## Table 918

Summary of Analysis of Variance, Example 9.5, a Turbidimetric Assay Using a 3 2 Dose Ratio Sum of Mean Variance Limiting Probability Calculated Source of Variance d.f. Squares Squares Ratio Value Level, P Probability Preparations 1 135.40 135.40 3.740 < 4.41 0.050 0.069 Regression 1 68,644.00 68,644.00 1,895.952 > 15.38 0.001 < 0.001 Parallelism 1 64.00 64.00 1.768 < 4.41 0.050 0.200 (deviations from) Quadratic curvature 1 660.10 660.10 18.232 < 4.41 0.050 0.001 Opposed curvature 1...

## The quantitative theory of microbial growth and inhibition

General experience of turbidimetric assays shows that when some measure of turbidity absorptiometric or nephelometric is plotted against logarithm of a dose of growth-inhibiting substance over a sufficiently great range of concentrations, the resulting line approximates to part of a sigmoid curve, as is suggested by Figure 1.3. Various mechanisms could be postulated to explain this sort of curve

## Which mathematical model

The foregoing descriptions show the principles of the two mathematical models. In fact, the calculation procedures used are complex. They take into consideration random error and assess the extent of deviations from the model. Deviations that are too great might suggest invalidity of the assay. The applicability of the two models is as follows 1. Agar diffusion assays, whether of growth-promoting or growth-inhibiting substances, give responses that are treated as parallel-line assays. The lines...

## Official reference materials

The ultimate reference materials are those established under the auspices of the World Health Organization (WHO). International Biological Reference Preparations were being established up to 1986. In some cases, potencies were assigned to these. Since 1986, only International Biological Standards are being established in all cases, a potency in terms of International Units (IU) is assigned, generally on the basis of an international collaborative study. An International Unit is defined as the...

## Concluding Thoughts

In the preceding chapters, we have considered the theory and practice of the three main types of microbiological assay. We have considered the objectives of the assay, the degree of accuracy, precision and specificity required and discussed choice of assay design appropriate to the objectives of the assay. Finally, we need to decide how we shall report the results of our work. Normally, assays are done at least in duplicate but sometimes the replication may be much higher, for example in the...

## Preface

One must have reasons for writing a book the preface provides an opportunity to explain those reasons. My background is that of an analytical chemist. By chance, I became involved in microbiological assay some decades ago and also became intrigued by the seemingly unusual calculations that were used to arrive at estimated potency and confidence limits for the estimate. I was fortunate in having excellent tutors in particular, Peter Tootill, who had been prominent in promoting the use of good...

## Assay for Pharmaceutical Analysis

Boca Raton London New York Washington, D.C. This edition published in the Taylor & Francis e-Library, 2005. To purchase your own copy of this or any of Taylor & Francis or Routledge's collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk. Library of Congress Cataloging-in-Publication Data Microbiological assay for pharmaceutical analysis a rational approach I William Hewitt, p. cm. Includes bibliographical references and index. ISBN 0-8493-1824-6 1. Microbiological...

## Table

Relative Potencies of Two Major Components of Polymyxin B1 as Determined Using Agar from Different Sources Agar Source Antibiotic Component Relative Potency Eiken Polymyxin B1 100 4.9 of agar, or with agar from a different source that was qualitatively different, then relative zone sizes for standard and unknown could vary according to composition of the medium. Such a case was reported by Fujihara et al. (1994). They observed that in the assay of polymyxin B1 there were significant differences...

## Measuring responses

The measured response is either the size of a zone of inhibition or exhibition in the agar diffusion assay or a measure of turbidity in a tube assay. Traditionally, the diameter of each zone is measured to the nearest 0.1 mm. A convenient way of doing this is to view the plate, illuminated from below, against a black background. A suitable plate-reading box is illustrated in Figure 6.1. Machinists' vernier calipers with needles soldered to the jaws may be used for measuring the points are used...

## Standard Reference Materials

In the early twenty-first century, we accept the need for reference standards for biological tests without question. It is interesting to look back at the rather slow development of that recognition. Burn (1950) drew attention to the early definition of the English standard for the yard. A yard was defined as the length of a king's arm. In a museum in Winchester, the ancient capital of England, there are examples of the standard yard. Unfortunately, due to changes of king, these standard yards...

## Info

A The degree of replication n can be increased to any number by using more plates. b These are asymmetrical designs. c The number of preparations m can be increased from 2 to any number by adding one more set of plates for each additional unknown. d The abbreviations LS and QLS represent Latin square and Quasi-Latin Square, respectively. a The degree of replication n can be increased to any number by using more plates. b These are asymmetrical designs. c The number of preparations m can be...

## The different sorts of microbiological assay

The main use of microbiological assay is in the determination of the potency of growth-inhibiting substances especially antibiotics and of growth-promoting substances amino acids and vitamins of the B group. There are three main assay procedures 1. The agar diffusion assay, which is applicable to both growth-inhibiting substances and growth-promoting substances 2. The flask or tube assay for growth-promoting substances 3. The tube assay for growth-inhibiting substances Although methods 2 and 3...

## What Do We Want of an Assay How Do We Attain Our Goal

The determination of sample potency may be required for a variety of reasons. It is important for the analyst to consider the purpose of the analysis so that he or she may then select a procedure that will give results of the required quality by a method that is cost effective. A large variety of experimental designs is available to the analyst these designs and their capabilities will be described elsewhere in this book. Suffice it to say here that they range from simple tests of low precision...

## Linearization of sigmoid curves

The need to transform sigmoid response lines into a more convenient form for arithmetical manipulation arose in connection with microbiological assays. Procedures for linearization were proposed independently by Hemmingsen (1933) and Gaddum (1933). They used the normal distribution curve in its cumulative form to manipulate quantal responses into a more manageable form. The two forms of the normal distribution curve are shown in Figures 4.5a and b. A quantal response is an all-or-none effect,...

## Critical factors in the assay of growthpromoting substances

These are the critical factors and less-critical factors Dose levels. These should be high enough to permit adequate growth of the organism so that turbidity can be measured accurately. They should not be so high as to necessitate prolonged incubation to attain linearity of the dose-response line. Variation in size of inoculum in individual tubes. This is not very serious. Provided that incubation proceeds long enough, small initial differences should not influence final cell concentration....

## General Practical Aspects of Microbiological Assays

Microbiological assay (MBA) is defined here as the estimation of potency of a growth-promoting (GPS) or growth-inhibiting substance (GIS) by comparing its quantitative effect on the growth of a specific microorganism with that of a reference standard of defined potency. It is common belief that being a biological assay, MBA is subject to biological error and is, therefore, inherently less reliable than chemical or physicochemical methods. However, according to Coomber et al. (1982), it is...

## Slope Ratio Assays Some Designs and Their Evaluation

It is generally in the case of turbidimetric assays of vitamins and amino acids that the slope ratio assay is applicable. There are official assay procedures described in the USP, United States Pharmacopoeia, but not in the EP, European Pharmacopoeia. Despite their absence from monographs, the EP, European Pharmacopoeia does give guidance on the evaluation of slope ratio assays. The ideal concepts on which slope ratio assays are based are 2. Responses that are directly proportional to dose...

## The Agar Diffusion Assay Its Quantitative Basis

Penicillin was discovered by Alexander Fleming in 1929. It was in 1939 that a program of research began with a view to possibly manufacture penicillin or other substances having antibacterial activity. This project was at the Sir William Dunn School of Pathology in Oxford, England and was directed by Howard Florey and Ernst Chain. Penicillin proved to be the most promising candidate substance. Norman George Heatley, a biochemist, was assigned the task of developing conditions of growth of the...

## Q

FIGURE 11.5 Illustration of the variation of bias due to curvature with changing true potency ratio of unknown to reference standard in a streptomycin assay using Bacillus subtilis in a 3 + 3 assay with 2 1 dose ratio. -0.4 -0.2 0.0 0.2 0.4 Logarithm of Known Potency Ratio

## Other sources of error

Apart from the general sources of error described in Chapter 6, there are some sources of error peculiar to tube assays for growth-promoting substances 1. Cleanliness of glassware is of paramount importance. Traces of adsorbed vitamin, antibiotic, or detergent may affect growth in individual tubes, leading to erratic responses. 2. Because of the very low concentration of active substance in test solutions in the case of some assays, adsorption onto glassware during preparation of the test...

## Bio Assay Calculation By Latin Square Plate

Absorbance, 40, 41 Absorptiometry, See Staphylococcus aureus, 38-39 Agar diffusion assays, 9-21 applicability, 7 aseptic techniques, 73, 74 description, 3-4 dose-response line, 3 experimental designs for, 183-187 historical introduction, 9-10 inoculum preparation, 65 large plate using four-dose level standard curve, 186-187 potency estimate calculation, 18-20 preliminary evaluation, 90 reference standards for, 79-80 response measurement, 75-76 substituting values, 95 two-dose level using petri...

## T4

Although ideally, the tubes should be placed in the rack in accordance with a randomized pattern, this may cause operational problems that are time consuming and accident prone. Problems of positional differences are perhaps better resolved by the use of a very well-stirred water bath. Kavanagh (1972) describes a high-precision water bath used in the Autoturb system. It is claimed that temperatures in different positions in the bath do not differ by more than 0.02 C. choosing a design for...

## National and regional reference materials

The effort involved in establishing a standard subsidiary to the international standard is very substantial. It entails the selection of suitable material, its packing in a form that will ensure its stability over many years, then a collaborative assay by several laboratories, followed by the statistical evaluation of the raw data and allocation of a potency in accordance with the findings of the statistical evaluation. Many countries have established national reference standards. However, the...