Contributing editor Tanuja Koppal, PhD, talks to Scott Sutton, PhD, principal of Microbiology Network, Inc., and Alison Buchan, PhD, associate professor, Department of Microbiology at the University of Tennessee, about the main sources and causes of contamination in a microbiology lab and how these problems can be minimized or eliminated. They discuss issues of setting up best practices and of availing themselves of new technologies and resources, while revisiting topics that seem to be more commonsense when dealing with general lab safety and hygiene.
Q: What are the main sources of contamination in a microbiology lab?
Sutton: My orientation is toward quality control for manufacturing, and in my opinion the main source of contamination of the product is the people working in the manufacturing areas— from those who are doing line operations to those collecting the samples. The other sources of microbial contamination are water that is used in the systems, improper cleaning procedures, and raw materials that carry a high level of the incoming bioburden. All of these can contribute to the contamination of the finished product.
Q: Can air quality contribute to contamination?
Sutton: The pharmaceutical area is guided by a lot of regulations, and one of them is the FDA’s aseptic guidance document, which details FDA expectations. It states that environmental monitoring is done in order to reflect the state of control of the facility and is not a measure of finished product quality. At least in a laboratory setting, outside of manufacturing, most contamination issues can be traced back to improper technique in the hood or the lab bench, and they aren’t due to the lab environment. It’s now an industry standard to perform all microbiological work in a hood. Although a well-qualified microbiologist can perform high-quality work on an open bench, if a problem did occur, it’s reassuring to know that the work was done in a closed and somewhat controlled environment in a hood. However, a lot of the time the distinction between a laminar flow hood and a biological safety cabinet [BSC] is lost. The purpose of the BSC is to protect the worker, and it is not necessarily designed to prevent contamination of the plates or product.
Q: How should lab managers go about setting up best practices and training personnel?
Sutton: The microbiology laboratory remains an area that is very operator dependent, and there is a lot of physical interaction between the technician and the sample. The operators are very involved in manipulating the plates, handling the samples, and interpreting the data obtained. The raw data is usually a technician’s interpretation of what is seen. This is very different from analytical chemistry, where the sample is often analyzed in a qualified piece of equipment. There is a lot more precision and accuracy in an analytical chemist’s world than there is in a microbiologist’s. Hence, the background, training and management skills that a manager brings to the microbiology lab largely determine how successful the lab is going to be. Especially in a microbiology lab, you need someone who can ensure that high-quality standards are being met on a daily basis. If the manager is strict and stringent about good microbiological techniques, then others take their cue from him or her. This is also reflected in an informational chapter in the U.S. Pharmacopeial Convention, USP Chapter 1117, called “Microbiological Best Laboratory Practices.” It includes a significant section on the training and academic qualifications of technicians and lab managers. They felt it important to insist that the lab manager be a subject matter expert in the field in order to ensure the success of the lab.
Q: Can you recommend some other resources that lab managers can go to for guidance?
Sutton: The Microbiology Network website does include a lot of resources such as white papers, a list of commonly used acronyms, and publications from consultants who are focused on regulatory microbiology and contamination control. If you are doing anything in a regulated industry such as pharmaceuticals, biotechnology, dietary supplements, personal care products, and cosmetics, I would first recommend reading Chapter 1117 in the USP to at least be aware of what the expectations are in a good manufacturing practices environment. If you are working in an aseptic arena or in environmental monitoring, I would definitely recommend the FDA’s aseptic guidance document. The USP has also recently published a draft of Chapter 1115, which is on the bioburden contamination control of non-sterile manufacturing. There is also the Book of Standards for microbiologists if you are in the personal care products or cosmetics industries. The medical device industry is influenced by CFR [Code of Federal Regulations] Part 820 and also by a lot of ISO [International Organization for Standardization] regulations. There are 21 CFR Part 11 for pharmaceutical, over the counter, and some of the biotechs and 21 CFR Parts 610 and 612 for biologics. There is so much information out there, and a good way to stay up to speed is by attending webinars and conferences.
Q: Are advances in technology making it easier for the lab manager?
Sutton: There is a big push toward rapid microbiology in terms of going away from tests that involve plate counts or growth and broth. However, they have not been very successful in getting into the pharmaceutical or medical device arenas. From a microbiology perspective they are very attractive, but from a corporate perspective their value and return on investment are unclear. Part of the problem is that frequently we don’t have the processes to really take advantage of the time savings in microbiological testing. We need to get to a point where we have our processes set to take advantage of the rapid tests, but I don’t think we are ready for them yet. However, technology can be very useful in some other areas. For instance, even a modest environmental monitoring program generates hundreds of data points per week, and trying to keep track of that on paper is impossible. There, technology such as databases can be used to analyze and trend environmental data in order to report the state of control of the facility. A well-designed laboratory information management system can also make a big difference in a microbiology lab.
Q: Do you think contamination will continue to remain a serious problem?
Sutton: I don’t see microbiological contamination as a serious problem across the pharma industry as a whole. Where there are insufficient resources and lab managers are not adequately trained or prepared, then it can be a problem, and those problems can be tracked by routine investigations. I am already seeing a great deal of improvement, particularly since the publication of USP Chapter 1117. We need to start paying more attention to the basic science of microbiology and techniques for aseptic manipulation of samples and tests. The way things are going to improve even further, in terms of contamination of the sample or the test, is when lab managers are better prepared to supervise the laboratories. The labs need the subject matter expertise of the managers [in addition to] their management skills.
Scott Sutton, PhD, is the principal of Microbiology Network, Inc. (http://microbiologynetwork.com), a company he started in 1996 as a means to encourage training and communications within the microbiological community. The Microbiology Network operates two email discussion groups—the PMFList (for pharmaceutical microbiology) and the PSGDList (for stability issues). With more than 80 publications and hundreds of presentations, Dr. Sutton is a recognized consultant and trainer with emphasis on GMP, investigations, environmental monitoring, and contamination control (for both aseptic manufacturing and non-sterile production facilities) as well as on microbiology laboratory audits and operations. He is an active author and speaker for the industry; supports ASM, PCPC, and PDA; and has served with the USP Analytical Microbiology Committee of Experts since 1993.
Alison Buchan, PhD, is an associate professor of microbiology at the University of Tennessee, Knoxville. Her research focuses on the roles of microbes in natural environments. Major objectives in Dr. Buchan’s lab include linking microbial taxa to critical ecosystem processes, exploring functional gene diversity and its relation to community structure and biogeochemical processes, and identifying novel enzymes and/or catabolic pathways. Much of her work focuses on the Roseobacter clade, an abundant, broadly distributed, and biogeochemically relevant group of heterotrophic marine bacteria. In addition, Dr. Buchan also examines viruses that infect the Roseobacter clade in order to understand how phage lysis influences Roseobacter populations and to understand the chemical nature of dissolved organic matter that is released due to phage activity. She is also on the faculty of UT’s Genome Science and Technology graduate program and the school’s Center for Environmental Biotechnology.
Contamination from the Lab into the Sample
“We are always worried about contaminants getting into our cultures,” says Alison Buchan, PhD, associate professor of microbiology at the University of Tennessee, Knoxville. “We often work with environmental samples and use molecular tools to ask what microbes are in our samples. So the last thing we want is airborne contaminants or nucleic acid contamination from the benchtop or pipettes getting into our samples and destroying the natural microbial community we are interested in investigating.”
Dr. Buchan’s lab works a lot with water samples collected from coastal and ocean environments, and they study the microbial makeup of these natural unmanipulated samples. She also uses environmentally relevant cultured bacteria and studies their genetics and physiology in the lab. “There is no one way that’s right to practice aseptic techniques, and often people feel strongly about a given technique,” says Dr. Buchan. “Probably the oldest and cheapest way to do things is to use an open flame on your bench to sterilize inoculating loops, openings of test tubes, etc. That’s what we use in our lab, and it is still common with a lot of microbiologists. As you move into larger labs, and in industry, they may use techniques that are more sophisticated, but it’s up for debate whether they are necessarily better. Biosafety cabinets that carefully control airflow are often used to minimize exposure to contaminants, but we use a laminar flow hood because the marine bacteria that we work with are nonpathogenic.”
Because the lab is worried about nucleic acid contamination from work surfaces or equipment, all samples are processed in the hood under an ultraviolet lamp to destroy any residual nucleic acids. “We are very careful in the field and in the lab when handling these environmental samples to make sure we don’t transfer materials or microbes from our hands into the samples.” (More details on the procedures used in Dr. Buchan’s lab are listed below.) People new to the lab have to work with the lab manager to learn and demonstrate the use of proper aseptic technique. “We do have specific guidelines set up depending on the types of samples people are working with. We always use pipette tips that have filters in them to avoid cross-contamination of samples. We wear gloves and use sterile forceps to collect and transport samples. Samples are frozen during transportation and once [they are] in the lab.”
The lab also works with viruses called phages that infect bacteria, and they have to make sure that these phages do not contaminate the lab environment and the bacterial cultures.
“Whenever we work on propagating the phage we use proper containment procedures and work in a hood wiped down with bleach and located in a separate room. We have found that the best way to minimize cross-contamination is to designate specific areas for specific practices. In our main lab we culture the bacteria, in an ancillary lab we propagate the phage, and then we have another lab where we do all our molecular work such as PCR. We also make sure that the lab notebook is not a source of contamination and have a separate area for record keeping.”
Dr. Buchan believes that being hands-on is the best way to make sure that nothing is jeopardizing the sample and what you do. “Think really hard about the types of samples you use and the technology you want to embrace and how you want to do things. Think about the space, the equipment, and all your options. You can walk into it being naïve, thinking there is one right way, but in fact the possibilities are endless. You need to spend time investigating what will work best for you and the people in the lab, because retrofitting things can be painful.”
Laminar flow hood: dos and don’ts
Keep the cabinet fully closed when not in use
Create significant turbulence in the cabinet with rapid motion
A good resource to refer to is http://www.cdc.gov/biosafety/publications/bmbl5/.