Pre-Analysis & Sample Control
Sample Tempering: Allow all samples to reach room temperature before starting any analysis.
Storage & Delay Protocol: If analysis cannot begin within 2 hours of sampling:
Store samples at 2–8°C and log the entry in the respective logbook.
Ensure testing is completed within the mandatory allowable hold times:
TAMC (In-house): Within 24 hours
TAMC (Off-site contract labs): Within 36 hours
BET Samples: Within 96 hours
Analytical Procedure (TAMC via Pour Plate Method)
Homogenization: Gently swirl the sample bottle before performing the microbiological analysis to ensure even distribution.
Plating: 1. Pipette 1 mL of the water sample into each of two sterile Petri dishes (in duplicate).
2. Pour approximately 20–25 mL of sterile R2A agar into each dish.
3. Crucial: Ensure the R2A agar has been previously sterilized and cooled to approximately 45°C before pouring to avoid killing any viable microorganisms.
Phase 4: Analytical Procedure (Continued)
Temperature Verification: Before pouring the R2A agar, check its temperature using an IR thermometer to confirm it has cooled to approximately 45°C.
Note: Operate the IR thermometer strictly according to SOP No.: BEL-MB048.
Mixing and Pouring: 1. Pour approximately 20–25 mL of the verified R2A agar into the plates containing your 1 mL samples. 2. Immediately cover the Petri dishes. 3. Mix the sample with the agar by gently rotating the dishes—first in a clockwise direction, and then in an anti-clockwise direction.
Solidification: Leave the plates undisturbed inside the Laminar Air Flow (LAF) or Biosafety Cabinet (BSC) and allow the media to solidify completely.
Phase 5: Quality Control (Negative Control)
Negative Control Setup: Prepare one Petri dish containing only 20–25 mL of the sterile R2A agar (sterilized and cooled to ~45°C) without any sample. This serves as your negative control to confirm media sterility and environmental cleanliness during testing.
Phase 6: Incubation
Placing Plates: Once solidified, invert the plates (both sample duplicates and the negative control).
Parameters: Incubate the inverted plates at 30°C to 35°C for Not Less Than (NLT) 5 days.
Phase 1: Equipment Setup
Environment: Perform the assembly entirely under a Biosafety Cabinet (BSC) to maintain sterility.
Filter Specification: Assemble the filtration unit and place a sterile membrane filter with a nominal pore size of not greater than 0.45 µm onto the filtration funnel.
Phase 2: Sample Filtration Quantities
Filter the exact volume required based on the specific water type being tested:
| Water Type | Preparation / Filtration Volume |
| Purified Water | First, transfer approximately 100 mL of 0.1% sterile peptone water into the sterile funnel, then add 3 mL of the sample, and filter the entire combined volume. |
| Water for Injection (WFI) | Filter 200 mL of the sample directly. |
| Pure Steam Condensate | Filter 200 mL of the sample directly. |
Phase 3: Quality Control (Negative Control)
Negative Control Setup: Using a completely separate, sterile membrane filter (nominal pore size $\le$ 0.45 µm), filter 100 mL of 0.1% sterile peptone water.
Purpose: This step must be performed alongside the samples to verify the sterility of the equipment, rinsing fluid (peptone water), and the testing environment.
Phase 4: Membrane Transfer & Incubation
CRITICAL NOTE: Use a dedicated filtration funnel and membrane filter for each individual sample or test to prevent cross-contamination.
Membrane Transfer: 1. Using sterile forceps, carefully remove the membrane filter from the filtration apparatus. 2. Place the filter onto the surface of a Sterile R2A agar plate using a smooth, rolling motion. 3. Precautions: Take extreme care to avoid entrapment of air between the membrane filter and the agar surface, as air bubbles will prevent nutrients from reaching any trapped microorganisms.
Incubation: Incubate all sample plates and the negative control plate in an upright position at 30°C to 35°C for Not Less Than (NLT) 5 days.
Phase 5: Interpretation of Results
Negative Control Validation: Inspect the negative control plate first. It shall not show any microbial growth. If growth is observed, the test is invalid due to potential contamination and must be investigated/repeated.
Results Calculations & Reporting Units
| Sample Type | Reporting Unit | Calculation Rule | Mandatory Action if Colonies are Found |
| In-Process Water | CFU/mL | Report raw colony count directly (since 1 mL was plated). | Standard reporting. |
| Purified Water | CFU/mL | Divide the raw colony count by 3 (since 3 mL was added to the peptone water). | Standard reporting. |
| Water for Injection (WFI) | CFU/100 mL | Divide the raw colony count by 2 (since 200 mL was filtered, reducing it back to a 100 mL baseline). | Perform organism identification |
| Pure Steam Condensate | CFU/100 mL | Divide the raw colony count by 2 (since 200 mL was filtered, reducing it back to a 100 mL baseline). | Perform organism identification |
⚠️ Zero Growth Reporting Rule: If no colonies grow on the plate, report the results as 'Nil CFU' on manual documentation. For electronic LIMS entries, input the numerical value as "0" CFU.
1. Sample Preparation (Preparation of Solution A)
Filtration: Transfer 100 mL of the sample to a membrane filter and filter it immediately.
Enrichment: After filtration is complete, use sterile forceps to transfer the membrane filter into 100 mL of Soybean Casein Digest Medium (SCDM).
Incubation: Mix the sample thoroughly and incubate at 30°C to 35°C for Not Less Than (NLT) 24 hours. This enriched broth is designated as Solution A.
2. Test for Escherichia coli (E. coli)
Step 1: Subculture Enrichment
After the incubation of Solution A is complete, transfer 1 mL of Solution A into 100 mL of MacConkey Broth (MCB).
Incubate the MCB at 42°C to 44°C for Not Less Than (NLT) 48 hours.
Step 2: Selective Streaking
Following the MCB incubation, take a loopful of the broth and streak it onto a MacConkey Agar (MCA) plate.
Incubate the MCA plate at 30°C to 35°C for Not Less Than (NLT) 72 hours.
Interpretation & Compliance:
Observation: The growth of colonies indicates the possible presence of E. coli. Suspect colonies must be confirmed by specific identification tests.
Compliance: The sample complies with the test if no colonies are present on the MCA plate, OR if the subsequent identification tests are negative.
3. Test for Salmonella
Step 1: Subculture Enrichment
After the incubation of Solution A is complete, transfer 0.1 mL of Solution A into 10 mL of Rappaport-Vassiliadis Salmonella Enrichment Broth (RVSEB).
Incubate the RVSEB at 30°C to 35°C for Not Less Than (NLT) 24 hours.
Step 2: Selective Streaking
Following the RVSEB incubation, take a loopful of the broth and streak it onto a Xylose Lysine Deoxycholate Agar (XLDA) plate.
Incubate the XLDA plate at 30°C to 35°C for Not Less Than (NLT) 48 hours.
Interpretation & Morphology:
Colonial Morphology: The observation of red colonies with or without black centers indicates the possible presence of Salmonella species.
Confirmation: Any such suspected colonies must be confirmed using specific identification tests.
Test for Staphylococcus aureus
Streaking: After the incubation of Solution A is complete, take a loopful from Solution A and streak it onto a Mannitol Salt Agar (MSA) plate.
Incubation: Incubate the MSA plate at 30°C to 35°C for Not Less Than (NLT) 72 hours.
Interpretation & Morphology: Look for the following characteristic colonial morphology:
Observation: Yellow or white color colonies surrounded by a yellow zone indicate the possible presence of Staphylococcus aureus.
Confirmation: Any such suspected colonies must be confirmed using specific identification tests.
Compliance: The sample complies with the test if:
No such described colonies are present, OR
The subsequent identification tests return negative results.
2. Test for Pseudomonas aeruginosa
Streaking: After the incubation of Solution A is complete, take a loopful from Solution A and streak it onto a Cetrimide Agar (CA) plate.
Incubation: Incubate the CA plate at 30°C to 35°C for Not Less Than (NLT) 72 hours.
Interpretation:
Observation: Any growth of colonies indicates the possible presence of Pseudomonas aeruginosa.
Confirmation: This must be confirmed by further identification tests.
Compliance: The sample complies with the test if:
No colonies are present on the CA plate, OR
The subsequent identification tests return negative results.
3. Enhanced Negative Control Procedure
The negative control must be run simultaneously alongside the sample for each media type used.
Filtration: Filter 100 mL of 0.1% sterile peptone water through a separate sterile membrane filter.
Critical: You must use a membrane from the same lot as the one used for sample filtration.
Inoculation: After filtration is complete, transfer the membrane filter into 100 mL of Soybean Casein Digest Medium (SCDM). Mix the sample thoroughly.
Incubation: Incubate the medium at 30°C to 35°C for Not Less Than (NLT) 24 hours.
Neutralization Protocols for Treated Water Samples
| Disinfectant Agent | Neutralizing Chemical to Add | Volume Per 250 mL Container | Addition Method (Choose One) | Container Labeling Requirement |
| Chlorine Dioxide Treated Water | 3% Sodium Sulfite | 2.8 mL | • Before Sterilization: Add directly to the bottles prior to autoclaving/sterilizing. • Post-Sterilization: If using pre-sterilized/sterilized bottles, add the solution strictly under a Biosafety Cabinet (BSC) before sampling. | Add a suffix to the container label clearly indicating the addition of Sodium Sulfite. |
| Chlorine Treated Water | 3% Sodium Thiosulphate | 0.2 mL | • Before Sterilization: Add directly to the bottles prior to autoclaving/sterilizing. • Post-Sterilization: If using pre-sterilized bottles, add the solution strictly under a Biosafety Cabinet (BSC) before sampling. | Add a suffix to the container label clearly indicating the addition of Sodium Thiosulphate. |
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