MICLAB-055 Microbiological Monitoring of Plant Water Systems

1. General

1.1.         Bioburden and Endotoxin testing is conducted on water collected from at least one sample point from the cold WFI loop each day, with each point of use tested at least weekly according to the schedule.  All other grades / types of water are to be sampled and tested once per week.

2. Sampling Procedure for Bioburden and Endotoxin Samples

2.1.         Bioburden and Endotoxin samples are to be collected at the same time.  This ensures bioburden and endotoxin sample results can be compared.

2.2.         Bioburden samples should be taken first.

2.3.         Bioburden Sampling

2.3.1.     Sterilized bottles should be labelled with the outlet point, the date and time the sample is collected.

2.3.2.     Sampling must be conducted in employing good aseptic technique.

2.3.3.     Spray inside and outside of sample port with 70% IPA.  If a hose is attached to the outlet, sanities the end of the hose using the same procedure.

2.3.4.     Allow 70% IPA 30 seconds contact time before flushing.

2.3.5.     For WFI (Distilled Water), Purified Water and pre-treatment water, open sample valve and flush at least 1 liter of water.  Collect flush water in a bucket or flush directly down the drain were possible.

2.3.6.     Where a Hose is attached to an outlet in production (the use point), the water should be sampled through that hose if possible.

2.3.7.     Without stopping the flow of water, throttle back the flow of water until a laminar flow is obtained and collect the required volume.

2.3.8.     Bioburden samples are to be collected in pre‑sterilized bottles

2.3.9.     If an endotoxin sample is required as well as bioburden sample, proceed as per Section 2.4 once bioburden sample has been taken.

2.3.10.   Spray the outlet port with 70% IPA after the sample is taken and leave to air dry.  The time the sample was collected should be recorded on the container label and on the schedule form.  Any observations or deviations should be recorded in the comments section in the schedule form.

2.3.1.     Bioburden samples must be refrigerated upon return to the laboratory and tested within 24hrs of sampling.

NOTE: Distilled water should not be sampled when the temperature of the water exceeds 50°C.  The exception could be Hot WFI.  When sampling from the Hot Water Loop in the always wear heatproof gloves.

NOTE: All samples taken from outlets on Purified water systems (i.e. pre-treatment water and purified water) must be sampled on the same day in order to obtain a clear snapshot of the water quality.

2.4.         Endotoxin Samples – Sufficient sample must be taken to enable a retest to be performed. Endotoxin testing will only be performed on WFI on routine basis.

2.4.1.     Label 7ml non- pyrogenic, sterile polystyrene vials with the outlet point, date and the time the samples are taken.

2.4.2.     Rinse the 7mL vial and cap once with the water to be tested before collecting the sample.

2.4.3.     Fill vial three-quarter full with water and cap.

2.4.4.     Where testing is conducted within 24 hours of collection, samples should be stored in refrigerator between 2-8°C.

2.4.5.     Samples should be stored frozen if testing is not going to take place within 24hrs. Samples must be thawed out prior to testing.

3. Bioburden Testing

3.1.         Test Method

3.1.1.     Sterilized equipment required for the test should be used no longer than 48 hours after autoclaving

3.1.2.     Conduct the testing in the laminar flow cabinet.

3.1.3.     Under the Micro lab laminar flow cabinet, aseptically transfer the required volume, from the sterile bottle to sterilized filter cups.

3.1.4.     Filter the sample through a 0.45m membrane filter, and rinse with 150mL of sterile Peptone water.  Place the whole filter carefully onto an R2A plate.

3.1.5.     Include a negative control for each test session by filtering 150ml of Peptone water through a 0.45m membrane filter and place onto a R2A plate

3.1.6.     Incubate all plates inverted at between 30^oC – 35^oC for 5 days and examine and count the number of colonies present.

3.2.         Results

2.3.1       Results and media batch details are to be entered into the log book. Enter the negative control result in the comments for each sample test.

2.3.2       Enter Time Tested under the Comments in the log book.

2.3.3       Growth on the sterile control is to be evaluated in the case where growth in a test sample meets or exceeds the alert limit.

2.3.4       When entering the results, the technician is to enter their signature (sign on) into the log book.

2.3.5       See section 5 for alert and action limits.

2.3.6       If any spore formers are isolated from a distilled water outlet, their heat resistance must be evaluated according to MICLAB 065 The Alert Level for D-value of 1.5min.

2.3.7       Manager or appropriate delegate will prepare weekly reports for review to ensure all results have been reviewed and approved.

4. Endotoxin Testing of WFI (Distilled Water)

4.1.         WFI is to be tested by either the LAL- Gel Clot Test Method (MICLAB 080) or the KCA Test Method (MICLAB 085)

4.2.         WFI has an endotoxin Action limit of ≤0.25 EU/mL (the Action level).  There is an Alert level of >0.125 EU/mL 

5. Bioburden and Bacterial Endotoxin Alert and Action Levels

5.1.         In the event that the Alert or Action Level is exceeded for either Bioburden or Endotoxin results, initiate a laboratory investigation as per MICLAB 110. 

6. An example of Diagrammatic Representations of a typical purified Water Systems

6.1.         Purified Water systems and location of Bioburden sampling points.

7. Bioburden Waste Tank Water Sampling

7.1.         Sampling

Waste tanks are usually in the non sterile area.  Trained Microbiology staff are responsible for sampling from these machines.  Sampling is to be carried out using a sterile 10mL pipette into a pre-sterilized 20mL bottle.  Sampling of machine waste water tanks may be conducted as part of a larger investigation.  Sampling of waste water tanks is not routinely required.

7.2.         Test Method

Filter appropriate volume in order to determine count from wast water. Follow procedures outlines for routine bioburden testing of water samples (Section 3.0)

7.3.         Results

After incubation, the total count for each machine is recorded in appropriate log book.

Alert level 2cfu/0.1mL

Action level: 5cfu/0.1mL

Further identification of isolates should be assessed on case by case basis, depending on the reason for conducting sampling in the first instance.

8. Clean Steam Sampling & Testing

8.1.         Sampling

8.1.1.     The microbiological quality of clean steam is to be tested on an annual basis as per validation protocols.

8.1.2.     Samples are to be collected by a trained and competent operator as listed below.

1 x 500mL condensed steam bioburden sample, in sterile Schott bottle.

1 x 3ml condensed steam pyrogen sample, in de-pyrogenated sample container.

8.2.         Test Method and Results

8.2.1.     Clean steam is to be treated as WFI for the purpose of testing and should be tested for Bioburden and Endotoxin as outlined in Section 3.0 and 4.0 above.

8.2.2.     Record results directly into Validation protocol. Any colonies if present are to be Gram stained and identified to at least Genus level. WFI Alert and Action levels apply to clean steam.

8.3.3       If spore forming organisms are isolated, their heat resistance must be evaluated according to MICLAB 065

Alert level: D-value of 1.5min.

9. OOL/OOS Result Actions

9.1.         Initiate a Laboratory Investigation as per MICLAB 110. Microbiology Laboratory Investigation and Retest Procedure for Atypical Results and Out-of-Specification Results. Initiate a phase 1 investigation and record details in the investigation form.

9.2.         In the initial instance of recording an OOL/OOS result, Allocate the plate and colony types a colony identification number as per procedure MICLAB 070. and record the Identification number in the comments. Record the identification numbers on the isolate identification form Form 675 and complete the initial required sections of the form.

9.3.         Colony types are to be gram stained and colony and gram morphologies recorded in form Form 675. Gram Negative rods isolated from Chiller, purified and WFI water are to be identified to species level.

9.4.         Alert the laboratory manager of the OOL/OOS breech

9.5.         Conduct 3 consecutive repeat/follow up samples.  In some cases due to a five-day incubation, follow-up sampling may occur as part of routine sampling. If this is the case then the collected sample results may be referred to as part of the repeat/ follow up investigation.

9.6.         Upon completion of Phase 1 Investigation, if the OOS result is deemed to be valid raise a Deviation Report (DR) and refer to the investigation report.

9.7.         A DR should also be raised if the following occur:

Three or more sample points on the same ringmain on the same sampling date meet or exceed ALERT Level (Purified and WFI water only)

Three consecutive results from a sample point meet or exceed ALERT level.

The sample point meets or exceeds the ACTION level.

9.8.         Under normal conditions Alert level excursions will only require organism identification, however action limit excursions require further investigation to determine a definitive cause and recommend corrective and preventative actions, (CAPA).

9.9.         Summary Sheet – Recording Quick Reference

10. Investigation task check list

11. Trending

11.1.      Graphs are to be generated for WFI on a quarterly basis and for all other sample points on a 6 monthly basis.

12. Summary of Changes