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Benjamin M.

de Vera III, RMT 1 | P a g e
M209 (Applied Virology)

1.0ml 1.0ml 1.0ml 1.0ml 1.0ml 1.0ml
1.0ml
Virus

9.0ml
Medium
+
M209: TCID50 and Neutralization Laboratory Activity
(SET 2)

A. TCID50 Determination

CASE: A 10-fold dilution of the virus isolate was used for the neutralization with a starting dilution
of 1:100. TCID50 determination was done first. The generated CPE ratio per dilution is as follows:
1:100 = 3/3, 1:1,000 = 3/3, 1:10,000 =3/3, 1:100,000 = 1/3, 1:10,000,000 = 0/3.

Answer the following:

1. Generate the diagram of virus dilution.










1X10
2
1X10
3
1X10
4
1X10
5
1X10
6
1X10
7

+ + + + + -
+ + + + - -
+ + + + - -
3/3 3/3 3/3 3/3 1/3 0/3
100% 100% 100% 100% 33% 0%

2. Generate the table for Reed and Muench method of TCID50 determination.

Virus
Dilution
Infections
per number
Inoculated
Observed Values Cumulative Values
Infection
Ratio
Percent
Infections
Positive Negative Positive Negative
10
2
3/3 3
0
13 0 13/13 100 %
10
3
3/3 3 0 10 0 10/10 100 %
10
4
3/3
3
0 7 0 7/7 100 %
10
5
3/3 3 0 4 0 4/4 100 %
10
6
1/3 1 2 1 2 1/3 33 %
10
7
0/3 0 3 0 5 0/5 0 %




Benjamin M. de Vera III, RMT 2 | P a g e
M209 (Applied Virology)

3. Compute for the following:
a. TCID50 using Reed and Muench method




=(


)

=(

)

= 6.33 (approx.) - 0.19

TCID50 = .
b. TCID50 using Karber method










=[ −

+ ∑

+

+

+

+

]

= [(7 - 0.5) + (1 x 0.33)]

=6.5 + 0.33

TCID50 = .

c. 100 TCID50
= 0.1 ml x 1
6.13

= 0.0163

= .

4. For actual neutralization assays, 100 TCID50 is the dilution used. Why is this so?
This dilution should be determined for each virus and / or study design but often is a substantial
excess of TCID50 (more often to be 100 TCID50). This is to ensure maximal cytopathic effect in the
absence of antiviral agents.

B. Neutralization
CASE: An African patient from a highly endemic country for poliomyelitis was suffering from acute
upper respiratory tract infection with GIT disturbance. After several days, the symptoms progressed
to loss of superficial reflexes with presentation of muscle spasms and uncoordinated non-paralyzed
muscles. The doctor requested several laboratory tests including neutralization. Two sets of
samples were submitted more than a week apart.

Note: When interpreting the results, be sure to compare against the recommended cut-off titers for
polio. You may also consider four-fold increase in titers if applicable to your case.

Benjamin M. de Vera III, RMT 3 | P a g e
M209 (Applied Virology)

TITERS
POLIO 1
ACUTE CONVALESCENCE
TRIAL 1 TRIAL 2 TRIAL 3 TRIAL 1 TRIAL 2 TRIAL 3
1:5 CPE CPE CPE CPE CPE CPE
1:10 CPE CPE CPE CPE CPE CPE
1:20 CPE CPE CPE CPE CPE CPE
1:40 CPE CPE CPE CPE CPE CPE
1:80 CPE CPE CPE CPE CPE CPE
1:160 CPE CPE CPE CPE CPE CPE

TITERS
POLIO 2
ACUTE CONVALESCENCE
TRIAL 1 TRIAL 2 TRIAL 3 TRIAL 1 TRIAL 2 TRIAL 3
1:5 CPE CPE CPE NO CPE NO CPE NO CPE
1:10 CPE CPE CPE NO CPE NO CPE NO CPE
1:20 CPE CPE CPE CPE CPE CPE
1:40 CPE CPE CPE CPE CPE CPE
1:80 CPE CPE CPE CPE CPE CPE
1:160 CPE CPE CPE CPE CPE CPE

TITERS
POLIO 3
ACUTE CONVALESCENCE
TRIAL 1 TRIAL 2 TRIAL 3 TRIAL 1 TRIAL 2 TRIAL 3
1:5 NO CPE NO CPE NO CPE NO CPE NO CPE NO CPE
1:10 NO CPE NO CPE NO CPE NO CPE NO CPE NO CPE
1:20 NO CPE NO CPE NO CPE NO CPE NO CPE NO CPE
1:40 NO CPE NO CPE NO CPE NO CPE NO CPE NO CPE
1:80 NO CPE NO CPE NO CPE NO CPE NO CPE NO CPE
1:160 NO CPE NO CPE NO CPE NO CPE NO CPE NO CPE


Answer the following:
1. Interpret the given virus neutralization readings against three polio strains.
CPE = Absence of the virus NO CPE = Presence of the virus
POLIO 1 – Negative. Observation of CPE in both acute and convalescent sample
POLIO 2 – Negative. NO CPE observed only in the lower titers of the convalescent sample
POLIO 3 – Positive. Consistent NO CPE in both acute and convalescent sample

2. Discuss the significance of sample collection in performing neutralization.
Increase in titer of virus can be observed during acute phase, while significant decrease of titer is
presumed on convalescence (mounting of immune response). Ideal sample fir virus isolation and
neutralization is acute phase. False positive (No CPE) result may be observed if sample is not collected
at appropriate time due to decrease in virus titer. It is vital that time of shedding is expected and should
be considered.