Biological Contaminants: Strategies of Investigation and Control
By Robert W. Powitz, Ph.D, MPH, RBSP
Microbial surveillance is a tool to evaluate the effect of controls on the program or process within a given environment. A program to assess the microbial environment of a given facility can serve as an adjunct to the sterilization or decontamination program as well as the microbial quality of products that are aseptically processed. Microbial surveillance can be targeted to systems involving air, materials, facilities, equipment, personnel, and water.
Aerobiology is the science of the aerial transport of microorganisms and other microscopic biological materials, together with their transfer to the air, their deposition and the ensuing consequences for life forms, including the microscopic entities themselves, as well as product contamination. Airborne microbes need to be considered in terms of their biological and physical attributes. The aerobiological pathway involves the take off, aerial transport and subsequent landing.
In the airborne state, the survival of microorganisms is affected by many environmental factors. Under natural conditions, many of these factors operate simultaneously. The fragility of microorganisms depends on species and physiological conditions, while the magnitude of stresses depends on the processes by which they become aerosol. Once airborne, microbes either become desiccated or hydrated, depending on whether they were originally wetter or drier in their initial growth site. Under natural conditions, microbial aerosols are also exposed to radiation, oxygen, ozone and various pollutants, all of which are potentially lethal. Therefore, an important feature of microbial assessment is that some microorganisms, injured as a result of these stresses, may recover when they are placed in a suitable growth environment.
The monitoring of aerosols occurs in virtually all aspects of work with the airborne state, whether measuring particle- size distribution, particle concentration, diffusion rates of aerosols through buildings, efficiency of safety systems, or quality assurance. These diverse needs are matched to aerosol monitoring methods. All aerosol samplers must be judged in terms of the ability to physically collect microbial aerosols, while minimizing sampling stresses so that biological activity is not impaired. Observed results of sampling will depend on the precise sampling method used. Therefore, it is necessary to investigate the effects of any given sampler in each situation where it is applied. In general, aerosol collection devices that have the lowest shear forces yield samples of microorganisms with the highest viability. However, these samplers usually have the lowest physical efficiencies with respect to numbers of airborne particles collected. All aerosol samplers need to be calibrated in terms of flow rate and collection efficiency as a function of particle size and shape.
The sampling strategy used to monitor microbiological aerosols differs from the accepted strategies for monitoring other environmental contaminants. While the objective of any sampling program is to produce a set of samples representative of the source under investigation and suitable for subsequent analysis, there are no duplicate samples in assessing the bioload of air. While a sample must by typical and representative, it needs to be chosen so that it has the same qualities or properties as the material under consideration, namely the intramural air. Sample size must be chosen with respect to parameters and the requirements and/or limitations of the analytical method. Therefore, to obtain a truly representative assessment of the microbial constituents of air, both viable and non-viable methodologies will have to be employed simultaneously.
Strategies for a microbiological surveillance program
(1) Define the problem. Recognizing and defining the problem is the first step toward building a surveillance program. (2) Define the environment, including primary and secondary containment, air balance zones, air filtration parameters, treatment of liquid and gaseous wastes, room layout and traffic control, construction materials and communication systems.
(3) Establish contamination control criteria. How many organisms are allowed? What type of organisms are they?
(4) Employ techniques of control. What are the facility design features: primary/secondary/tertiary? How is equipment contained?
(5) Employ microbial testing and surveillance methods. These include: air sampling, surface and component sampling, physical and chemical tests and measurements, testing filters and water and finally, leak testing. To select a site for routine surveillance consider the following:
Specifically where could microbial contamination most likely affect product or process quality?
Which sites would be most likely to have the heaviest microbial proliferation during actual production?
Should judgmental, systematic or random sampling design determine site selection?
Which sites would represent the most inaccessible or difficult areas to decontaminate?
Can areas adjacent to target sites serve as indicator sites?
How can microbes be dispersed in the environment?
Environmental bioloads
There is no uniformity in environmental bioloads. Each environment differs in the quality and quantity of microbes, which do not survive well outside their natural environment or growth sites. Each environment can be considered a separate biosphere; that is, each has its own characteristic bioload, which is in a constant state of flux caused by death, growth and environmental forces such as humidity.
To sample the environmental bio-loads, or bacteria, a sampling device must be able to capture bacteria ranging in size from 0.5 to 5.0 microns. An air sampler must be gentle and compatible with viability. Microbes should not be subject to mechanical disruption and desiccation. Most microbes are usually associated with other particles, therefore, the sample must be able to collect up to 10-micron size particles. Particles can have one bacterium or many bacteria. Because variations in microbial types and fluctuations in numbers happen over a short period of time, air samples must be taken in a systematic and sequential manner. There is no such thing as a “duplicate” air sample. A few rules that should be followed when conducting environmental sampling are:
Samples that are not representative of the population are of little use.
Poor sample collection procedures yield unrepresentative samples and contribute to the uncertainty of the analy-tical results.
Sampling errors and analytical errors occur independently of each other, so sampling-related errors cannot be accounted for by laboratory blanks or control samples.
Contamination is a common source of error in all types of environmental measurements.
Bioaerosol sampling devices
For viable samplers, the devices listed below can be used.
Impingers. Simulate nasal passage; efficient for respiratory range of 0.8 to 15 microns; good for fragile microbe recovery; some losses through splashing and impact trauma.
Sieve samplers. Particle size differentiation, the single stage allows particles of all sizes to be sampled together; more than one viable particle can be impacted on the same sites; the agar, directly under each hole of a stage, can rapidly dry–needs short sampling time or high RH; Petri dishes need to be filled precisely to give correct plate-to-agar surface distance.
Sequential samplers. Slit-to-Agar. Colony counts may be correlated to sampling rate and time; no size differentiation; drying of agar likely with long sampling runs.
Centrifugal samplers. Questionable flow rate and efficiency; they are used as their own control; limited to the types of organisms studied.
For non-viable samplers, there is an impaction sampler, such as the Burkard hand-held device for volumeric spore assays and light microscopic examination. n
Robert W. Powitz is the principal sanitarian at R.W. Powitz & Associates, P.C. (Old Saybrook, CT), an environmental health and safety consultant firm.
Powitz is presenting a seminar on biological contaminants at CleanRooms `97 East in Boston, March 3-5. For information on the CleanRooms `97 East Conference Program, please call (603) 891-9267.
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Figure 1. A) All known bacteria can be categorized into one of three “basic shape” groups as shown. B) The basic shapes of bacteria can vary. Shown are the coccobacillus, or ovoid spheres, and vibrios, or curved rods.