Microbiological Environments

MICROBIOLOGICAL ENVIRONMENTS Consultation / Laboratory Services

Expertise and reliability in Production/ Environmental Monitoring and General Microbiological Testing

Addressing questions from e-mail May 2, 2005:

1) Is there an acceptable value for the baseline that is recommended?

Baseline contamination levels are derived over an extended period of time. The data should reflect conditions that result from normal changes and operational variation, including shift and personnel changes, seasonal changes and routine re-positioning of equipment and personnel. A baseline therefore is essentially different for each facility/ site location and there is no “acceptable” baseline; your baseline counts will define the normal level of contamination at which your operation typically runs and against which excursions and trends above that norm will be measured.

Because the baseline sampling sessions intend to learn as much as possible about the facility and its contamination levels, the sampling plan should also be expansive– beyond the scope of what will eventually be the ongoing sampling plan; this is done so that all possible venues and harbors of contamination can be realized at this early stage. From the baseline data we will then select the ongoing critical or characteristically problematic sites for routine monitoring;

It is important that during collection of baseline data, glitches and spikes in data be carefully looked at before inclusion with the baseline data since to include them may falsely raise it; if there is a discernable cause for an aberrant data point, note should be made of it, the cause corrected and the point replaced with a statistically appropriate replacement value, such as the average of the other data points.

“ Zero” is not acceptable, even though nothing but zero’s is gathered over a considerable period. Statistical methods may be applied which deal with large numbers of zero’s; e.g. the Non-parametrics Tolerance Limits approach; here, using a large number of data points, Alert Levels are calculated at the 95th percentile by multiplying the mean times 3 and Action Levels at the 99th percentile by multiplying the mean by 4.6. Where even over an extended period of sampling only “0”’s have been obtained, a count of “1” may be assigned as the Alert Level and the U.S.P. – advised “3” as an Action level.

Although compendial data may suffice for the interim, empirical data should be used as soon as possible, since it may reflect that the operation is capable of running at levels consistently lower than the compendial limits and the Alert and Action levels should so be adjusted.

Note: Data uniformity is important; if during the collection of baseline samples, site-to-site data scatter or session-to-session variation is extreme, this may be an indication of either an uncontrolled air handling system or inconsistencies in operator habit/ behavior; this should be addressed by air-handler inspection/ correction and/or further training in gowning and aseptic technique before continuing with data collection. Generation of smoother data represents a true

177 North Commerce Way, Bethlehem, PA, 18017; Phone: 610-866-7272; Fax: 610-866-7287; E-mail: micenv@ microbioenv .com; www.microbioenv.com MICROBIOLOGICAL ENVIRONMENTS Consultation / Laboratory Services

controlled operational baseline level of contamination against which deviations and out-of- norm trends can be measured.

2) How is the baseline to be calculated?...consecutive days, every other day, weekly, monthly?

As mentioned, the baseline should include extensive data representing all inherent variability in the normally functioning facility over as long a period as is possible/ practical. The baseline is essentially the set of average data points for all points sampled over time under those conditions. The critical calculation involving the baseline data is the setting of Alert and Action levels based on it.

3) Do we need to establish standard deviation and confidence intervals?

Standard deviations and confidence limits are applied in calculating Alert and Action levels. With uncontrolled facilities that routinely give high counts, the data distribution is more likely to be normal and the Alert and Action levels are calculated as the mean plus 2σ and mean plus 3σ respectively. In tightly controlled facilities under Class 10,000 and lower, many “0”’s are generated and the data is skewed – as discussed in 1), the Alert Levels are calculated at the 95th percentile by multiplying the mean times 3 and Action Levels at the 99th percentile by multiplying the mean by 4.6.

4) Do the CFU’s have to be identified to determine type of organism? Or are we just counting colonies?

We like to profile the microbial population on each strip or plate by characterizing each colony type as to it’s Gram’s stain/ morphology; therefore, when there’s an Exceeded Concern Level, we have someplace to look to start our investigation –

Indication Staphylococcus/ Micrococcus – > personnel habits or gowning problems Gram negative rods – > water condensation, leaking, aerosols; possible hygiene problem Bacillus species – > dust, dirt/ floor traffic; possible air handling Molds – > influx of unfiltered air, mold from street clothing or mold-contaminated cardboard; water reservoir, e.g. incubator humidification Yeast – > possible outdoor air influx or clothing-borne, especially in late summer/ fall; possible human contaminant Diptheroids/ coryneforms – > poor air conditioning = uncomfortable, sweating personnel discharge from gowns

5) Is it recommended to benchmark the baseline? 177 North Commerce Way, Bethlehem, PA, 18017; Phone: 610-866-7272; Fax: 610-866-7287; E-mail: micenv@ microbioenv .com; www.microbioenv.com MICROBIOLOGICAL ENVIRONMENTS Consultation / Laboratory Services

If by “benchmarking”, you mean permanently establishing your first set of data as your baseline, the answer is “No”. As your facility grows in experience, training/ re-training, validating and re-validating its processes and experiences ongoing problem-solving, your baseline contamination level– that level at which your operation can consistently run– will over time.

Many facilities use a moving average, which leaves the high counts associated with of the “era of learning and inexperience” behind; as your cleanliness and aseptic capabilities increase, the baseline values against which you judge yourself must reflect that; as you continuously improve the cleanliness capability of your operation you must establish new, tighter levels against which to measure them so you can realize when you’re becoming microbiologically out of control.

6), 7) N/A

8) Addressing the portion of the question regarding “which location” should stand as the benchmark – and with the understanding that benchmarking is not recommended:

Your question tells me that you don’t understand that in most programs, each sample site has it’s own baseline – there isn’t one baseline value that’s established for all surface samples or another that’s for all air samples. In some cases for uncontrolled facilities, this may be applicable, but in controlled and critical operations, each routine site is judged against its own history – not collectively with others. It may be expressed “Laminar Flow Hood samples” collectively must meet a certain criterion, but in a typical data base, sites are treated individually. Note that this way, an out of spec result is an independent event, not a collective one and may be so addressed.

177 North Commerce Way, Bethlehem, PA, 18017; Phone: 610-866-7272; Fax: 610-866-7287; E-mail: micenv@ microbioenv .com; www.microbioenv.com