|
Karber Formula
Karber Method
Cytopathic Effect in
Tissue Culture
Conversion Chart
|
|
Interpretation
Positive results are shown by protected wells in which there is no microscopic evidence of CPE (Cytopathic Effect in Tissue Culture) or, if the plates have been stained, where the blue cell sheet remains. Negative results are shown by unprotected wells in which there is microscopic evidence of CPE or, if stained, where wells are empty.
1. Check the quality of the cells in wells C 10-12 on one VC (virus control) plate. Then read the VC titrations for the presence of CPE in appropriate wells and record it in terms of numbers of wells showing CPE out of eight, per virus dilution, per plate. Calculate the virus titre using the conversion chart. The virus dose is established by subtracting the CD (challenge dilution) at which test sera had reacted from the virus titre. Values between log10 1.5-2.5 TCID50 are acceptable. (An alternative dilution series and replicate numbers can be used to those proposed here. In this case the end point titre can be calculated from the Karber Formula.)
2. Record the presence of CPE in the RS (reference serum) plates, by noting the wells showing no CPE out of four, per serum dilution, per plate, at each CD. Calculate the RS titre at each virus CD using the conversion chart. This should normally be within log10 0.3 (i.e., two-fold) of the running mean.
3. Read the TP (test plates) and calculate titres of test sera using the Karber Method. Definitive titres are usually taken as those derived from plates (or wells) where the actual challenge dose used was as close as possible to 100 TCID50.
4. Test sera are considered positive, negative or inconclusive based on the number of wells showing CPE or protection from viral attack, provided that the acceptance criteria defined above for the virus dose and RS titre are met. On occasion, sera may also be considered negative if the virus dose is too low, and/or if the RS titre is too high. This is based on the grounds that the conditions have been biased in favour of neutralisation occurring.
5. Repeat the test if the cells in the controls are of such poor quality or cover that they cannot readily be distinguished from those where CPE is present. Re-test sera that show gross toxicity, such that so few cells are left they are not identifiable, or when CPE-like effects are produced. Sera are classified according to OIE criteria. Titres less than or equal to 1/11 are negative and values greater than or equal to 1/45 are positive. Values between 1/16 and 1/32 are considered inconclusive and a repeat sample is requested. Titres greater than or equal to 1/16 in the repeat sample indicate that the animal is considered positive for antibody to FMD virus.
|
