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ANNEX

Appendix 5Determination of contamination level in IF and FISH tests

1.

Count the mean number of typical fluorescent cells per field of view (c).

2.

Calculate the number of typical fluorescent cells per microscope slide window (C).

C = c × S/s

whereS=surface area of window of multispot slide
ands=surface area of objective field
s = πi2/4G2K2wherei=field coefficient (varies from 8 to 24 depending upon ocular type)
K=tube coefficient (1 or 1,25)
G=magnification of objective (100 ×, 40 × etc.).
3.

Calculate the number of typical fluorescent cells per ml of re-suspended pellet (N).

N = C × 1 000/y × F

wherey=volume of re-suspended pellet on each window
andF=dilution factor of re-suspended pellet.