Training within the Microbiology Laboratory Dr. Tim Sandle Timsandle@btinternet.com www.pharmig.blogspot.

com Introduction The training and assessment of staff working in a microbiology laboratory is of great importance, both to give confidence in the results produced by the laboratory and to satisfy regulatory inspectors. Guidance in developing a suitable training and assessment scheme is limited. There is a mass of published material relating to training in general, and there is a reasonable amount of published material related to training of staff in clean room behavior. There is little, however, devoted to the training and qualification of staff working in the microbiological laboratory itself. The importance of training is outlined by a number of regulatory documents, including the EU GMP Guide and by the FDA. With EU GMP, Chapter Two (2.8.12) emphasizes the importance of training: Basic training and practice on GMP Newly appointed personnel require basic training Training should be periodically assessed Training programs must be approved by the Head of Quality Training records must be kept Special training is required for those working in cleanrooms and for microbiology

With the latter point, special importance is given to the need for microbiologists to be trained and for those working in cleanrooms, where knowledge of disinfection practices and hygiene is paramount. Training is referenced in the FDA Code of Federal Regulations Part 21, as well: Training must be for particular operations Training must be undertaken by qualified individuals Training must be on a continuous basis More specifically for Microbiology, the 1993 guide for the inspection of microbiology laboratories indicates that microbiologists need extensive training and experience, especially due to difficulties in interpreting data and the methodological limitations of the common test methods.
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As for the training program itself, a recent conversation between the author and an MHRA inspector led to the inspectorates view of best practices for a training program consisting of: Documentation A formal program A mix of theory and practice To be competency based To be ongoing To be interactive To be challenging To have set pass and fail criteria

These references to training notwithstanding, there are a general lack of practical advice, which results in a variety of different training schemes across microbiology laboratories. The training of microbiology staff is a subject worthy of greater research. There is probably a need for a survey of industry common practices and standards. The aim of this article is to initiate the process and to consider some areas of training within the pharmaceutical microbiology laboratory. Before examining specific techniques, the following is taken for granted: All staff have a training folder in which to record the outcome of training (that is, all training is documented) Training is against a procedure Training is delivered by staff who are competent to do so

In designing a training scheme, a consistent approach is desirable. This will allow processes to be completed and to allow staff to understand what is involved. In terms of a training process, the following can be adopted as a model (assuming a satisfactory pass is obtained at each stage): Trainee observes test being conducted by a qualified staff member Trainee performs a number of practice sessions, using trial material, where a pass or fail can be obtained Trainee is assessed Trainee performs tests on live material under supervision, and each test record is counter signed

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Trainee undergoes a final assessment by an independent person (that is, someone other than the trainer)

Such a model may not be suitable for every technique or for each situation. The term under supervision is one which will vary from laboratory to laboratory, from close scrutiny to a supervisor being based on another bench in case of any problems arising. Assessments can take the form of: Observation against a series of set criteria (such as, if a procedure states a requirement for vortex mixing for one minute, is such an activity correctly followed?) By way of written assessment (such as, a questionnaire or multiple choice)

Several laboratories will give each trainee a rating or ascribe him or her level of training. This allows an auditor to understand what the trainee can and cannot do. An example of such a scheme is: Level 1 Level 2 Level 3 Level 4 = = = = Training has started. Trainee is observing only. Trainee is currently practicing with trial material only. Trainee has passed the first assessment. Trainee is testing the Product under supervision. Trainee has passed final assessment.

General Microbiology It may be self-evident, but staff working within a microbiology laboratory must have knowledge of basic microbiology. The extent of the knowledge may depend upon the level of experience and qualifications of the staff employed. It cannot be taken for granted, from the authors experience, that all graduates are of equal knowledge. A microbiology technician should have basic knowledge of the following:

Knowledge of bacteria and fungi commonly found in clean rooms. Knowledge of disinfection and sterilization and the appropriate handling and disposal of infectious materials. Knowledge and ability of basic laboratory techniques: swabbing; pour plating; subculturing; taking finger plates; settle plates; and active air samples.

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Knowledge of how to prepare and interpret Grams and other basic stains. Knowledge of how to recognize common bacterial species using conventional biochemical tests and commonly used kits.

The list can be added to in order to meet the needs of particular organizations. Turbidity observation Microbiology staff is often required to examine culture media for microbial growth. Arguably, the most difficult is the examination of broth cultures for turbidity, especially when the growth is not copious. The need is therefore for both an eyesight test and a demonstration that staff can distinguish microbial growth from other sediments, deposits, protein flakes and so on. The test is particularly important for staff that is required to inspect and read off sterility tests and other broth cultures. The need for a growth eyesight test is listed in the PIC / S 1999 guideline. In the context of the document, it refers to a test for personnel who inspect final products and containers from media simulation trials. A turbidity observation test could consist of spiking several bottles of broth with a range of different bacteria and fungi and different concentrations (from a concentration of >1 x106, which is just about discernable with the naked eye). The bottles would require inactivating for health and safety reasons and to suspend growth which could be performed by autoclaving. Care needs to be taken that this does not render the broth medium a different color or appearance. These spiked bottles would then be marked in some way by the trainer and mixed with a much larger number of unspiked bottles. The trainee would need to inspect all bottles against an artificial light source and successfully detect the ones which contain the microbial cultures. A suitable acceptance criteria should be set. This could be 100 110%, where the argument would be that all spiked bottles must be detected, but if the trainee was to detect a low number that were not spiked, this would not be as big an error as missing any potentially contaminated product. Bottles wrongly identified could then be checked with a supervisor.
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Repeatability A repeatability test is a test of pipetting technique. This involves performing several replicates of a microbial suspension at a countable test range. A statistical test is then used to assess the closeness of the results to an expected value or the degree of deviation from a mean (such as chi-squared or standard deviation) (Sandle, 2001). Sterility Testing A review of training in sterility testing is one of the most commonly asked for areas by regulators. In constructing a training program--once the basics of the test have been mastered outside of the sterility test facility--the trainee should perform trials on a minimum of three previously tested products (therefore, already passed as sterile) or three lots of sterile broth. The trials should include all manipulations of the product required, including reconstitution, neutralization, rinsing, and so on. All of this should be carefully observed by the assessor. The sterility test trials should also include full environmental monitoring and negative controls. Each trial must pass the sterility test in order for the final competency to be approved. Environmental Monitoring An assessment of environmental monitoring techniques is primarily by observation and an assessment of any unusual results. Observations will include: The loading and positioning of an active air sampler (particularly ensuring that fingers do not touch the agar plate or strip) The removal of the lid from a settle plate When taking a contact (or RODAC) plate, ensuring that the plate remains on the surface for the correct time and that an appropriate amount of pressure is applied (or if a mechanical sampler is used, that this is operated correctly) The correct surface area is taken when swabs are taken, and that they are plated out correctly (especially the rotation of the cotton bud across the surface of the

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plate) or dissolved sufficiently prior to membrane filtration. Using sterilized surfaces can be used to observe swabbing and contact plates in relation of aseptic technique. However, this can involve further investigation should the trainee recover one or two colonies post-incubation. Plate counting The simplest way to assess plate counting is for an experienced technician to first count a minimum of three plates with a relatively high number of counts (and then to wipe away any markings made from the surface of the petri dish). The trainee would then recount the plates. This can be performed using a colony counter, automated plate counter or simple tally counter and an artificial light source. The respective counts can then be compared and acceptance criteria (such as 95 105%) applied to the trainees results. The test can be combined with the test for repeatability described above and can be further enhanced by the trainee performing serial dilutions and preparing the plates to be counted.

Aseptic Operations in Clean Rooms For staff involved in aseptic manipulations (and many microbiology staff are frequently required to enter and operate within higher-graded clean rooms in addition to the basics of aseptic technique required for sampling and testing), the draft FDA Guidelines (2003) set out the elements of a training package. These include: Aseptic technique Clean room behavior Basic microbiology (including sources of contamination and the importance of sanitization techniques) Hygiene Gowning
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Much of this is assessed by observation from an experienced member of the staff. It is also important to emphasise that this training requires regular updating. The criticality of such training is further reinforced in the PIC/S guidelines, which state: The routine training of personnel who work in a controlled environment needs special emphasis as people are potentially one of the main sources of micro-organisms in the environment. (PIC / S, 1999)

The important areas of training in aseptic techniques are: the sanitization of gloves; moving slowly and deliberately; avoidance of disrupting the path of a unidirectional airflow; approaching products from sideways and not from above, and so on (Kadidlo and Loper, 2003). The use of video recordings is useful when demonstrating clean room behavior (such as avoiding turbulence within the critical zone). Sampling Techniques One of the most fundamental aspects of microbiological testing is, because it is the basis of obtaining accurate test results. A good definition of aseptic sampling is provided by Singh (2004): Aseptic technique is the technique involved in removing a sample as well as preventing contamination of sample and remnant simultaneously. The importance of aseptic sampling is detailed in the Code of Federal Regulations: Sterile equipment and aseptic sampling techniques shall be used when necessary. The main reason for aseptic sampling failing is inadequate training. A training scheme will need to cover avoidance of poor sampling habits and means to avoid the shedding of microflora from the body. Poor sampling habits can include the use of nonsterile equipment or the improper use of the equipment. Items to include are: Use of sterile equipment Use of containers and bottles with narrow openings Transfer equipment should not touch the side of the containers during transfer Sample containers should not be overfilled Where items are placed in bags, there must be room within the bag to fold the back over two to three times before closing Conversations must be avoided whilst sampling

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Further guidance is contained within CFR 211.84.c(3). It is essential that the concept of aseptic technique is taught first, and that protective measures (UDAF, masks, gloves, and so on) are seen as additional protective measures and not things that protect against poor technique. One training method is using sterile broth to mimic aseptic transfer. Many commercial test kits are available, and these provide a qualitative pass or fail indicator. Where microbiology staff are required to take environmental monitoring samples in clean rooms, the primary method for qualification is by participation in media simulation trials. Gowning Gowning tests are important to assess access to a sterility testing facility, process area or aseptic filling suite. Before a gowning test is conducted, the trainee will require a medical assessment. This should be performed by a qualified occupational health practitioner and may involve taking swabs of the trainee in order to assess if they are a high shedder of objectionable microorganisms. A typical gowning test would consist of an observational assessment and environmental monitoring samples. The observational assessment will consist of a check list of behavioral items. The environmental monitoring samples will include air samples (active air samples and may be settle plates), gown contact plates and finger dabs. Microbial Identifications The main way to assess microbial identification techniques is by using cultures unknown to the trainee. Once the trainee has been deemed to have adequately mastered the techniques, they can be presented with a number of unknown cultures (the cultures being known to the trainer). The trainee would then proceed to perform Grams stain, associated tests and biochemical tests, in order to speciate the cultures. In practice, the use of typed cultures from a culture collection is preferred over environmental isolates, because they are less prone to phenotypic or genotypic variations, which provides greater consistency when comparing test results. Many automated biochemical tests provide data relating to the closeness of the obtained profile to a database. The acceptance criteria from this can be part of the competency assessment.

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Some test centres offer identification proficiency schemes. Many of these are food research centers, although some of the preparations are not always suitable to pharmaceutical microbiology laboratories. Water Testing Water samples are tested using the membrane filtration method. In assessing membrane filtration techniques, the UK Drinking Water Inspectorate Guidelines (2000) details the assessment of the technique by comparing one hundred duplicate tests against a table of 95% confidence intervals. The test results from plate 2 should fall within the expected range of test 1. Therefore, the trainees technique for mixing, diluting and filtering test samples can be assessed. The water used for the evaluation should yield counts in order for the results to be statistically significant. The use of WFI is not, therefore, appropriate due to the expected low counts. Similar tests are recommended by the US EPA and the AOAC. Pour Plates An assessment of pour plate technique can be performed by testing in-process or raw material samples previously tested by a competent technician. This can be a very variable approach, given the distribution of microorganisms in liquids and solids, and the fact that populations may have grown or died over time. This has also been a problem in attempts to develop proficiency schemes for some years where cultures with a known count range are difficult to stabilize. An alternative approach would be to use sterile solutions, although these will only test the trainees aseptic technique and not their ability to recover known counts. A better assessment is from the repeatability test (see above) or by performing duplicate plate counts. These can be compared using 95% confidence intervals (such as the table in USP <1227>) and give an indication of pipetting technique. Retraining One of the more contestable aspects of training is the frequency of retraining and requalification. Some laboratories consider this automatically annual, whereas others use negative (and sometimes positive) controls and see this as a sufficient form of an ongoing check of a technicians technique.

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A further point to consider is what is meant by retraining. Is this something that can be reliably covered by a questionnaire? Is it the full training repeated? Can certain areas be deemed mandatory? Of the areas explored above, pipetting technique and eyesight tests could fall into this category. Another approach is to re-rain with each new version of an SOP. With most documents requiring review once every one to three years, this could be used to determine the frequency of any retraining. Retraining need not be full training (as in use of trial material and observation) but can be by way of questionnaire or some other test whereby the supervisor can be satisfied that the technician retains a good understanding of the technique. Summary and the Way Forward This article has briefly surveyed some of the training considerations for staff working within a pharmaceutical microbiology laboratory. The points raised in the article are designed to promote ideas for constructing a training scheme. They are not intended to be prescriptive. Following on from general training are wider issues pertaining to recruitment and selection. There is a need to develop a scheme for a registered or chartered Pharmaceutical Microbiologist. This could be gained by competency (for long time experienced staff) or by course work (for new entries into the pharmaceutical microbiology community). There could also be two levels for technical staff and managerial staff. Similar schemes exist for other disciplines that have professional bodies (such as the PMFG for food microbiology).

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References Drinking Water Inspectorate (2000): Comparison of Methods for Drinking Water Bacteriology Cultural Techniques at: http://www.dwi.gov.uk/regs/infolett/2000/pdf/il500pro.pdf Environmental Protection Agency, http://www.epa.gov/nerlcwww/index.html microbiological methods at:

FDA (2003, draft): Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing Current Good Manufacturing Practice Kadidlo, D. and Loper, K. (2003): Aseptic Processing, Tech Talk, Volume 10, No.4, publication of the US Cell Therapy organisation PIC/S Draft Recommendation on Aseptic Training / 17-Feb-1999 Professional Food Microbiology Group (PMFG) at http://www.ifst.org/pfmg.htm Dalhouse University, Halifax, Nova Scotia, Canada http://microbiology.medicine.dal.ca/people/forward/kf_train.htm Sandle, T. (2001): Repeatability and Precision - Necessary Factors in Standard Testing?, PharMIG News, No.5, August 2001 Sandle, T. (2010): Best practices in microbiology laboratory training in Hodges, N. and Hanlon, G. Industrial Pharmaceutical Microbiology: Standards and Controls, Supplement 11, ppS11.1 to S11.16 Singh, G. G. (2004): Advantages of Practising Aseptic Technique When Distributing Test Samples, American Pharmaceutical Review, internet article reprint: www.americanpharmaceuticalreview.com

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