Ljungqvist B, Reinmüller B. Interaction between air movements and the dispersion of contaminants: clean zones with unidir-ectional air flow. J Parenter Sci Technol. 1993; 47(2):60–69.
Ljungqvist B, Reinmüller B. Airborne viable particles and total number of airborne particles: comparative studies of active air sampling. PDA J Sci Technol. 2000; 54:112–116.
Maruyama M, Matsuoka T, Deguchi M, Akers J. The application of robotics to aptic surface monitoring. Pharm Technol. 2007; 32(7):40–44.
Process simulation testing for sterile bulk pharmaceutical chemicals. PDA Technical Report No. 28. J Parenter Sci Technol. 1998; 52 S3.
Reinmüller B. Dispersion and risk asssment of airborne contaminants in pharmaceutical cleanrooms. Building Serv Eng Bull (Sweden). 2001; Bulletin No. 56.
Stewart SL, Grinshpun SA, Willeke K, Terzieva S, Ulevicius V, Donnelly J. Effect of impact stress on microbial recovery on an agar surface. Appl Environ Micro. 1995; 61:1232–1239.
Whyte W. Reduction of microbial dispersion by clothing. J Parenter Sci Technol. 1985; 39(1):51–60.
á1117ñ MICROBIOLOGICAL BEST LABORATORY PRACTICES
INTRODUCTION
Good laboratory practices in a microbiology laboratory consist of activities that depend on veral principles: aptic techni-que, control of media, control of test strains, operation and control of equipment, diligent recording and evaluation of data, and training of the laboratory staff. Becau of the inherent risk of variability in microbiology data, reliability and reproducibili-ty are dependent on the u of accepted methods and adherence to good laboratory practices.
MEDIA PREPARATION AND QUALITY CONTROL
冰是睡着的水Media Preparation
Culture media are the basis for most microbiological tests. Safeguarding the quality of the media is therefore critical to the success of the microbiology laboratory. Media preparation, proper storage, and quality control testing can ensure a consistent supply of high-quality media.
It is important to choo the correct media or components in making media bad on the u of accepted sources or refer-ences for formulas. The manufacturer's formula and instructions for preparation routinely accompany dehydrated media and ready-made media. Becau different media types may have different preparation requirements (e.g., heating, additives, and pH adjustment), it is important to follow the instructions to ensure preparation of acceptable media quality. A certificate of analysis describing expiration dating and recommended storage conditions accompanies ready-made media, as well as the quality control organisms ud in growth-promotion and lectivity testing of that media.
Water is the universal diluent for microbiological media. Purified Water is most often ud for media preparation, but in cer-tain cas the u of deionized or distilled water may be appropriate. Water of lesr quality should not be ud for microbio-logical media preparation. The volume of the water ud should be recorded.
Consistent preparation of media requires accurate weighing of dehydrated media or media constituents. A calibrated bal-ance with the appropriate weight range for the ingredients should be ud (See Weighing on an Analytical Balance á1251ñ). Clean weighing containers and tools (such as spatulas) should be ud to prevent foreign substances from entering the formu-lation. The weigh
轻袅袅t of the components should be recorded.
Dehydrated media should be thoroughly dissolved in water before dispensing and sterilization. If heating is necessary to help dissolve the media, care should be taken not to overheat media, becau all culture media, to a greater or lesr extent, are heat-nsitive. Equipment ud in the preparation of media should be appropriate to allow for controlled heating, constant agitation, and mixing of the media. Darkening of media (Maillard-type reaction or nonenzymatic browning) is a general indi-cation of overheating. When adding required supplements to media, adequate mixing of the medium after adding the supple-ment should be performed.
Preparation of media in poorly cleaned glassware can allow inhibitory substances to enter the media. Inhibitory substances can come from detergent residue after cleaning glassware or from prior materials ud in the glassware. Be sure that the clean-ing process removes debris and foreign matter, and that the detergent is thoroughly rind out with Purified Water. See Clean-ing Glass Apparatus á1051ñ for additional guidance.
Sterilization of media should be performed within the parameters provided by the manufacturer or validated by the ur. Commercially prepared media should provide documentation of the sterilizatio
n method ud. Autoclaving by moist heat is the preferred sterilization technique, except in instances when boiling is required in order to avoid deterioration of heat-labile
components of the media. Sterilization by filtration may also be appropriate for some formulations.
The effects of the sterilization method and conditions on the media should be validated by sterility and growth-promotion testing of the media. In addition, if sterilized by moist heat, the autoclave cycle should be validated to ensure proper heat dis-tribution for lected loads and volumes. Typically, manufacturers recommend using an autoclave cycle of 121° for 15 minutes using a validated autoclave. The conditions apply to time at temperature of the media. As container size and the load config-uration of the autoclave will influence the rate of heating, longer cycles may be required f
or larger loads. However, the sterili-zation time will be dependent on the media volume and autoclave load. Sterilization cycles in which the autoclave is slow to come up to temperature may result in overheating of the media. Therefore, care must be taken to validate a sterilization cycle,balancing the need for sterile media against the tendency of the media to degrade under excessive heating. Storage of the media in the autoclave after the liquid cycle is completed is not recommended after cooling, as it may damage the media.Improper heating or sterilizing conditions—for commercially prepared or internally prepared media—may result in a difference in color change, loss of clarity, altered gel strength, or pH drift from the manufacturer's recommended range, as well as re-duced growth-promotion activity and/or lectivity.
The pH of each batch of medium should be confirmed after it has cooled to room temperature (20°–25°) by aptically withdrawing a sample for testing. Refrigerated purchad media should be allowed to warm up to ambient room temperature if it is to be checked for pH confirmation. A flat pH probe is recommended for agar surfaces, and an immersion probe is rec-ommended for liquids. See pH á791ñ for guidance with pH measurement and instrument calibration. The pH of media should be in a range of ±0.2 of the value indicated by the manufacturer, unless a wider range is acceptable by the validated method.Prepared media should be checked by appropriate inspection of plates and tubes for the following:ri过去式
•Cracked containers or lids
•Unequal filling of containers
•Dehydration resulting in cracks or dimpled surfaces on solid medium
•Hemolysis
•Excessive darkening or color change
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•Crystal formation from possible freezing
•Excessive number of bubbles
•Microbial contamination
•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 dish)
Media Storage
阴阳不调It is prudent to consider how the manufacturer or supplier transports and stores media before distribution to the end ur.Manufacturers of media should u transport and storage conditions that minimize the loss of moisture, control the tempera-ture, prevent microbial contamination, and provide mechanical protection to the prepared media.
Media should be labeled properly with batch or lot numbers, preparation and expiration dates, and m
edia identification.Media should be stored according to the manufacturer's instructions. Media prepared in hou should be stored under valida-ted conditions. Do not store agar at or below 0°, as freezing could damage the gel structure. Protect stored media from expo-sure to light and excessive temperature. Before prolonged storage, agar plates should be placed into a aled package or con-tainer to retard moisture loss.
Remelting of an original container of solid media should be performed only once to avoid media who quality is compro-mid by overheating or potential contamination. It is recommended that remelting be performed in a heated water bath or by using free-flowing steam. The u of microwave ovens and heating plates is common, but care should be taken to avoid damaging media by overheating and to avoid the potential injury to laboratory personnel from glass breakage and burns. The molten agar medium should be held in a monitored water bath at a temperature of 45° to 50° for not more than 8 hours.Caution should be taken when pouring the media from a container immerd in a water bath to prevent water from the bath commingling with the poured sterile media. Wiping the exterior of the container dry before pouring may be advisable.Disposal of ud cultured media (as well as expired media) should follow local biological hazard safety procedures.
Quality Control Testing
Although growth media can be prepared in a laboratory from individual components, many laboratories, for ea of u, u dehydrated media or purcha commercially prepared media in plastic plates or glass containers. Manufacturers of media at-tempt to standardize raw materials from biological sources, but must constantly deal with unavoidable differences in raw ma-terials obtained from natural sources, and therefore, lot-to-lot variability of media must be considered. In addition, the per-formance of media prepared in a laboratory or by a manufacturer is highly dependent on preparation and storage conditions.Improper media preparation can cau unsatisfactory conditions for microbial growth or recovery and unreliable results.
Therefore, quality control tests should be performed on all prepared media, including media associated with swabs or media in strips and other nontraditional formats. Tests routinely performed on in-hou prepared media should include pH, growth
promotion, inhibition, and indicative properties (as appropriate), and periodic stability checks to confirm the expiration dating.
When in-hou prepared microbiological media are properly prepared and sterilized using a validated method, the growth-promotion testing may be limited to each incoming lot of dehydrated media, unless otherwi instructed by the relevant com-pendial method. If the media preparation procedure was not validated, then every batch of media should be subjected to growth-promotion testing. Test organisms may be lected from the appropriate compendial test chapter. In addition, micro-organisms ud in growth-promotion testing may be bad on the manufacturer's recommendation for a particular medium, or may include reprentative environmental isolates (but the latter are not to be construed as compendial requirements). Expiration dates on media should have supporting growth-promotion testing to indicate that the performance of the media still meets acceptance criteria up to and including the expiration date. The length of shelf life of a batch of media will depend on the stability of the ingredients and formulation under specified conditions, as well as the type of container and closure. When a batch of media does not meet the requirements of growth-promotion testing, an investigation should be initiated to identify the cau. This investigation should include a corrective action plan to prevent the recurrence of the problem. Any batch of media that fails growth-promotion testing is unsuitable for u. [NOTE—Failed growth-promotion test results may not be ud to negate positive test results.]
Some reagents are ud for diagnostic purpos to help support identification of microbial organisms, e.g., Gram stain and oxida test reagents. The may have attributes that can be quality control tested similar to microbiological media. Select the correct quality control standard microorganisms, following the manufacturer's instructions, and perform the testing before un-known sample diagnostic testing. All relevant diagnostic reagents should be subjected to incoming quality confirmation before u.
Special care should be taken with media that is ud in sterility tests (e Sterility Tests á71ñ for requirements) and in environ-mental monitoring studies. Media ud for environmental monitoring of critical areas should preferably be double-wrapped and terminally sterilized. If terminal sterilization is not performed, media should be subjected to 100% pre-incubation and in-spection before u within a critical area. [N OTE—Growth-promotion testing for this media must be performed after the pre-incubation stage.] This will prevent extraneous contamination from being carried into controlled environments and will pre-vent fal-positive results. A raid agar level for surface contact plates should be verified.
MAINTENANCE OF MICROBIOLOGICAL CULTURES
Biological specimens can be the most delicate standards to handle becau their viability and characteristics are dependent on adequate handling and storage. Standardizing the handling and storage of cultures by the ur laboratory should be done in a way that will minimize the opportunity for contamination or alteration of growth characteristics. The careful and consis-tent treatment of stock cultures is critically important to the consistency of microbiological test results. Cultures for u in com-pendial tests should be acquired from a national culture collection or a qualified condary supplier. They can be acquired fro-zen, freeze-dried, on slants, or in ready-to-u forms. Confirmation of the purity of the culture and the identity of the culture should be performed before its u in quality control testing. Ready-to-u cultures should be subjected to incoming testing for purity and identity before u. The confirmation of identity for commonly ud laboratory strains should ideally be done at the level of genus and species.
Preparation and resuscitation of cultures should follow the instructions of the supplier or a validated, established method. The “Seed-Lot” technique is recommended for storage of stock cultures.
The original sample from the national culture collection or a qualified condary supplier is resuscitated and grown in an appropriate medium. Aliquots of this stock culture (the first transfer or passage) are suspended in a cryoprotective medium, transferred to vials, and frozen at –30° or belo
w, until u. If stored at –70°, or in lyophilized form, strains may be kept indefi-nitely. The frozen stocks can then be ud to inoculate monthly or weekly working cultures. Once opened, do not refreeze unud cell suspensions after culturing a working suspension. The unud portion should be discarded to minimize the risk of loss of viability and contamination of the stock.
The number of transfers of working control cultures should be tracked to prevent excessive subculturing that increas the risk of phenotypic alteration or mutation. The number of transfers allowable for specific compendial tests may be specified in that test. One passage is defined as the transfer of organisms from a viable culture to a fresh medium with growth of the mi-croorganisms. Any form of subculturing is considered to be a transfer/passage.
LABORATORY EQUIPMENT
Most equipment (incubators, water baths, and autoclaves) is subject to standard validation practices of incoming qualifica-tion, operational qualification, and performance qualification. Additionally, periodic calibration (generally annually) is com-monly required. New equipment, critical to the operation of the laboratory, should be qualified according to a protocol ap-proved by the quality assurance unit (QAU). In addition, regular cleaning and sanitization of equipment such as incubators,
refrigerators, and water baths should be performed to minimize the potential for contamination in the laboratory. Door als of incubators and refrigerators should be cleaned and checked for state of repair.
Instruments (pH meters and spectrophotometers) ud in a microbiology laboratory should be calibrated on a regular schedule and tested to verify performance on a routine basis. The frequency of calibration and performance verification will
vary bad on the type of instrument and the importance of that equipment to the generation of data in the laboratory.
Equipment that is difficult to sanitize (such as refrigerators and incubators) should be dedicated to aptic operations (such as storage of media for testing and incubation of sterility test samples) and live culture operations to minimize the potential for inadvertent contamination of the tests.
Autoclaves are central to the operation of the laboratory and must have proper validation in place to demonstrate adequate sterilization for a variety of operations. Autoclave resources must be available (and validated) to sterilize waste media (if per-formed in that laboratory) as well as the media prepared in that laboratory. The choice of one or veral autoclaves is not driv-en by a need to parate aptic and live operations (everything in the properly maintained autoclave is sterile after the cycle)but rather driven by resource considerations (e below).
LABORATORY LAYOUT AND OPERATIONS
Laboratory layout and design should carefully consider the requirements of good microbiological practices and laboratory safety. It is esntial that cross-contamination of microbial cultures be minimized to the greatest extent possible, and it is also important that microbiological samples be handled in an environment that makes contamination highly unlikely.
In general, a laboratory should be divided into clean or aptic areas and live culture areas. Areas in
which environmental or sterile product samples are handled and incubated should be maintained completely free of live cultures, if possible. If com-plete paration of live and clean culture zones cannot be accomplished, then other barriers and aptic practices should be employed to reduce the likelihood of accidental contamination. The barriers include protective clothing, sanitization and dis-infection procedures, and biological safety cabinets designated for clean or aptic operations only. Procedures for handling spills or mishaps with live cultures should be in place, and all relevant technical personnel should be trained regarding the
methods.
Some samples will demonstrate microbial growth and require further laboratory analysis to identify th
e contaminants. When
growth is detected, the sample should be taken from the clean ction of the laboratory to the live culture ction without undue delay. Subculturing, staining, microbial identification, or other investigational operations should be undertaken in the
live culture ction of the laboratory. If possible, any sample found to contain growing colonies should not be opened in the
clean zone of the laboratory. Careful gregation of contaminated samples and materials will reduce fal-positive results.
Staff engaged in sampling activities should not enter or work in the live culture handling ction of a laboratory unless spe-cial precautions are taken, including wearing protective clothing and gloves and careful sanitizing of hands upon exiting. Ideal-
ly, staff assigned to sampling activities, particularly tho in support of aptic processing, should not work in the vicinity of live culture laboratory operations.皮卡丘怎么折
It is important to consider that microbial contamination of samples, which leads to fal-positive results, is always possible unless careful aptic precautions are taken. Facilities should be designed so that raw material and excipient sampling can be done under controlled conditions, including proper gowning and sterilized sampling equipment. It may not always be possible to sample utility systems, such as water systems, under full aptic conditions; however, it should be noted that when samples are not taken aptically, their reliability is inevitably compromid.
Environmental sampling methods should require minimal aptic handling in loading and unloading sampling instruments.Whenever possible, sampling equipment should be loaded with its microbiological recovery media in the environment that is to be sampled.
All testing in laboratories ud for critical testing procedures, such as sterility testing of final dosage forms, bulk product,ed cultures for biological production, or cell cultures ud in biological production, should be performed under controlled conditions. Isolator technology is also appropriate for critical, sterile microbiological testing. Isolators have been shown to have lower levels of environmental contamination than manned clean rooms, and therefore, are generally less likely to produce fal-positive results. Proper validation of isolators is critical both to ensure environmental integrity and to prevent the possibili-ty of fal-negative results as a result of chemical disinfection of
materials brought into or ud within isolators (e Sterility Testing—Validation of Isolator Systems á1208ñ).
SAMPLE HANDLING
Viable microorganisms in most microbiology samples, particularly water, environmental monitoring and bioburden samples,are nsitive to handling and storage conditions. Critical parameters in the conditions include product (or sample) composi-tion, container composition, time of storage, and temperature of storage. Therefore, it is important to minimize the amount of time between the sampling event and the initiation of testing and to control, as much as possible, the conditions of storage. If the sample is to be transported to a distant location for testing, then the conditions of transport (time, temperature, etc.)should be qualified as suitable for that test and sample. Guidance for water testing in this regard can be found in Water for Pharmaceutical Purpos á1231ñ. Product mixing before sampling may need to be evaluated and applied in order to ensure mi-crobial disperment and reprentation in the sample aliquot.
All microbiological samples should be taken using aptic techniques, including tho taken in support of nonsterile prod-ucts. If possible, all microbiological samples should be taken under full as
eptic conditions in specialized sampling areas. The areas should be as clo to the point of u as possible to minimize contamination during transit.
Samples submitted to the microbiology laboratory should be accompanied by documentation detailing source of the sam-
ple, date the sample was taken, date of sample submission, person or department responsible for the submission, and any
黑杞子功效potentially hazardous materials associated with the sample. The testing department should acknowledge receipt of the sample and reconcile the identity and number of samples as part of this sample documentation.
MICROBIOLOGICAL MEDIA INCUBATION TIMES
Incubation times for microbiological tests of less than 3 days' duration should be expresd in hours: e.g., “Incubate at 30°to 35° for 18 to 72 hours”. Tests longer than 72 hours' duration should be expre
sd in days: e.g., “Incubate at 30° to 35° for 3 to 5 days”. For incubation times expresd in hours, incubate for the minimum specified time, and exerci good microbio-logical judgment when exceeding the incubation time. For incubation times expresd in days, incubations started in the morning or afternoon should generally be concluded at that same time of day.
TRAINING OF PERSONNEL
Each person engaged in each pha of pharmaceutical manufacture should have the education, training, and experience to do his or her job. The demands of microbiological testing require that the core educational background of the staff, supervi-sors, and managers be in microbiology or a cloly related biological science. They should be assigned responsibilities in keep-ing with their level of skill and experience.
A coherent system of standard operating procedures (SOPs) is necessary to run the microbiology laboratory. The proce-dures rve two purpos in a training program. Firstly, the SOPs describe the methodology that the microbiologist will fol-low to obtain accurate and reproducible results, and so rve as the basis for training. Secondly, by tracking the procedures in which a particular microbiologist has demonstrated proficiency, the procedure number or title also rves to identify what training the microbiologist has received specific to his or her job function.
Training curricula should be established for each laboratory staff member specific to his or her job function. He or she should not independently conduct a microbial test until qualified to run the test. Training records should be current, documenting the microbiologist's training in the current revision to the particular SOP.
Periodic performance asssment is a wi investment in data quality. This performance testing should provide evidence of competency in core activities of the microbiology laboratory such as hygiene, plating, aptic technique, documentation, and others as suggested by the microbiologist's job function.
Microbiologists with supervisory or managerial responsibilities should have appropriate education and in-hou training in supervisory skills, laboratory safety, scheduling, budgeting, investigational skills, technical report writing, relevant SOPs, and other critical aspects of the company's process as suggested in their role of directing a laboratory function.
Competency may be demonstrated by specific cour work, relevant experience, and routinely engaging in relevant con-tinuing education. Achieving certification through an accredited body is also a desirable credential. Further, it is expected that laboratory supervisors and managers have a demo
nstrated level of competence in microbiology at least as high as tho they supervi. Experti in microbiology can be achieved by a variety of routes in addition to academic cour work and accredita-tion. Each company is expected to evaluate the credentials of tho responsible for designing, implementing, and operating the microbiology program. Companies can thus ensure that tho responsible for the program understand the basic principles of microbiology, can interpret guidelines and regulations bad on good science, and have access to individuals with theoreti-cal and practical knowledge in microbiology to provide assistance in areas in which the persons responsible for the program may not have adequate knowledge and understanding. It should be noted that microbiology is a scientifically bad discipline that deals with biological principles substantially different from tho of analytical chemistry and engineering disciplines. Many times it is difficult for individuals without specific microbiological training to make the transition.
烟英文LABORATORY RESOURCES
The laboratory management is responsible for ensuring that the laboratory has sufficient resources to meet the existing test-ing requirements. This requires some proficiency in budget management and in determining appropriate measures of labora-tory performance. A measure of laboratory performance is the number of investigations performed on tests conducted by the laboratory, but this
measure alone is not sufficient. In addition to tracking investigations, the period of time between sample submission and initiation of testing should be tracked, as well as the period of time between end of test and report relea (or test closure). Significant delays in the measures are also indications of an under-resourced laboratory staff.
The laboratory management should have sufficient budget to meet testing requirements. Particular measures of budgetary requirements will be specific to the given laboratory, but budgetary considerations related directly to the need of the laborato-ry for sufficient resources must be addresd to ensure reliable testing results.
DOCUMENTATION
Documentation should be sufficient to demonstrate that the testing was performed in a laboratory and by methods that were under control. This includes, but is not limited to, documentation of the following:
•
Microbiologist training and verification of proficiency
