Potenciales de embedding para la perovskita CsCaBr 3

Laboratory equipment should be maintained so that it will perform properly.

All performance deficiencies and corrective actions taken should be docu-mented. Some of the items requiring validation and documentation include buffer solutions for pH meters, thermometers, temperature gauges, and ste-rility indicators such as spore strips for autoclaves. A file should be kept for each piece of equipment to be validated and should contain the following information: model and serial numbers, purchase date, manufacturer, main-tenance and operation manuals, and information for contacting technical service representatives.

The operator's manual on each piece of equipment should establish control steps for carrying out validation. Documentation should include the frequency and criteria for acceptance, performance standards, deficiencies, and corrective actions taken. Specialized checks, such as calibration of cen-trifuges, balances, and scintillation counters, should be performed by an authorized representative of the relevant manufacturer.

Installation of a piece of equipment should be validated in order to establish basic operational requirements, specifications, and tolerances. Most equipment manufacturers provide recommended installation procedures, but do not

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chapter six: Validation of methods 155 ply qualification instructions to validate proper installation of their equipment.

The user is then left to determine what validates proper installation.

In validating installation, a user should identify the critical features of operation that might affect function, variability, data, and records and also consider the effects that usage patterns have on the equipment; usage may be continuous, intermittent, variable, or involve long down times between uses. Idle equipment usually needs more attention than devices in constant operation.

Calibration of the installed equipment should involve particular care in selecting the reagents used with the machine, the techniques for detection of variances, and the measurement of what constitutes control. Wherever possible, a calibration program should use recognized standards, especially those from recognized standard-setting agencies or associations.

After a piece of equipment is validated as operational, it will require periodic monitoring to detect variation in set standards of performance.

Major variations of these standards may require shut-down and revalidation.

Use of control charts is a good statistical control process to verify that the equipment continues to operate within limits. Written standardized proce-dures are required for all laboratory equipment. Of course, all validation procedures require attestation of at least two signatures (with dates) to the records kept in a bound laboratory notebook or on appropriate forms. These validation records should be placed on the equipment or instrument or retained in a centralized location.

Also included in validation of equipment is the need for preventative maintenance to provide evidence that the equipment is in a state of control.

Preventative maintenance helps minimize malfunction and performance variation. It allows stable laboratory operations and is preferable to sporadic, uncontrolled major maintenance. Routine maintenance involves daily, weekly, or monthly activities such as lubrication and cleaning and replace-ment of recording paper. A written record should show the date and time when the maintenance was performed. When more skill and technical knowledge is required for maintenance activities than an average technician can handle, specially trained individuals should perform this function.

The key element to controlling and validating refrigerators and freezers is that these devices should maintain the temperatures required. The sophis-tication of their electronic controls will decide the validation steps needed.

Simple temperature controlling rheostat devices only set the instrument at a certain temperature point and provide no feedback.

Refrigerators and freezers with these controls are dependent somewhat on the ambient temperature of the environment into which they are placed.

Validation must take into account the potential for variation of the ambient temperature, the frequency with which the doors are opened and closed, and the temperature gradients that may exist within the refrigerator or freezer when empty and full.

Validation procedures should include a check of the internal chamber temperature with a calibrated thermometer traceable to a standard of the NBS

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156 Cosmetic Microbiology: A Practical Approach (National Bureau of Standards) or with calibrated temperature sensors inde-pendent of the equipment’s operational devices. Alarms are helpful for warn-ing of equipment malfunctions. When very precise temperatures are needed, more highly specialized control devices and feedback systems can be used.

Centrifuges may be used to harvest inocula for preservative efficacy tests or cells required to set up seed-lot cultures for maintaining the organisms.

One of the key areas for control is the velocity of spin. This velocity should be kept even and consistent at each of the speeds to be used. Therefore, tachometers must be kept calibrated in order to check the dial settings of the centrifuge against the actual spin velocity. In refrigerated centrifuges, the temperature variability within the chamber must be determined. In addition, procedures that specify which materials and solvents may be used in a centrifuge and which are prohibited should be followed, along with proce-dures that ensure that such materials do not remain in the centrifuge head after use. Use of timing devices on a centrifuge must also be validated for reproducibility and reliability (see below).

The elements of blending and mixing devices that require control include velocity and timing. The measurement of blending and mixing capabilities should be performed at several velocities. Equipment timers should be checked against calibrated timers.

Maximum–minimum thermometers may be used to control and monitor equipment temperatures. An operator can use this type of thermometer to manually check the thermometer and determine the maximum temperature reached in the instrument over a period of time. Maximum–minimum ther-mometers may be used in rooms, chambers, freezers, refrigerators, incuba-tors, and water baths. One criticism is that these thermometers do not show how long a maximum temperature is held. A thermometer can be calibrated by comparison with an NBS-calibrated thermometer at two control temper-atures that span the range desired.

Balances used in microbiology labs range from simple triple beam types to electronic balances that can tare and calibrate at the push of a button.

Regardless of type, all balances must be properly calibrated with certified weights. Some balances must be calibrated daily. Modern electronic balances require calibration less frequently, but usually at least monthly with a single weight.

pH meters also come in a diversity of designs. The manufacturer's rec-ommendations for calibration should be followed. At a minimum, daily calibration against two buffer solutions should be done, one at pH 7 and the other (the slope) at either pH 4 or 10, depending on whether the solutions to be measured are acidic or basic.

Timers are used in all kinds of laboratory processes and for all kinds of equipment and instruments. Calibration of timers is often overlooked. How-ever, certified calibrating stop watches can be used to check timers.

Instruments that measure turbidity include spectrophotometers, Klett meters, and nepholometers. These instruments are complex enough to demand that the operations manuals are carefully followed regarding

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chapter six: Validation of methods 157 lation, operation, and calibration. Typically for a spectrophotometer, a blank is used to provide the standard for 100% transmittance (or 2.0 optical density) and complete blocking of the light at a specific wavelength is provided as the standard for 0% transmittance (or 0 optical density).

Incubators provide controlled temperatures and moisture for the pro-motion of bacterial growth in inoculated media. They have specific require-ments for precise operation. Installation, operation, and calibration should consider the variability acceptable for reproducible reliable data collection.

For example, temperature variations within an incubator should be docu-mented because extremes from the top to the bottom of an incubator can occur and can differ based on how full or empty the incubator is. Many incubators resolve this problem via fan circulation of the air inside the incubator. Use of multiple thermocouples installed throughout the incubator and hooked to a computer is an efficient way of determining the temperature profile of an incubator. If this type of monitoring is beyond the ability of a laboratory, the manufacturer of the incubator should have such capacity. In sophisticated incubators, temperature data can be recorded continuously on charts or via computer data systems.

In a sense, water baths are similar to incubators. The amount of permis-sible variation in temperature depends upon the intended use of the bath.

Another area where the analogy to incubators is accurate is that circulation of water in a water bath (rather than air circulated in an incubator) will provide better temperature control; calibration of the thermal sensors and fluid flow control devices are the basic elements of control. In addition, thermal mapping should also be performed during installation. Alarm sys-tems are available and continuous recording syssys-tems can be integrated into a water bath.

Colony counters require minimal controls. Nevertheless, written proce-dures covering proper use and maintenance are required. The most critical element of a colony counter is the mechanism for recording the counts.

Manual tabulators are still very common but are being replaced by touch probes that eliminate human variability. With manual tabulation, a plunger is pressed with the thumb each time a colony is counted. The touch probe records a count each time a probe is pressed to a colony. Variability is controlled by providing a standard plate with a known number of colonies and having a technician count them.

The more sophisticated automated surface colony readers eliminate the human elements of fatigue and errors due to poor visual acuity. They are used where high numbers of plate counts are routine. The automated colony counters are based on either image analysis or interruption of a laser beam as it passes through a colony. These counters must be standardized using plates with painted patterns of spots to mimic colonies. Installation qualifi-cation and employee training are critical for operating scanning recording colony counters.

Laminar air flow hoods are used for culture transfers and for putting up cultures permanently to ensure purity. Standards for these types of hoods

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158 Cosmetic Microbiology: A Practical Approach focus on maintaining laminarity and, of course, sterility. Both of these param-eters can be checked daily. In addition, detection of leaks in HEPA filters is critical to operation of laminar flow hoods. A simple mechanism to validate the sterility of a hood is to use either settling plates or air samples from within the hood. In addition to having written procedures for validation and the operation of laminar air flow hoods, detailed instructions for the person-nel operating the hoods should be provided so the operators do not com-promise the flow of air.

Sterilizers

Laboratories require the use of sterilizers to provide sterile media and mate-rials for assaying the microbial contents of products. The most commonly used sterilizer is the autoclave. It operates by killing the microbes through exposure to moist heat generated by steam under pressure. The critical function is to ensure that this moist heat penetrates throughout the autoclave load to destroy all organisms. The two key variables to consider are size and type of load. Thus, validation of an autoclave should include instrument or component calibration, installation qualification, operational qualification, and certification under a diversity of loads.

During installation and operational qualification, thermal profiles of the empty chamber and during performance under various loads obtained with thermal sensors and biological indicators will provide the validation needed.

As these operations are performed, one should identify the chamber area that takes the longest to reach temperature before sterilization takes place.

The simplest way to do this is to use thermocouples that are passed into the autoclave through the chamber door. These are connected to microprocessor recording devices with computer tie-ins.

After this initial thermal mapping, one should mimic typical loads expected in the planned sterilizer operations. The type of load will impact the thermal profile. Absorptive loads may cause steam to condense and thus increase the time required for the chamber to become saturated with steam.

Fluid and solid loads may take longer to heat up. The type of container surrounding the material to be sterilized will also affect the efficacy of an autoclave. For example, a plastic flask will not permit penetration of heat to the media as quickly as a glass flask.

The most common standard in microbiological laboratory operations is the one for making microbiological media in an autoclave: sterilize for 15 min at 121°C. Unfortunately, this standard does not ensure sterility. The neophyte often misinterprets this standard as a sterilizing cycle of 15 min at 121°C instead of 15 min of thermal exposure of the center of the item to be autoclaved.²

This problem is especially acute during sterilization of large volumes of liquids. These have a high capacity for absorbing heat and must be heated to the boiling point before being autoclaved. For smaller volumes, a single liter for example, a microbiologist can often incorporate this step into the

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chapter six: Validation of methods 159 autoclave cycle by extending it to 30 min. More often, however, the volume is so great that incorporating the heating time into the sterilizing cycle is not possible because once inside an autoclave, a material cannot be stirred. As a result, uneven heating and burning of the medium may take place.

The most expedient way to validate a sterilizing cycle in a microbiology laboratory is the use of a biological indicator (BI).³ We have not discussed sterilizers other than autoclaves, but BIs are useful for all sterilizer cycle validation methods including steam, dry heat, and ethylene oxide. For more extensive discussion of various other sterilizing devices, refer to Gardner and Peel.⁴ Biological indicators come in a variety of forms, including inoc-ulated products with sterilant-resistant spores, spore paper strips, and disks and commercially available self-contained BI systems. The BIs should be placed in each corner of a load and in the center of an autoclave. The BIs are then incubated after exposure to the autoclave to see whether sterilization was effective.

Decontamination

Relatively little attention is given to decontamination procedures for micro-biological cultures and lab benches and materials that may become contam-inated during testing of products. The goal of decontamination is usually defined as rendering an area and materials within it safe to handle. When validating the ability of an autoclave to sterilize spent cultures, the same types of guidelines used to validate the autoclave can be employed: using the cultures or using a BI in the center of the load or area.

Essentially, the same principles used to attain sterility should be applied to decontamination processes. Decontaminating the lab benches can be val-idated using contact plates or swabs to monitor the efficacy of the process used. In all cases, documentation is required in order to track the results.

Summary

While this chapter has of necessity been very general, the guidelines described should provide at least a model for developing a laboratory’s own specific procedures for validation. There really is no single way to accomplish validation. However, a five-step process can be followed to validate any system:

1. Define what the system, method, or procedure is supposed to do.

2. Identify and control the variables in the system, method, or proce-dure.

3. Establish acceptance criteria for the system, method, or procedure before beginning the validation process.

4. Develop and execute a protocol to determine whether the procedure meets the criteria.

5. Document the procedures and results.

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160 Cosmetic Microbiology: A Practical Approach Regardless of the methods used to validate a process, the one common requirement is documentation. Documentation provides the evidence that justifies the methods used. A simple way to accomplish this aim is to use a validation register. External documentation in the form of published, peer-reviewed scientific journal articles should be gathered whenever pos-sible.

References

1. Brannan, D.K., Dille, J.C., and Kaufman, D. J., Correlation of in vitro challenge testing with consumer-use testing for cosmetic products, Appl. Environ. Mi-crobiol., 53, 1827, 1987.

2. Halleck, F.E., Thermal solution sterilization, Pharm. Technol., 2, 48, 1978.

3. Pflug, I.J. and Smith, G.M., The use of biological indicators for monitoring wet-heat sterilization processes, in Sterilization of Medical Products, Gaughran, E.R.L. and Kereluk, K., Eds., Johnson & Johnson, New Brunswick, NJ, 1977, p. 19.

4. Gardner, J.F. and Peel, M.M., Introduction to Sterilization, Disinfection, and In-fection Control, 2nd ed., Churchill Livingstone, Melbourne, 1991.

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