Validation of Microbiological Methods

To provide the guideline for validation of the microbiological methods to ensure analytical accuracy and precision and that the methods are suitable for the intended use 

It is the responsibility of the quality control manager to follow the procedure. The quality assurance manager is responsible for SOP compliance. 

The main objective of validation of an analytical procedure is to demonstrate that the procedure is suitable for its intended purpose. The procedures presented in this SOP provide basic guidelines for the validation of methods for microbiological assay, estimation of the number of microorganisms, detection of indicators of objectionable microorganisms, validation of preservative efficacy testing, and validation of the sterility testing and endotoxin test (LAL test). 

It is an essential condition of biological assay methods that the tests on the standard preparation and on the sample whose potency is being determined should be carried out at the same time and, in all other respects, under strictly comparable conditions. The validation of microbiological assay method includes performance criteria (analytical parameters) such as linearity, range, accuracy, precision, specificity, etc. 

Specificity is usually difficult to assess with microbial assay methods, because the tests measure the total activity and this will represent the synergetic action of all active components in the mixture under test. 

---

The work in one central laboratory could be considered for the study of sampling techniques, in particular for environmental or workplace hygiene problems. Finally, a new project using the same approach allowed to set up a standardised procedure for the validation of microbiological methods, compared to a reference method. 

USP 24 Testing Chapters (51) Antimicrobial Effectiveness Testing, (61) Microbial Limit Tests and (71) Sterility Tests, United States Pharmacopeial Convention, Inc., Rockville, MD, 2000. USP 24 Informational Chapters (1116) Microbiological Evaluation of Clean Rooms and other Controlled Environments, (1111) Microbiological Attributes of Pharmaceutical Articles, (1151) Pharmaceutical Dosage Forms, (1225) Validation of Compendial Methods, and (1231) Water for Pharmaceutical Purposes, United States Pharmacopeial Convention, Inc., Rockville, MD, 2000. 

Informational Chapters Microbiological Evaluation of Clean Rooms and Other Controlled Environments, Ch. 1116 Microbiological Attributes of Pharmaceutical Articles, Ch. 1111 Pharmaceutical Dosage Eorms, Ch. 1151 Validation of Compendial Methods, Ch. 1225 Water for Pharmaceutical Purposes, Ch. 1231. In U.S.P. 24. 

It must be stressed here that method validation is as important and complex in microbiology. Similar steps and precautions exist. An important part of the book edited by Lightfoot and Maier [6] deals with all these aspects from development and validation of the method, validation of instruments and supplies up to the use of control charts. The reader is referred to these guidelines and to sections 2.3.3 and 2.4.3. 

Supported by the overall development in all fields of analysis during the past few decades, a precise analytical methodology has been developed for the different aspects of quality control, comprising physicochemical, biotechnological, sensory and microbiological methods. In order to meet the sense of the quality control system and by that the customers requirements, all methods applied have to be validated by adequate quality assurance tools. 

U.S. Pharmacopeia (USP) (2007), Validation of alternative microbiological methods, general chapter <1223>, U.S. Pharmacopeial Convention, Rockville, MD. 

It may be important to consider the variability of the matrix due to the physiological nature of the sample. In the case of LC-M/MS-based procedures, appropriate steps should be taken to ensure the lack of matrix effects throughout application of the method, especially if the nature of the matrix changes from the matrix used during method validation. For Microbiological and immunoassay, if separation is used prior to assay for study samples but not for standards, it is important to establish recovery and use it in determining results. In this case, possible approaches to assess efficiency and reproducibility of recovery are ... 

Work Item K validation of alternative microbiological methods. 

Analytical method validation—Aprerequisite for any validation involving the analysis of the microbiological, physical, or chemical aspects of materials is the use of analytical methods that have been demonstrated to be reliable and reproducible. No meaningful assessment of product or material quality can be made without the... 

This effort must extend to the microbiological laboratory as well, in which validation of methods is essential to assure confidence in the results. This will include appropriate testing environments, laboratory sterilization/depyrogenation validation, equipment qualification and calibration, use of standards, and positive and negative controls. 

Test procedures should include all parameters that are susceptible to change during storage and that are likely to influence quality, safety, and/or efficacy. These include, as appropriate, physical, chemical, biological, and microbiological characteristics, and for drug products, loss of preservative and functionality. Validated, stability-indicating methods should be used. 

The science that underpins steam sterilization is well known and has been long established. It is the preferred method of sterilization in the pharmaceutical industry it is used for sterilization of aqueous products in a wide variety of presentations, for sterilization of equipment and porous materials required in aseptic manufacture, in microbiology laboratories for sterilizing media and other materials, and for sterilization of massive systems of vessels and pipework [steam-in-place (SIP) systems]. Numerous rules and guidelines have been published on the topic, yet steam sterilization and particularly bio-validation of steam sterilization is still a subject for controversy and debate. 

As regards food and water microbiology, current outbreaks in Europe have made apparent the necessity for official inspection agencies or ministry departments and for food industries or water agencies to get laboratories accredited quickly to perform tests in accordance with new test parameters, or to apply current methods to new ranges of food products. Accredited laboratories must be able to develop and validate new methods, or to derive methods from standards. Official recognition of these new test methods or their official incorporation into the scope of accreditation takes a long time, and audits are required for validation of these test methods. 

Its implementation is easy in small countries due to centralization of management and ease of surveillance of this new form of accreditation by a national accreditation body. Due to the long-standing establishment of validation of food microbiological methods in some countries, the beginning of validation of water microbiological methods, and establishment of European standards for the validation of food microbiological test methods (Microval project), the flexible-scope type of accreditation must be adapted to certain national requirements, but could prove less complex than the standard requirements. 

It is very important to select promptly the most effective antibiotic for successful therapy of infectious diseases, and wound and post-surgical infections. The duration of standard microbiology assays applied in clinical practice exceeds 3-5 days since preliminary isolation of the pathogen from the clinical sample is required. In the present study we optimized a rapid bio luminescent antibiotic susceptibility assay based on comparison of bacterial ATP concentrations (bioluminescent signals) in a control (aliquot of the sample, free of the antibiotic) and probe (aliquot of the sample, supplied with antibiotic examined) after short-time incubation. For validation of the proposed assay, bacteria strains isolated from clinical samples were analyzed in parallel by the Bioluminescent Assay and Standard Microbiology Assay - Disk Method or Serial Dilutions Method. 

---