Embryo Infective Dose 50 (EID50)

Dr.Hadeel M. F. Al-Aany Clin.
VIROLOGY
PhD.Med.Microbiol. 25March 2019 sun
Viral titration in tissue culture
Titration of virus mean approximately evaluation of viral units or viral

particles that can cause death in animals or infect the cells of tissue
culture by inducing pathological effects or plaque formation or by
inducing infection in chicken embryos as pustule on the embryo natal
membrane. The procedure of titration include preparation of tenfold
dilution of viral suspension then inject each dilution in pairs number of
animals , chicken embryos or tissue culture falcons such as 4, 8 or 10.
Evaluate the end point of reaction by evaluation of
Minimum dose of virus can killed half number of injected animals which
called Lethal Dose 50 (LD50).
Or minimum dose of virus can infect half number of injected animals
which called Infective dose50 ( ID50).
Minimum dose of virus can killed half number of chicken embryos which
called Embryo Lethal Dose50 (ELD50).
Minimum dose can infect half number of injected chicken embryo or
Embryo Infective Dose 50(EID50).
Minimum dose can induce pathololgical effect on half number of cells of
viral injected cell culture which is called Tissue Culture Infective
Dose50 (TCID50).
Titration of virus may be gained by plaque assay.
Viral titration Method by cell culture:
Materials
1.microtiter plate composed of 96 wells (flat bottom) contain confluent

monolayer. (falcon can be used but it is expensive ).
2.sterilized test tubes.
3.Culture media for viral dilution
4.microtiter micropipette
5.sterilized adhesive band
6.virus needed titration.
Dr.Hadeel M. F. Al-Aany Clin.VIROLOGY
Methods:
1.Make tenfold titration in sterilized test tubes by adding of 0.9ml of
preserve culture media to each test tube then added 0.1ml of virus to
first test tube mix well then transfer 0.1ml of this first tube to second tube
…then complete in the same way reaching 10th tube; discard last 0.1ml.
The tips must be changed with each transferring . this procedure made in
sterilized condition. To prevent contamination.
2.inject 0.05ml of each dilution of the 10 dilution in wells used as

control.
3.covering the plate by definite cover and use sterilized adhesive band for
strict covering.
4. incubate at 37c. notice the pathological changes for the duration of

7days for evaluation of TCID50.
Evaluation of viral titer:
End point is reciprocal dilution can cause pathological effects to 50% of

viral injected cell culture.
For example the last dilution cause pathological effect of 50% of cell
culture is 10−5 the end point is 105 that mean the virus titer is 105
.i.e.0.1ml of original viral suspension contain 100000 viral unit can
cause pathological effect in 50% of cell culture.
%of infected No.of normal No.of infected Viral

cells cells cells dilution
100 0 4 10−1
100 0 4 10−2
100 0 4 10−3
75 1 3 10−4
50 2 2 10−5
25 3 1 10−6
25 3 1 10−7
0 4 0 10−8
0 4 0 10−9
0 4 0 10−10
End point =105 viral titer=105 in this table the result restricted in one
dilution but if the result appear in 2 dilution in this case use equation for
measurement of titer this equation called proportional distance (P.D)
%𝑜𝑓 𝑚𝑜𝑟𝑒 𝑡ℎ𝑎𝑛 50% 𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑖𝑜𝑛−50%

(P.D) =
%𝑜𝑓 𝑚𝑜𝑟𝑒 𝑡ℎ𝑎𝑛 50% 𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑖𝑜𝑛−%𝑜𝑓 𝑙𝑒𝑠𝑠 𝑡ℎ𝑎𝑛 50%𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑖𝑜𝑛
For examble in this following table:

%of Mediu Summatio Summatio No. of No.of Viral
infecte m of n of n of norma infecte dilutio
d cells infecte normal infected l cells d cells n
d cells cells cells
100 20/20 0 20 0 4 10−1
100 16/16 0 16 0 4 10−2
100 12/12 0 12 0 4 10−3
88 8/9 1 8 1 3 10−4
71 5/7 2 5 1 3 10−5
28 2/7 5 2 3 1 10−6
11 1/9 8 1 3 1 10−7
0 0/12 12 0 4 0 10−8
71−50 21
P.D = 71−28 = 43=0.48
105 +0.48→titer of virus =10548
TCID50= 10548 TCID50/0.05ml
Another example
ACCUMULATED VALUES
VIRUS MORTALITY
DILUTION RATIO
DIED SURVIED DIED SURVIVED RATIO
(%)
10-3 6 0 17 0 17/17 100
10 -4 6 0 11 0 11/11 100
10 -5 4 2 5 2 5/7 71
10-6 1 5 1 7 1/8 13
10-7 0 6 0 13 0/13 0
PROPORTIONATE
DISTANCE = (% mortality at dilution next above 50%) - 50%
----------------------------------------------------------------
(% mortality at next __ (% mortality at next
Dilution above 50%) dilution below 50%)
EX.: (71 - 50) 21
------------ = ----- = 0.36 or 0.4
(71 - 13) 58
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Embryo Infective Dose 50 (EID50)

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Dr.Hadeel M. F. Al-Aany Clin.

Viral titration in tissue culture

Titration of virus mean approximately evaluation of viral units or viral

1.microtiter plate composed of 96 wells (flat bottom) contain confluent

2.inject 0.05ml of each dilution of the 10 dilution in wells used as

4. incubate at 37c. notice the pathological changes for the duration of

Evaluation of viral titer:

End point is reciprocal dilution can cause pathological effects to 50% of

%of infected No.of normal No.of infected Viral

%𝑜𝑓 𝑚𝑜𝑟𝑒 𝑡ℎ𝑎𝑛 50% 𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑖𝑜𝑛−50%

For examble in this following table:

DISTANCE = (% mortality at dilution next above 50%) - 50%

(% mortality at next __ (% mortality at next

Dilution above 50%) dilution below 50%)

EX.: (71 - 50) 21

------------ = ----- = 0.36 or 0.4

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