Islamic Emarat of Afghanistan SOP NUMBER
Ministry of Public Health
Issued Date
Drug and Food Directorate
Page No.
STANDARD OPERATING PROCEDURE
TITLE: Detection of Total Coliforms and E. coli by Colilert-18 in Water (colilert test)
1. Scope and Application
This method is intended for use in the simultaneous detection and confirmation of total
coliforms and E. coli in water. Any positive sample for total coliforms is an indication of
contamination. Any positive sample for both total coliforms and E. coli is an acute
violation.
The minimum, non-zero number of bacterial counts detectable with this method is a
function of the dilution scheme used when processing the sample.
The Colilert-18 method can be applied to fresh waters, drinking waters, ground waters,
reuse waters, waste waters and marine waters. It can be used as a Presence/Absence test
or quantification with the Quanti -Tray system or with 5 X 20 mL, 10 X 10mL, 15 tubes
serial dilution (MPN).
Since there can be wide range of coliform levels in surface waters and wastewaters,
dilutions can be used with this method for detecting and enumerating the actual level.
Make at least a 1:10 dilution using sterile non-buffered oxidant free water for E. coli. In
some sub-tropical waters, a 1:20 dilution may be necessary.
2. Responsibility
Microbiologist and Technical Manager
3. Accountability
Quality manager or Quality assurance
Review No. Review Date Review Due Date
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Islamic Emarat of Afghanistan SOP NUMBER
Ministry of Public Health
Issued Date
Drug and Food Directorate
Page No.
STANDARD OPERATING PROCEDURE
4. Principal
4. 1
Total coliform bacteria chromogenic substrates, such as ortho-nitrophenyl-β-D-
galactopyranoside (ONPG) or chloro phenol red-β-D-galactopyranoside (CPRG), are
used to detect the enzyme β-D-galactosidase, which is produced by total coliform
bacteria. The β-D-galactosidase enzyme hydrolyzes the substrate and produces a
color change, which indicates a positive test for total coliforms at 24 h (ONPG) or 28
h (CPRG) without additional procedures. Non coliform bacteria, such as Aeromonas
and Pseudomonas species, may produce small amounts of the enzyme β- D-
Galactosidase, out are suppressed and generally will not produce a positive response
within the incubation time unless more than 104 colony-forming units (CFU)/mL (106
CFU/100 mL) are present.
4 .2
Escherichia coli: A fluorogenic substrate, such as 4-methyl- umbelliferyl-β-D-
glucuronide (MUG), is used to detect the enzyme β-glucuronidase, which is produced
by E. coli. The β-glucuronidase enzyme hydrolyzes the substrate and produces a
fluorescent product when viewed under long-wavelength (365-366 nm) ultraviolet
(UV) light. The presence of fluorescence indicates a positive test for E. coli. Some
strains of Shigella spp. also may produce a positive fluorescence response.
5. Interferences
5.1. Some samples containing humic material may have an innate color and a control
blank of the same water sample may be required for comparison to the inoculated
sample or a dilution may be made.
5.2. Heterotrophic bacteria greater than 1,000,000/100mL could yield a positive coliform
reaction for coliforms.
6. Material and Equipment
6.1. Sterile Pipettes, appropriate volume pipettes, and sterile loops.
6.2. Sterile vessels, glass or plastic (free from fluorescence) with or without sodium
thiosulfate. Any water containing an oxidizing agent such as chlorine must be
neutralized with sodium thiosulfate.
Vessels containing sodium thiosulfate must neutralize up to 5 mg/L of chlorine
for drinking water samples and up to 15 mg/L for waste water effluents.
Review No. Review Date Review Due Date
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Islamic Emarat of Afghanistan SOP NUMBER
Ministry of Public Health
Issued Date
Drug and Food Directorate
Page No.
STANDARD OPERATING PROCEDURE
Vessels must be at least 120 mL or of larger capacity to hold 100 mL sample to
allow for proper mixing of sample.
6.3. 51 well Quanti-Tray or Quanti-Tray/2000
6.4. Quanti-Tray Sealer.
6.5. Incubator maintained at 35 ± 0.5°C.
6.6. 6 Watt 365-366 nm UV light. (12.1)
7. Reagents
7.1. Sterile, non-buffered, oxidant-free water for dilutions.
7.2. Presence /Absence, 51 Well Quanti-Tray or Quanti-Tray 2000 Comparator.
7.3. Antifoam reagent (optional).
7.4. Sodium thiosulfate reagent Standard Methods for the Examination of Water and
Wastewater, or sterile vessels containing sodium thiosulfate to neutralize up to
15mg/L chlorine.
7.5. Store Colilert-18 at 2-25°C away from light. The expiration date is indicated on the
package (15 months from the date of manufacture).
8. Sample Preservation and Storage
8.1. Preservation and Storage
8.1.1. Storage Temperature and Handling Conditions: The bacteriological samples at a
temperature less than < 10°C (2-10°C) during transit to the laboratory. Use
insulated containers to assure proper maintenance of storage temperature. Ensure
that sample vessels are not totally immersed in water during transit. Do not allow
samples to freeze. If frozen, sample cannot be thawed and a new sample is
required.
8.1.2. Holding Time Limitations: Examine samples as soon as possible after collection.
For drinking water samples do not exceed 30 hours hold time from collection to
incubation. For non-potable water for compliance, do not exceed 8 hours from time
of collection to incubation.
Review No. Review Date Review Due Date
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Islamic Emarat of Afghanistan SOP NUMBER
Ministry of Public Health
Issued Date
Drug and Food Directorate
Page No.
STANDARD OPERATING PROCEDURE
9. Multi well procedure
9.1. Carefully separate one blister pack from the strip taking care not to accidentally open the
adjacent pack.
9.2. Ensure the powder is in the bottom of the blister pack.
9.3. Hold the blister pack face down (paper side up) at the top and towards the bottom and
snap back at the score line, with the opening facing into the open vessel.
9.4. Add the reagent to 100ml water sample contained in the sterile, non-fluorescent vessel.
9.5. Aseptically cap and seal the vessel.
9.6. Mix well until dissolved.
9.7. Incubate for 18 and up to 22 hours at 35 ± 0.5°C.
9.8. Read the results at 18 hours. In addition, laboratories may incubate samples for
additional time (up to 22 hours total for their convenience). Compare each result against the
comparator dispensed into an identical vessel.
9.9. If the sample has a yellow color equal to or greater than the comparator, the presence of
total coliforms is confirmed. If color is not uniform, mix by inversion, then recheck.
9.10. If yellow is observed, check vessel for fluorescence by placing a 6-watt, 365-366 nm
UV light within five inches of the sample in a dark environment. Be sure the light is facing
away from your eyes and towards the vessel. If the fluorescence is equal to or greater than the
comparator, the presence of E. coli is confirmed.
9.11. Positives for both total coliforms and E. coli observed before 18 hours and negatives
observed after 22 hours are also valid.
10. Result
Color changes for various media
substrate Total coliform positive E – coli positive Negative result
ONPG-MUG Yellow Blue fluorescence Color less/no
fluorescence
CPRG-MUG Red or magenta Blue fluorescence Yellow/no
fluorescence
Review No. Review Date Review Due Date
Prepared By Checked By Approved By
Islamic Emarat of Afghanistan SOP NUMBER
Ministry of Public Health
Issued Date
Drug and Food Directorate
Page No.
STANDARD OPERATING PROCEDURE
Review No. Review Date Review Due Date
Prepared By Checked By Approved By
Islamic Emarat of Afghanistan SOP NUMBER
Ministry of Public Health
Issued Date
Drug and Food Directorate
Page No.
STANDARD OPERATING PROCEDURE
Review No. Review Date Review Due Date
Prepared By Checked By Approved By
Islamic Emarat of Afghanistan SOP NUMBER
Ministry of Public Health
Issued Date
Drug and Food Directorate
Page No.
STANDARD OPERATING PROCEDURE
12. References
12.1. Colilert-18 Package Insert from IDEXX.
12.2. 1998 Standard Methods for the Examination of Water and Wastewater, 20th Edition
12.3. Quanti-Tray Package Insert from IDEXX.
12.4. APHA 1998, 20th
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