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Diagnostic Procedure
Colour Development in Plates
Trouble Shooting
Typical Photometer Print Out
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1. If colour development (Colour Development in Plates) occurs in the control wells of the microtitre plates (rows A to H, columns 1 to 4 on plate 1, checked before reading of plate) this indicates that the wells have been correctly coated with reagents. Colour reaction in the test wells should be estimated approximately by eye, and can be compared with optical density (OD) values, particularly as an aid to assessing any discrepant readings.
2. Calculate the mean background reactions for each plate by adding the the OD values of wells 5 and 6 for each row (serotype) and dividing by two; these values are due to the reagents and not to a specific reaction between antigen and antisera. Subtract each mean background OD for each serotype from the recorded OD for that serotype to obtain the 'corrected' value. For example, for type O row A, add the OD values for wells 5 and 6, and divide by two to give the mean background OD for row A. Subtract this figure from all recorded OD values in row A. Repeat for each row (serotype). A mean of each group of two wells for each test sample can thus be obtained to give a mean 'corrected' OD value for each sample against each antiserum serotype.
These calculations can usually be performed automatically by the photometer, and an example of a typical photometer print out, giving both recorded and 'corrected' ODs, is shown.
3. Plates are accepted if the mean corrected OD of the strong and weak positive controls is greater than a value of 0.1 above background. For test samples a mean corrected OD of > 0.1 above background indicates a positive result and the serotype can be read. Values close to 0.1 should be treated with caution before assuming the serotype of a test sample, and may need to be confirmed by either re-testing or by amplification of antigen through passage and testing of fresh supernatant fluid.
If there are problems (Trouble Shooting) tests may have to be repeated.
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