Microbial Limit Test of Water (MLT)- Raw, Treated and Purified Water

Microbial Limit Test of Water, a practical guide to the microbial limit test of raw, treated and purified water used in pharmaceutical manufacturing and microbiology laboratories.

Objective for Microbial Limit Test of Water

To establish a standardized procedure for performing Microbial Limit Testing (MLT) of raw water, treated water, and purified water.

This procedure describes the requirements for sample handling, microbial enumeration, membrane filtration, pour plate testing, detection of specified pathogens, result interpretation, documentation, and microbial trend analysis.

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Media Preparation and Sterilization SOP

Scope

This SOP applies to the microbiological examination of raw water, treated water, and purified water used at company.

The procedure covers the determination of total aerobic microbial count and the examination of water samples for specified microorganisms, wherever applicable.

Responsibility

Designation	Responsibility
Officer/Executive – Microbiology	Responsible for carrying out the MLT procedure as described in this SOP and recording the test results accurately.
Department Head – Microbiology	Responsible for reviewing the test results, approving or rejecting the results as applicable, and ensuring compliance with this SOP.
Quality Assurance Department	Responsible for reviewing and approving the SOP and issuing controlled copies to the concerned department.

Requirements for Microbial Limit Test of Water

The following equipment, instruments, materials, and microbiological media shall be available before starting the test:

• 1 mL and 10 mL micropipettes
• Sterile 1 mL and 10 mL micropipette tips
• Sterile Petri plates
• Sterile test tubes
• Incubator maintained at 30–35°C
• Incubator maintained at 42–44°C
• Calibrated weighing balance
• Autoclave
• Calibrated pH meter
• Appropriate microbiological culture media
• Sterile/pre-sterilized 0.45 µm membrane filters
• Membrane filtration assembly
• Sterile forceps
• Other accessories required for microbiological testing

Sampling of Water

General Sampling Requirements

1. Water samples shall be collected aseptically from the designated sampling points as per the applicable approved sampling SOP.
2. The sample container shall be properly identified with the sample location, sample identification number, date, and time of sampling.
3. Microbiological analysis should preferably be started as soon as possible after collection of the sample.
4. If testing cannot be performed on the day of sampling, the sample shall be stored at 2–8°C and tested within 24 hours, as applicable to the approved procedure.
5. Care shall be taken during collection, transportation, and handling to prevent external contamination.
6. Microbial enumeration may be performed by either the pour plate method or the membrane filtration method, as applicable.

Microbial test by Pour Plate Method

1. Transfer 1.0 mL of the water sample aseptically into a sterile Petri plate.
2. Add approximately 15–20 mL of R2A agar maintained at a suitable temperature for pouring.
3. Gently mix the sample and medium by moving the Petri plate on a smooth, level surface in a suitable pattern, such as a figure-eight motion.
4. Prepare the test in duplicate.
5. Allow the medium to solidify completely.
6. Incubate the plates at 30–35°C for 5–7 days.
7. After completion of incubation, examine the plates for microbial growth.
8. Count the colonies and calculate the microbial count as colony-forming units per millilitre (CFU/mL).
9. Record the observations and calculated results in the appropriate test record.

Calculation

For duplicate plates:

Average CFU/mL = (Count of Plate 1 + Count of Plate 2) / 2

Where dilution is applicable:

Total Aerobic Microbial Count (CFU/mL) = Average CFU/mL × Dilution Factor

For the specified dilution in this procedure:

Dilution Factor = 10

Microbial test by Membrane Filtration Method

1. Sterilize the membrane filtration assembly and required accessories before use, or use a validated pre-sterilized filtration assembly where applicable.
2. Use a sterile 0.45 µm membrane filter.
3. Transfer the required R2A agar plates to the LAF, ensuring that aseptic conditions are maintained.
4. Assemble the membrane filtration unit and sterile filter holder.
5. Using sterile forceps, carefully place the membrane filter on the filter support.
6. Transfer 10 mL of the water sample to the filtration assembly.
7. Filter the sample through the membrane.
8. Where required, rinse the membrane with an appropriate quantity of sterile diluent or buffered solution.
9. After filtration is complete, carefully remove the membrane using sterile forceps.
10. Place the membrane aseptically on the surface of the appropriate R2A agar plate, ensuring that there are no air pockets between the membrane and agar surface.
11. Incubate the plates at 30–35°C for 5–7 days.
12. Following incubation, examine the membrane for microbial colonies.
13. Count the colonies and calculate the microbial count in CFU/mL.
14. Record the results in the applicable laboratory record.

Microbial Limits for Water Samples

The applicable alert, action, and specification limits shall be as defined in the approved site specification.

Type of Water Sample	Alert Limit	Action Limit	Specification Limit
Raw Water	NMT 55 CFU/mL	NMT 59 CFU/mL	NMT 500 CFU/mL
Treated Water	NMT 31 CFU/mL	NMT 35 CFU/mL	NMT 100 CFU/mL
Purified Water	NMT 23 CFU/mL	NMT 27 CFU/mL	NMT 100 CFU/mL

Note: Alert, action, and specification limits should always be verified against the current approved site specification and applicable pharmacopoeial requirements before implementation.

Test for Specified Microorganisms

Where specified by the approved water specification or applicable test requirement, the sample shall be examined for the following microorganisms:

• Escherichia coli
• Salmonella species
• Pseudomonas aeruginosa
• Staphylococcus aureus

All testing shall be performed using approved microbiological methods and suitable positive and negative controls.

Test for Escherichia coli

Enrichment

1. Transfer 10 mL of the water sample into 90 mL of SCDM.
2. Incubate at 30–35°C for 18–24 hours.
3. After incubation, proceed with the primary test.

Primary Test

1. Transfer 1 mL of the enrichment culture into 100 mL of MacConkey broth.
2. Incubate at 42–44°C for 24–48 hours.
3. Observe the culture for characteristic changes.
4. Where the specified reaction indicates a presumptive positive result, proceed to the secondary test.

Secondary Test

1. Using a sterile loop, take a portion of the enrichment culture.
2. Streak the culture onto MacConkey agar.
3. Incubate at 30–35°C for 18–72 hours.
4. Observe the plates for characteristic colonies.
5. Pink, non-mucoid colonies may indicate the possible presence of E. coli and shall be subjected to confirmation.

Confirmatory Test – Indole Test

1. Inoculate the suspected culture into nutrient broth and incubate for the required period.
2. Add approximately 0.1 mL of Kovac’s reagent to the incubated culture.
3. Mix gently and allow the tube to stand.
4. Formation of a characteristic pink/red layer in the reagent layer indicates a positive indole reaction.
5. Record the result and interpret it according to the approved test method.

Positive Control

Use a suitable E. coli positive control culture to verify the performance of the test system.

Transfer the required quantity of the positive control culture into MacConkey broth and process it along with the test as per the approved procedure.

Test for Salmonella Species

Enrichment

1. Transfer 10 mL of the water sample into 90 mL of SCDM.
2. Incubate at 30–35°C for 18–24 hours.
3. Proceed with the primary enrichment test.

Primary Enrichment

1. Transfer 0.1 mL of the enrichment culture into 10 mL of Rappaport-Vassiliadis Salmonella Enrichment Broth (RVSEB).
2. Incubate according to the approved method.
3. Proceed to the secondary test after incubation.

Secondary Test

1. Using a sterile loop, take a portion of the enriched culture.
2. Streak it onto Xylose Lysine Deoxycholate Agar (XLDA).
3. Incubate at 30–35°C for 24–48 hours.
4. Examine the plate for colonies characteristic of Salmonella.
5. Suspected colonies shall be subjected to confirmatory testing.

Confirmatory Test

1. Select suspected colonies from the selective agar plate.
2. Inoculate the selected colonies onto Triple Sugar Iron (TSI) agar slants using an appropriate stabbing and streaking technique.
3. Inoculate the suspected culture into urea medium as required.
4. Incubate at 30–35°C for 18–24 hours.
5. Observe the biochemical reactions and interpret the results according to the approved test method.
6. Confirm the identity of the suspected organism based on the complete set of characteristic reactions.

Positive Control

A suitable positive control culture of Salmonella shall be processed along with the test to demonstrate the suitability of the test conditions.

Test for Pseudomonas aeruginosa

Enrichment

1. Transfer 10 mL of the water sample into 90 mL of SCDM.
2. Incubate at 30–35°C for 18–24 hours.
3. Proceed with the primary test.

Primary Test

1. Using a sterile loop, streak the enrichment culture onto a suitable pre-incubated Cetrimide agar plate.
2. Incubate at 30–35°C for 18–72 hours.
3. Examine the plate for characteristic growth.
4. Greenish colonies may indicate the possible presence of P. aeruginosa and shall be subjected to confirmation.

Confirmatory Test – Oxidase Test

The oxidase test is used as a biochemical confirmation test for suspected P. aeruginosa.

1. Perform the oxidase test using a suitable approved oxidase reagent.
2. Observe the reaction within the specified time.
3. A characteristic rapid colour change indicates a positive oxidase reaction.
4. Interpret the result together with the colony characteristics and other applicable confirmation tests.

Positive Control

A suitable positive control culture of P. aeruginosa shall be streaked onto Cetrimide agar and incubated under the specified conditions to confirm the suitability of the test system.

Test for Staphylococcus aureus

Enrichment

1. Transfer 10 mL of the water sample into 90 mL of SCDM.
2. Incubate at 30–35°C for 18–24 hours.
3. Proceed with the primary test.

Primary Test

1. Streak a loopful of the enrichment culture onto a suitable pre-incubated Mannitol Salt Agar (MSA) plate.
2. Incubate at 30–35°C for 18–72 hours.
3. Examine the plate for characteristic colonies and colour changes.
4. Suspected colonies shall be subjected to the coagulase confirmation test.

Confirmatory Test – Coagulase Test

1. Dispense approximately 0.5 mL of reconstituted plasma into a sterile test tube.
2. Add approximately 0.05 mL of the 18–24-hour culture to be tested.
3. Mix gently to obtain a uniform suspension.
4. Incubate under the conditions specified in the approved test method.
5. Examine the tube periodically for clot formation.
6. Any definite clotting observed within the specified test period shall be interpreted according to the approved method.

Limits for Specified Pathogens

The specified pathogens shall be absent from the applicable water sample.

Specified Microorganism	Requirement
Escherichia coli	Absent
Salmonella species	Absent
Pseudomonas aeruginosa	Absent
Staphylococcus aureus	Absent

Water Quality Trend Analysis

Monthly and annual trend analysis shall be performed for the microbiological and chemical quality of water, as applicable.

The trend analysis shall be prepared using the approved formats:

• Monthly Water Quality Trend
• Annual Water Quality Trend

Trend data shall be reviewed periodically to identify any increasing microbial count, recurring excursions, or adverse trends that may require investigation or corrective action.

Safety and Aseptic Precautions

1. Handle all water samples carefully to minimize the possibility of contamination.
2. Follow appropriate microbiological safety practices during sample handling and testing.
3. Disinfect work surfaces before and after testing.
4. Use sterile materials and equipment wherever required.
5. Perform applicable manipulations under a qualified Laminar Air Flow (LAF) unit or other suitable controlled environment.
6. Use appropriate disinfectant, such as IPA, for sanitizing suitable surfaces and gloves/hands in accordance with the laboratory’s approved safety procedure.
7. Handle cultures and contaminated materials as potentially hazardous microbiological material.
8. Dispose of used culture media, plates, membranes, and other contaminated materials through the approved biohazard waste disposal procedure.
9. Follow the laboratory’s approved biosafety and waste-management practices at all times.

Abbreviations

Abbreviation	Full Form
QC	Quality Control
SOP	Standard Operating Procedure
QA	Quality Assurance
NA	Not Applicable
CFU	Colony Forming Unit
NLT	Not Less Than
NMT	Not More Than
MLT	Microbial Limit Test
SCDM	Soybean-Casein Digest Medium
SDA	Sabouraud Dextrose Agar
XLDA	Xylose Lysine Deoxycholate Agar
RVSEB	Rappaport-Vassiliadis Salmonella Enrichment Broth
LAF	Laminar Air Flow
mL	Millilitre
µm	Micrometre
g	Gram

Change History

Details of Revision	Change Control No.	Reason for Revision

References

• Approved in-house microbiological testing methods.
• Current approved specifications for raw, treated, and purified water.
• Applicable pharmacopoeial requirements for microbiological examination of water.
• Current manufacturer’s instructions for applicable microbiological media and reagents.

Frequently Asked Questions (FAQs)

1. What is the Microbial Limit Test (MLT) of water?

The Microbial Limit Test (MLT) is a microbiological examination performed to determine the level of microbial contamination in water. It helps assess the microbiological quality of raw water, treated water, and purified water used in pharmaceutical operations.

2. Why is microbial testing of water important?

Water can support the growth of microorganisms and may become a source of contamination that is why microbial testing water is important.

3. Which types of water are tested for microbial limits?

Testing may be performed on raw water, treated water, purified water, and water from different points of the distribution system.

4. What parameters are generally tested during MLT of water?

Typical microbiological testing may include Total Aerobic Microbial Count (TAMC) and, where applicable, Total Yeast and Mold Count (TYMC).