by William Hewitt

• Explores how to design an efficient assay that minimizes sources of error
• Facilitates real understanding of the validity of an assay rather than just acceptance of the results of a statistical evaluation
• Discusses the criteria for making the decision to accept or reject the results of an assay
• Covers issues of sensitivity, specificity/selectivity, accuracy, and precision
• Illustrates the wide range of experimental designs available to the analyst and compares their properties
• Includes inspection techniques from European and United States pharmacopoeias

A user-friendly guide for the evaluation of microbiological assays, Microbiological Assay for Pharmaceutical Analysis: A Rational Approach provides a lucid explanation of the sources of error in microbiological assay and helps analysts choose efficient assay designs that will minimize those sources of error.

Beginning with a review of the theoretical basis for the quantitative aspects, it discusses microbiological assay as a branch of pharmaceutical analysis and distinguishes it from biological assay in general. The book draws attention to the microbiological aspects that may not be so obvious to the chemical analyst and to the analytical aspects that may not be so obvious to the microbiologist.

This reference contains detailed evaluations of assays that illustrate typical experimental designs and addresses how to present a realistic assessment of the best potency estimate from a series of assays. Although there are other valuable books available in this area, they do not address evaluation.

Microbiological Assay for Pharmaceutical Analysis: A Rational Approach expands on the guidance given in pharmacopoeias and helps you choose the assay design most appropriate for the purpose of your assay.

Contents

1. MICROBIOLOGICAL ASSAY IN PERSPECTIVE
   • Biological Assays in General
   • The Different Sorts of Microbiological Assay
   • The Basis of Calculation of Potency Estimates
   • Which Mathematical Model?

2. THE AGAR DIFFUSION ASSAY - ITS QUANTITATIVE BASIS
   • Historical Introduction
   • The Theory of Zone Formation
   • Principle of Calculation of Potency Estimate

3. THE THEORY AND PRACTICE OF TUBE ASSAYS FOR GROWTH PROMOTING SUBSTANCES
   • Introduction
   • The Mode of Action
   • Critical Factors in the Assay of Growth-Promoting Substances
   • Other Sources of Error

4. THE THEORY AND PRACTICE OF TUBE ASSAYS FOR GROWTH INHIBITING SUBSTANCES
   • Historical Introduction
   • Measurement of Response
   • The Form of Response Line
   • Historical Development of the Turbidimetric Method
   • Linearization of Sigmoid Curves
   • The Quantitative Theory of Microbial Inhibition
   • A Practically Determined Log Dose-Response Curve
   • Factors Affecting Final Cell Count
   • Summary and Conclusions

5. WHAT DO WE WANT OF AN ASSAY? HOW DO WE ATTAIN OUR GOAL?
   • Introduction
   • Pharmacopoeial Intention
   • Control of Antibiotic Bulk Materials
   • Control in Routine Manufacture
   • Research and Development

6. GENERAL PRACTICAL ASPECTS OF MICROBIOLOGICAL ASSAYS
   • Introduction
   • Inoculum
   • Test Solutions
   • Weighing
   • The Assay Medium
   • Selection of Latin Squares and the Plating Routine
   • Aseptic Techniques
   • Measuring Responses
   • Calculation of Potency Estimates and Confidence Limits

7. STANDARD REFERENCE MATERIALS
   • Historical Introduction
   • "Official" Reference Materials
   • National and Regional Reference Materials
   • In-House Standards

8. PRELIMINARY EVALUATION OF DATA
   • Introduction
   • Common Sense Inspection
   • Specific Tests for Abnormality
   • Detection of Outliers
   • Replacement of Missing Values
   • Summary and Conclusions

9. PARALLEL LINES ASSAYS - SOME DESIGNS AND THEIR EVALUATION
   • Introduction
   • A 3-Dose Level Assay Using Petri Dishes
   • A 3-Dose Level Assay for One Unknown Using a Large Plate and Latin Square Design
   • A 2-Dose Level Assay for Four Preparations [three unknowns] Using a Large Plate and 8 x 8 Latin Square Design
   • A 2-Dose Level Assay for Two Preparations [one unknown] Using a Large Plate and 8 x 8 Latin Square Design with
   • Two Weighings of Each Preparation
   • A 4-Dose Level Turbidimetric Assay for One Unknown
   • A Small Plate Assay Using a 5-Dose Level Standard Curve

10. SLOPE RATIO ASSAYS, SOME DESIGNS AND THEIR EVALUATION
    • Introduction
    • Some Possible Designs
    • Preliminary Evaluation
    • General Statistical Procedures
    • The Procedure of Bliss
    • Evaluation of a 2-Dose Level Assay (1) Using the Bliss Procedure (2) The Procedure of the European Pharmacopoeia

11. CHOICE OF EXPERIMENTAL DESIGN
    • Introduction
    • Available Experimental Designs for Agar Diffusion Assays
    • Regulations and Options
    • Factors Influencing Width of Confidence Limits
    • Bias Due to Curvature of the Response Line
    • Non-Paralellism Due to Curvature
    • Choosing a Design for a Turbidimetric Growth Inhibiting Substance Assay
    • Choosing a Design for Turbidimetric Assay of Growth Promoting Substance
    • Summary and Conclusions

12. CONCLUDING THOUGHTS
    • Introduction
    • Calculation of a Weighted Mean Based on Internal Assay Variation
    • Comparison of the European and International Pharmacopoeia Methods
    • Application of the Various Formulae to Some Practical Results

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