It is difficult to fully explain the need for knowledgeable microbiologists in the field of industrial microbiology. There are many microbiologists who have become testing experts, due to the many hours of time performing tests in their laboratory. This is a critical first step in developing true expertise; however, this kind of experience does not, in and of itself, develop true industrial expertise. Without exposure to other methods, alternate approaches, and regulatory challenges (external to the laboratory), true expertise cannot be gained. Indeed, the term expert is used too often by those who have become an expert at one aspect of microbiology in their industry.

True expertise is developed by the additional experience gained outside of the laboratory. Spending time in the manufacturing environment is necessary to help the microbiologist understand all that can transpire in the microbiological world. Microbiology tests performed in a laboratory often could be modified to be more appropriate for a specific product from a specific manufacturer once a better background of the manufacturing process is gathered. Involvement in industry associations, conventions and meetings also helps to develop expertise. There are few things as helpful to building expertise as working together on industry standards or guidance documents, sharing ideas and new technologies in a forum, or giving and observing presentations on the relevant topics.

It seems to be that many industries are losing needed microbiology expertise on the manufacturing side due to microbiology aspects becoming one of many routine assignments that is folded into quality, regulatory, or manufacturing. In some cases, this may be necessary due to the inability to maintain a full-time person devoted to microbiology, especially for smaller manufacturing firms. The disadvantage is that manufacturing (or industrial) microbiology as a discipline is consequently not addressed, unless an outside expert is involved in such cases. This often results in manufacturers relying on laboratory personnel for guidance on performing tests and interpreting data. Many laboratories are willing to provide this assistance, but it is often given based on what is commonly known or done rather than what is best for the specific product/process. Nonetheless, it is best if product manufacturers develop a good relationship with their laboratories, whether they are internal or external. Developing appropriate test methods and correctly interpreting bioburden and sterility test data must be a collaborative effort with the viewpoints of both the laboratory and manufacturing represented.

Disinfection, sterilization, and preservation processes do not always address all microbial issues that might be present in the production of acceptable product; some degree of control over the product components and manufacturing processes is required to allow the processes to yield consistent results. Bioburden testing is commonly performed on product obtained under standard manufacturing conditions in order to gather information (quantitative and qualitative) on the typical microbial load during or after manufacturing and packaging. Obtaining and trending these data help the manufacturer to demonstrate continued microbial control over an entire process; however, it is important to note that merely performing bioburden testing alone on a limited basis, for example, semiannually, does not constitute a robust bioburden control program.

Bioburden testing of finished product often results in reactive activities, meaning that the bioburden test data are used to determine if the process has been under some semblance of control over a specified time. The bioburden test on finished product is commonly implemented without any preliminary understanding of the contribution of microbial contamination from subprocesses or components during manufacturing. Use of bioburden testing in this manner is considered a single “end-point” test. If the data are acceptable, it is assumed that the entire process is under reasonable control. If the data are not acceptable, it could mean that any of the many upstream components and processes might have changed, or that certain processes—single or multiple—might be out of control. A thorough bioburden control program uses bioburden testing to understand and qualify higher risk components and steps of the overall process. These higher risk components and processes can usually be identified by a knowledgeable microbiologist who is familiar with the pertinent aspects of product manufacturing. The microbiologist establishes a testing scheme and gathers data either to demonstrate that the current practice is acceptable or to identify where attention is needed or corrections are appropriate.

An understanding of bioburden can be used in several situations such as the following:

Bioburden on a finished product is the sum of the microbial contributions from several sources, including raw materials, manufacturing of components, assembly processes, personnel, manufacturing environment, assembly/manufacturing aids (eg, compressed gases, water, lubricants), cleaning processes, and packaging of finished product. To control product bioburden, attention must be
given to the microbiological status of these and other relevant sources. Strategic implementation of bioburden testing allows the microbiologist to correct practices or issues before they affect finished product, and to go into full-scale manufacturing with a degree of knowledge that it will be successful. When this approach is taken, the bioburden test of the finished product consequently becomes confirmation of the effort previously completed, rather than a hopeful exercise that will demonstrate control.

Bioburden testing is commonly performed on solid products (eg, plastics and metals) by performing an extraction with a water-based solution, followed by testing all or a portion of the extraction solution using membrane filtration, pour plating, or spread plating. For liquid products, bioburden testing is commonly performed by using membrane filtration, pour plating, or spread plating. When using the membrane filtration method, the membrane through which the liquid has been filtered is placed onto an agar plate or an absorbent pad saturated with liquid medium. After filtration and plating, or pour or spread plating, the agar plates are incubated at a specified temperature for a specified period to obtain visible colonies, followed by counting the colonies (Figure 64.1). Membrane filtration is often a preferred method because it allows for testing a larger volume of extraction solution or product. Pour or spread plating are good methods for bioburden enumeration but are limited by the quantity of solution or product that can be tested per plate. Bioburden testing is commonly performed in an environment to prevent contamination, usually a laminar flow hood in a controlled environment room, but usually not in a cleanroom because that level of control is not necessary.

It is sometimes assumed that bioburden testing will provide results of all types of microorganisms present on or in the product. This is not realistic because there are some types of microorganisms that require specific or unique growth nutrients, conditions, or incubation times to replicate and form a visible colony. For this reason, it is important that a microbiologist who is knowledgeable in the specific raw materials and manufacturing processes be involved in test development. Based on that information, growth media type(s) and incubation conditions can be selected that are best suited for the various microorganisms expected to be on the product. Most often, this assessment results in selection of media and incubation conditions that are ideal for the majority of bacteria (eg, those from surfaces, humans, and water) and environmental fungi (ie, molds and yeasts). Cultivation and detection of more unique bacteria and fungi, specific viruses, protozoa, etc, is often not practical nor indeed necessary in most situations. Specific applications, such as bioburden testing for allograft tissues and animal serum for human use, might need to consider the detection of certain material-related pathogens.⁷

Change is ever-present in most manufacturing environments. With multiple personnel involved in different aspects of product and processes, and each one of the personnel tasked to improve what is in their control, it is inevitable that change will happen. In many cases, change is for the better, but it must be controlled such that other aspects of the product or process are not inadvertently, negatively impacted. Bioburden tests, along with some degree of microorganism characterization (eg, morphology, selective culturing, Gram stain), are commonly employed as part of the change control process and are effective in understanding if the change has an unintended impact on the process or product.

The validation of a bioburden test, in its strictest interpretation, is not usually necessary if classical microbiological methods are being used. The terms qualification or verification usually apply, but if bioburden validation is to be considered, as with most microbiological tests, it should be assessed in two different manners. Validation of a bioburden test method is conducted as part of the
laboratory’s preparation to perform bioburden testing. This type of validation is not specific to a product type but can include several general product types to understand the variability that occurs in the parameters of the overall test method. At this stage, items such as the limits of detection (LOD) and limits of quantification as well as other components of validation such as ruggedness, precision, and accuracy can be addressed. This validation provides the laboratory with valuable information regarding the different parameters of the bioburden test method and the variability that can occur. In microbiology, it is not expected that the variability be as small as might be expected in more precise sciences such as chemistry or physical analysis.

Once a bioburden test method has been validated, and when applying the test for a specific product, the product qualification process becomes the key factor, rather than the traditional components of a complete validation exercise. When addressing a bioburden test for a specific product, topics such as method suitability and recovery efficiency (see the following text) play a larger—and often exclusive—role compared to the other aspects of validation that are addressed in general validation of the test method. In the stage of bioburden qualification, it is usually also determined whether the entire product can be tested, which is called a sample item portion (SIP) of 1.0, or whether it is necessary to test a portion of the product (an SIP of <1.0). Most often, when testing a portion of the product becomes necessary, it is due to the large size of a product or to overcome inhibition of microbial growth in the test system. Whenever possible an SIP of 1.0 should be tested, and only when it is not practicable should an SIP <1.0 be considered. Some approaches (eg, when used in support of testing associated with radiation sterilization) require verification of the appropriateness of the SIP to ensure that the SIP is not too small to accurately represent the entire product, based on the purpose for the test.

Bioburden characterization is an underused practice in most industries. Where bioburden data are being gathered for health care products, it is often assumed that numerical data is the primary factor for evaluation rather than characterization data. Often, the need for, or the usefulness of, characterization is not fully understood, especially when microbiologists are not involved in the process. Characterization also requires an additional expense without—as it might appear—a direct, immediate correlation to outcome. Lastly, characterization data can be difficult for a nonmicrobiologist to interpret and trend, and therefore, the benefit can be difficult to demonstrate. Characterization should normally occur more frequently in the earlier stages of a manufacturing setting or process, to establish a baseline of the numbers and types of microorganisms present in the normal manufacturing scenario. Following that, characterization is an important key to demonstrating a controlled, stable process and product.

An understanding of the bioburden count alone is sometimes not enough to make an educated decision regarding interpreting microbiological data, assessing the potential impact of the bioburden to the product or process, or understanding the general microbiology of a product or process. Bioburden characterization is a broad term used to describe tests performed on the microorganisms after they have been recovered from the product or process. The most common methods of characterization are selection of specific growth conditions for groups of microorganisms, determination of colony morphology, Gram or other staining methods, and identification to genus and species.

The first step of characterization can include selection of specific growth conditions for groups of microorganisms. In some industries, it is common to employ use of selective or differential media to exhibit growth of specific microorganism types. Although this is considered characterization, it is sometimes too broad to be meaningful, depending on the purpose of the test. Certain types of media (Figure 64.2) or incubation temperatures can result in very specific outcomes. The important issue in using growth conditions as a means of characterization is to understand the desired end points and to ensure that the level of characterization provided meets those end points.

In the health care product industry, it is common to perform bioburden testing using different media and/or temperatures to encourage growth of both aerobic bacteria and fungi (molds and yeasts). This form of characterization helps the manufacturer gain a knowledge of the breakdown of microorganisms that are likely bacteria compared to molds. There is some usefulness in this knowledge, especially for trending bacteria and mold contamination over time. Less overlap and more differentiation will result when a different medium, specific for selection, is used for each microorganism type (eg, tryptic soy agar or nutrient agar for bacterial growth and potato dextrose agar or rose bengal agar for fungal growth). For bioburden testing in the health care industry, information regarding characterization to detect the presence of spores and anaerobes can also be considered. Testing for the presence of spores in product or environmental bioburden is sometimes performed in support of moist heat or ethylene oxide (EO) sterilization, where spores are considered more resistant to the process. Typically, spore testing is performed by applying high heat for a short period of time (eg, a heat shock at around 80°C for at least 10 min) to the bioburden extraction fluid to inactive vegetative bacteria, followed by plating and incubation.⁹ If testing for aerobic bacteria is also being performed on the same products, it must be understood that spores that grow after heat shocking will generally also grow without the heat shock in the aerobic bacteria test. Thus, although the spore selection test helps to distinguish the percentage of microorganisms that are spores, the spore test results should not automatically be summed with the aerobic bacteria test results, as the total number could likely be overestimated because the spores can be a subset of the aerobic bacteria.

Testing for the presence of anaerobes often results in a similar issue as with spores. Most health care products are composed of different types of synthetic materials, such as plastic and metal, where there is, first, no potential source for strict anaerobes (termed obligate anaerobes) and, second, little potential for survival of obligate anaerobes. With these products, an anaerobe test, if not properly conducted, will result in the growth of microorganisms that are facultative rather than obligate anaerobes, meaning that they can also grow in the aerobic bacteria test. Again, summing these two results, without a screening or differentiating step, could result in over estimation of the total bioburden as well as possibly the false assumption that the product contains obligate anaerobes. In the case of tissue or tissue-based products, there is a potential source for obligate anaerobes, therefore anaerobic testing could be warranted. Even in this case, the presence of facultative anaerobes is just as likely as that of strict anaerobes, so an evaluation is recommended prior to summing the results or assuming the presence of obligate anaerobes.

Another means of characterization involves documentation of the colony morphology, which includes visible aspects of the microorganism growth such as colony size, shape, and color (Figure 64.3). Colony morphology by itself is not usually sufficient for making decisions or trending, as there are both Gram-positive and Gram-negative microorganisms that are, for example, of similar size, shape, and pigmentation. Morphology comparisons are useful when looking for a specific microorganism, or for other general activities such as noting predominant colony types. Colony morphology is often accompanied by staining (most commonly a Gram stain) and microscopic inspection (cell morphology), which can prove to be a useful data point.
A microbiologist can use colony morphology and Gram stain information, along with an in-depth knowledge of the components and manufacturing processes, to assess potential source(s) of contamination, and, if needed, determine what kinds of mitigating activities can be employed. For example, Gram stains can provide information to assist a microbiologist in understanding whether the microorganisms comprising the product bioburden likely came from water, humans, or the environment.

Microbial identifications to the genus and species level are usually performed as part of investigations, for specific trending purposes, or to characterize predominant microorganisms for a general characterization program. There are many systems available for the specific identification of microorganisms, and it is not the intent of this chapter to go into any detail regarding these systems.¹⁰ These include various types of biochemical, lipid, and genetic identification techniques, where the genetic methods are currently generally considered necessary when establishing critical information that might be reviewed by regulatory bodies.

It should be mentioned that even though a quick search of a microbial genus and species might show that the microorganism in question can originate from a specific source, for example, animals or birds, it should not be assumed that such sources automatically relate to components or are present in the manufacturing area. Microorganisms have a way of populating multiple habitats, and of being spread from one place to another. Truly, they are a perfect example of how “life finds a way.” A microbiologist, armed with the genus and species information of a microorganism, is usually able to determine a likely source and, if needed, a potential means of prevention or elimination. For example, bacteria such as the Gram-positive cocci Staphylococcus epidermidis and Staphylococcus hominis are common bacteria found on human skin, whereas strains of Gram-positive rods such as Bacillus species are common dust or surface contaminants (Figure 64.4). Equally, many types of fungi (eg, species of Aspergillus and Penicillium) can be associated with airborne/dust contamination but may also indicate more serious problems such as water damage. Gram-negative rods, such as strains of Pseudomonas and Escherichia coli, are often associated with water contamination during the manufacturing process.¹¹,¹²,¹³