How To Control Media And Test Strains For Medical Device Testing
What are common microbiology tests? Why are microbiology tests important for your medical device or product?
Microbiology testing identifies the presence and the type of microbes in a manufacturing environment, medical product, or medical device. Microbiological testing is both a regulatory requirement and a risk mitigation strategy for aseptic manufacturing environments. Specific regulatory testing for microbiology may include microbial enumeration testing, particulate analysis, yeast analysis, antifungal activity assessment, growth promotion testing, microbial limits testing, zone of inhibition testing, water analysis, bacteriostasis/fungistasis testing, bacteria identification, yeast identification, fungi identification, gram-negative staining, product inoculations, biological indicator tests, and BI incubation time reduction studies. As medical devices and products must be sterile to be used within a human body, microbiological testing is imperative for preventing product-induced infections at the time of use.
There are two methods used to create a sterile medical product. Firstly, a final chemical or heat sterilization process is used on packaged medical products or devices to kill any live microorganisms picked up during the manufacturing process for terminal sterilization. The second method utilizes aseptic processing through the entire medical device or product manufacturing process. Aseptic processing requires the exclusion of microorganisms from the manufacturing methods. Thus, microorganisms must be prevented from entering open containers or product materials during aseptic processing. As a result, microbial tests are used to monitor clean rooms and facilities that manufacture products using aseptic processing.
How is media for microbiological testing or cell culture controlled?
Culture media are the cornerstones for microbiological tests. Maintaining media quality is therefore critical to the success of any microbial assay. Media preparation, storage, and quality control (QC) testing ensure consistent, high-quality media for testing. All three components of media control (preparation, storage, and QC) are described in detail below.
Media Preparation
Each microorganism prefers a specific type of media to support its growth. Thus, preparing the correct type of media for the experiment or microorganism in question is imperative to the microbiological assay’s accuracy. Different media types (let’s call them media bases) have variants based on preparation requirements (e.g., heating, additives, and pH adjustment). To use a cooking analogy, the media base (broth of the soup) will have different preparation requirements (veggies and spices you add to the soup) depending on the microorganism(s) being assessed.
A certificate of analysis describing a media’s expiration date and recommended storage conditions accompanies ready-made media purchased from a vendor to ensure product quality. Water is the universal diluent for manufacturer’s media in a dehydrated form. Purified, deionized, or distilled water is used to prepare media from a powdered formulation. Lesser quality water should not be used for microbiological media preparation, and the volume of the water used should be recorded.
If a media undergoes a sterilization method, the effects of the sterilization method must be validated by sterility and growth-promotion testing of the media. Additionally, the pH of each media batch must be confirmed after it has cooled to room temperature (20°–25°C). Media from dehydrated preparations may require heating to support the thorough dissolution of the dehydrated concentrate in water. The heating of media should be gradual as media is heat sensitive and can be overheated. A calibrated balance is used to prepare media from dehydrated concentrate consistently. When using a calibrated balance, the weights of all media components are recorded. When preparing media, ensure that all glassware is thoroughly cleaned and sterilized.
Prepared media should be inspected for the following:
- Cracked containers or lids
- Unequally filled containers
- Dehydration (cracks or dimpled surfaces on solid medium)
- Hemolysis
- Noticeable media darkening or color change
- Crystal formation from possible freezing
- Excessive number of bubbles
- Microbial contamination (cloudiness)
- Status of redox indicators (if appropriate)
- Lot number and expiration date checked and recorded
- Sterility of the media
- Cleanliness of plates (lid should not stick to the dish)
Media Storage
Storage conditions differ between media types. Considerations for media storage include temperature control, humidity control, and container packaging that prevents microbial contamination during transport. Media should be labeled with batch or lot numbers, preparation date, expiration dates, and identification about the type of media and media additives. Remelting of an original container of solid media should be performed only once. A single remelt preserves media quality by avoiding overheating the media or microbial contamination. Media heating should be performed in a heated water bath instead of over a hot plate or in a microwave. Protect stored media from light exposure and excessive temperature fluctuations. Overall, media should be stored according to the manufacturer’s instructions.
Media Quality Control
Due to ease of use, many laboratories use dehydrated media or purchase commercially prepared media in plastic plates or glass containers. Manufacturers of media deal with unavoidable differences in raw materials obtained from natural sources. These differences in raw materials from natural sources create lot-to-lot media variability. Thus, it is important to track media by lot or batch number and to use the same batch or lot of media for microbiological tests.
Improper media preparation or storage can cause unsatisfactory microbial growth and microbial recovery conditions, leading to unreliable results. Therefore, quality control tests should be performed on all prepared media. Media quality control tests include routine evaluation of pH, growth promotion, inhibition, and indicative properties. Periodic stability checks to confirm that media performance lasts until labeled expiration dating should occur as well. Special care should be taken with the sterility of media used in sterility tests and environmental monitoring studies. Media used for environmental monitoring of critical areas should be double-wrapped and terminally sterilized. If terminal sterilization is not performed, media should be subjected to 100% pre-incubation and inspection before use.
How are microbiological test strains controlled?
The viability and characteristics of biological specimens (bacteria, yeast, mold, etc.) are dependent on appropriate handling and storage. The handling and storage of microbial cultures must be standardized such that contamination or alteration of microbe growth characteristics is prevented or minimized. Careful and consistent treatment of stock cultures is critical for consistent and accurate microbiological test results. Stock cultures are to be acquired from a national culture collection or a qualified secondary supplier. Confirmation of the purity of the culture and the identity of the culture (genus and species) is performed before use. Preparation and resuscitation of cultures should follow supplier instructions or an established and validated method.
The “Seed-Lot” technique is recommended for the storage of stock cultures. With the “Seed-Lot” technique, the original sample from a national culture collection or a qualified secondary supplier is resuscitated and grown in an appropriate medium. Aliquots of stock culture (the first passage) are suspended in a cryoprotective medium and frozen at –30° or below until use. Cryopreserved stock cultures stored at –70° (or in lyophilized form) may be kept indefinitely. The frozen first passage stocks can then be thawed and used for assays. If using the “Seed-Lot” technique, unused cell suspensions will not be frozen again and should be appropriately discarded once opened. The number of transfers (passages) of working control cultures should be tracked in a laboratory notebook to prevent excessive subculturing. Subculturing increases the risk of phenotypic alteration or mutation of microorganisms. Note that one passage is defined as transferring organisms from a viable culture to a fresh growth medium. Any form of subculturing is considered a passage.
Summary
Overall, the control of media and test strains for microbiology testing is imperative for consistent and accurate microbial testing. Media control is accomplished through careful media preparation, monitored media storage, and periodic media QC testing. Test strains for microbiological testing are controlled through careful sourcing, identification, and culturing of the test strains. Passage and subculturing of test strains are carefully monitored to ensure no phenotypic alterations or mutations of the stock microorganisms occur. Whether you choose to do microbiology testing in-house or outsource your regulatory testing, make sure you establish procedures that monitor and control media and microbial strains used for microbial assays. Should you decide to outsource your microbiological or aseptic environmental monitoring testing for your medical device or medical product, choose a contract testing organization that can support your needs.
Ethide Labs is a contract testing organization that specializes in Microbiology Testing and Environmental Monitoring. Ethide Labs provides in-vitro cytotoxicity tests in-house and outsources in-vivo cytotoxicity work for toxicity testing of medical devices, products, and drugs. Ethide Labs also offers Sterilization Validations, Bioburden Testing, Bacterial Endotoxin Testing, EO Residual Testing, Package Integrity Testing & Cytotoxicity Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <1117> Microbiological Best Laboratory Practices. Rockville, MD, USA. 2021. (USPC <1117>).
