Cleanrooms play a vital role in a world filled with microbes because they help maintain product sterility and safety from the outside. Even with the use of a cleanroom, however, it is important to constantly verify a product’s sterility as well as the contamination levels that exist within the production plant. This verification is where microbiological culture media play an important role in ensuring product safety.
Research in developing media to isolate and identify microbes first began in the late 1800s when scientists realized their potential to spur disease. The first attempts to isolate bacteria were carried out in a liquid medium that, unfortunately, yielded a negative outcome due to the prevalence of microbial contamination and the difficulty in isolating individual microbes. In the years following these early efforts, German physician Robert Koch postulated that using a solid medium could yield more favorable results. To test this theory, he used the surface of a potato exposed to air to observe spore growth of an individual microbe species.
While his initial attempt at individual isolation was successful because of the restricted potential of a potato as a suitable growth medium, Koch was unable to isolate a wide variety of microorganisms. Koch avoided other culture media surfaces, remaining faithful to the idea of solid media; eventually, he used the properties of gelatin, or agar, as the foundation for solid microbiological culture media.
Under many conditions, agar proved itself the most suitable growth medium for isolating and identifying a multitude of microorganisms. These conditions ranged from a melting temperature of approximately 100°C to a solid phase of about 40°C. Agar was also found to have a low potential for degrading microbes; the capacity to collaborate with various nutrients, vitamins, enzymes, carbohydrates, and sugars; the ability for incubation and refrigeration at various temperatures without decomposition; and the suitability of manipulation.
Agar’s innovation led to the development of widely used methods and products that paved the way to advanced microbiology and uncovered the world of microorganisms as it is known today. Both quantitative and qualitative techniques arose following the discovery of an agar medium. Separation techniques used broth media inoculated with microorganisms that could afterward be placed on agar and further isolated to grow individual colonies of one type of microbe, thus allowing full identification of the organism’s morphology.
Ensuring that pure microbial cultures are contained within their environment, the aseptic technique not only prevents outside contamination from entering the culture, but also keeps microbes from spreading to the outside world. Today, this technique can be found within every regulatory and compliance guideline document pertaining to work with microorganisms and is incorporated into every standard operating procedure within all genres of microbiology laboratories, including research and development, quality control, and environmental monitoring. As the only technique allowable for handling microorganisms, it is strictly enforced.
Instances of contamination deriving from sources such as air, utensils, glassware, and laboratory surfaces have led to the use of heat to kill off unwanted microorganisms and maintain a sterile environment. Robert Koch observed this effect in his work with the potato; by heating the potato and keeping it in a glass jar, he prevented contamination. According to Johnson and Case, "The destruction of microbes by heat was employed by Lazzaro Spallanzani in the 1760s. He heated nutrient broth to kill preexisting life in his attempts to disprove the concept of spontaneous generation. In the 1860s, Pasteur heated broth in specially designed flasks and ended the debate over spontaneous generation."1
The acceptance of culture media advanced the creation of media products for the optimum growth of targeted microorganisms. Developing media was no longer an arbitrary task but included precise research into the correct nutrients, energy sources, and growth factors necessary for microbial growth. Microbial metabolism, protein catabolism, respiration, temperature, and oxygen were all factors taken into consideration when developing microbial media. Derived sources enabled optimum microbial growth, proving that the microbes contained on the media surface were a true representation of the microbial presence in the sampling environment.
Culture Media in Cleanrooms
Standards for manufacturing cleanrooms are based on the sterility levels outlined by regulatory agencies such as the United States Pharmacopeia, European Pharmacopoeia, Japanese Pharmacopoeia, and the U.S. Food and Drug Administration. Regulating bodies have determined not only the sterility classifications necessary for cleanrooms but also the isolators and gloveboxes required based on the types of products manufactured. For products to maintain compliance to regulatory standards, corresponding levels of sterility must be maintained inside these controlled environments. Because these areas serve to ensure safe and sterile environments through microbial control, their environmental monitoring is non-negotiable.
Within cleanrooms, culture media are used to prove required sterility levels on surfaces such as bench tops, equipment, and personnel. By using a contact method in which the surface is contacted with the agar and then incubated, sterility is enforced by determining organism recovery on the surfaces of the agar media. Culture media are also used to maintain air microbial control: larger agar media surfaces serve as runways for the settling of microbes found in the air. The air quality of controlled environments may be determined by incubating these media surfaces.
Culture media are also used to demonstrate proper disinfection of controlled environmental surfaces. When effective neutralizers are incorporated within the media composition, organism detection is achieved, eliminating the possibility of false negatives that may occur as a result of inadequate neutralization.
Contact plates and settling plates are the main agar surfaces used in cleanroom monitoring. Contact plates are convex agar surfaces that are poured with approximately 17 mL of media into 50-mm dishes. The convex shape allows for easy sampling of surfaces. Settling plates are used to monitor both the air quality in critical areas by sedimentation and the sterility of surfaces that are touched by the fingertips of associated personnel. They usually contain about 20 mL of agar media and can be purchased in either 90-mm or 150-mm dish sizes, depending on the standard operating procedure requirements.
Requirements for a media supplier are simple: safety and sterility. These two areas are greatly influenced by the environment to be monitored. Regular hygiene monitoring may utilize single-packaged media; isolators that are gas impermeable may use double packaging for media, while cleanrooms or isolators may require triple packaging.
From packaging and storage conditions to shelf life and filling volume, culture media today pose a number of challenges for production facilities and personnel. One of the biggest challenges is the fact that most agar media must be stored at temperatures between 2°C and 8°C to prevent the loss of water and the degradation of media growth properties. Staying within this temperature range is believed to maintain a medium’s growth ability and to prevent any degradation of its formula. Loss of water within the current media packaging can lead to a loss of media growth potential and possible contamination issues. In many cases, this results in a large amount of product waste at a high cost.
Current media packaging allows for a shelf life of approximately two months. Using media after the expiration date can lower growth potential and compromise the integrity of the results. Manufacturers must turn over their media inventory six or more times a year, incurring all of the costs associated with frequent inspection and testing of incoming material.
Currently, agar media must be incubated for a period of time ranging from one to seven days, depending on the organism. The filling volume of both contact plates (17 mL) and settling plates (20 mL) does not accommodate the longer incubation periods that might occur on weekends, holidays, or during emergency breakdowns. Longer incubation can cause the media to dry out, making results unobtainable.
Other problems include illegible lot numbers and expiration dates, as well as plates falling off. In addition, double packaging has been shown to be less effective when going from a less controlled to a more controlled area. With the lack of research and development being done by companies into the overwhelming disinfectants available and minimal investigation into neutralization studies, inadequate neutralization is more likely to occur.
The New Generation
For the most critical manufacturing areas, it is necessary to have a safe and reliable product that features a defined performance throughout its shelf life. The new generation of media must not only meet the quality standards for the pharmaceutical industry and hold ISO 9001-2000 certifications, but must also be manufactured within strict good manufacturing practice guidelines that include a comprehensive quality management system to ensure that the end user receives media of superior quality.
The new generation of culture media is manufactured under ISO Class 5 conditions and is regulated by international guidelines. A thorough quality management system is defined by three components:
• The system includes documentation, deviation management, change control, trainings, audits, and certifications.
• Each system used in the development of the culture media is placed under validation, qualification, and calibration.
• The production facility is maintained under guidelines that include environmental monitoring and cleaning as outlined in the requirements for each class. In addition, products are validated under stressed conditions to allow for worst-case scenarios.
The benefits of this new generation of media to the microbiology industry include:
• Shelf life expectancy is extended to more than six months through optimal microorganism density in the media’s raw materials; special triple- bagged, gamma-irradiated packaging; and a desiccant to absorb condensation.
• Refrigeration is not required, saving valuable refrigeration space. This allows the media, which are fully validated at temperatures between 15°C and 25°C, to be stored at the place of usage, thus minimizing cross contamination, reducing storage costs, and increasing productivity.
• The new volumes withstand longer incubations and can sample high volumes of air. Contact plates contain 20 mL of agar media, and settling plates contain 30 mL of poured media. These volumes have consistently provided excellent recovery following long incubations.
• Locking lids enable the user to place the plate in either an open or closed position, decreasing the need for laboratory tape and further enabling the plate to be stored in both aerobic and anaerobic conditions.
The new generation of culture media is paving the way to an increased level of safety and quality that has not yet been achieved within environmental monitoring. Biopharmaceutical and medical device product manufacturers now have the ability to improve quality while significantly reducing costs and increasing productivity. n
Bogicevic is product manager at Biotest Diagnostics Corp. For more information, visit www.biotestusa.com.
1. Johnson TR, Case CL. Laboratory Experiments in Microbiology. 6th ed. Menlo Park, Calif.:Benjamin/Cummings Publishing Co.; 2000.