Typhoid, etc.
Poster at Gutu District Laboratory

8. Bacteria Concentration

9. 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

10. Section 2
Collection of Bacteria Samples in the Field

11. Examples of Potential Sampling Locations

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

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

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

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

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

17. (Gutu, Zimbabwe 7-7-2016)
Using Whirl-Pak Bags to Collect Bacteria Samples
Photos by T. Mahin

18. 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?

19. Section 3
Using DelAqua Kits for Bacteria Analysis of Water

20. 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.)

22. Incubator and On/off Switches
and Indicator Lights and Fuse

24. Filtration Components (in DelAqua Kit)

25. Bacteria Analysis with the DelAqua –
Making the Culture Media

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

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

28. Mixing the MLSB (continued)

29. Pouring into Small Bottles and Sterilising the MLSB

31. Making MLSB Medium
(Gutu Training)
Photo by T. Mahin

32. Sterliising Step

33. Sterilising Petri Dishes and the Media
Possible methods to use to strerilise petri dishes and media:
1. Autoclave
2. Pressure cooker

34. Sterilization of Filtration Equipment Step

35. Dry the Sample Cup

36. Pour a Few Drops of Methanol into the Sample Cup

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

38. Methanol Dispenser

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

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

41. 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.

42. Absorbent Pads Step

43. 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

44. Pour the Media onto the Absorbent Pad
Pad
Petri Dish Cover

45. Using Pipette to Pour Media onto Absorbent Pad

46. Handling Petri Dishes

47. Filtering the Sample Through The
Membrane Filter

48. Assemble the Filtration
Apparatus

49. Remove Membrane Filter from Packet
with Sterilised Tweezers

50. To Sterilise Tweezers, Flame the Tip with a Lighter

51. Place the Membrane Filter Onto the Bronze Disc

53. Fit the Filter Funnel and Plastic Collar
Filter Funnel
Plastic Collar

54. The 3 Plastic Collar Adjustment Positions

55. Pour the Sample into
the Filter Funnel

56. 100 ml Sample

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

59. From www.delaqua.org

60. Creating a Vacuum (Wagtech Potatest)

62. After Filtering the Sample, Remove the Membrane Filter

63. 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.

64. Place the Lid and Mark with non-Permanent Pen

65. Incubation of Samples

67. 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

68. Reading and Interpreting the Samples

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

70. From Wagtech Potatest
Users Manual

71. From Wagtech Potatest Users Manual

73. Sample on the right = 0 CFU/100 but how many thermotolerant
coliform bacteria you think are on the sample on the left?

74. 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?

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

76. 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?

77. 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?

78. Section 4
Compartment Bag Test for Bacteria (E. coli) Analysis

79. Compartment Bag Test
for E. coli Bacteria Testing

80. CBT Results (from Nepal after the earthquake)
Photo by T Mahin

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

83. Section 5
Chlorination of boreholes/wells

84. Chlorinating Wells
after Rehabilitation

85. Ground Water
Source for above -https://microbewiki.kenyon.edu

87. 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.

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

90. Use Proper Protection When Handling
High Strength Chlorine (HTH)
Photo by T. Mahin

91. Turbidity
High turbidity requires more chlorine to oxidise organic
matter and iron

92. Alternate Approach to Measure Turbidity

93. 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

94. Sealing the Top of a Well

95. 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

96. Use of Turbidity Tubes

97. Use of Turbidity Tubes

98. Use of Turbidity Tubes

99. From Wagtech Potatest Users Manual

100. Section 6
Chlorine Residual Testing in the Field

101. Examples of Chlorine Residual
Field Testing Equipment
• Comparator (“pool tester”)
• Chlorine test strips

102. 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?

103. 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

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

105. Invert the Comparator Until the Tablet is Dissolved

106. Photo by T. Mahin

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

108. Chlorine Test Strips
A simple easy to read way to test chlorine

110. Photo by T. Mahin

111. Measuring pH

112. Photo by T. Mahin
Measuring pH at a School in Gutu, Zimbabwe

113. For Discussion: What is the pH of this Sample?

114. 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

115. User Manuals
• For the DelAqua Manual
http://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 Manual
http://www.palintest.com/application/files/9514/5546/6923/Potatest_User_Manual_EN_LR.pdf

116. 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.

117. Photo by T. Mahin
MoHCC Presentation at Matobo Training

118. Thank you for your
participation!
For questions about this presentation you can email Tom Mahin at:
tom.mahin@gmail.com