Fish 424
Virology
Laboratory Exercises (Lab 6)
Introduction
Viral culture is very important for diagnostics and experimental research with
regards to fish health. To culture, quantify, and often identify viral pathogens an
appropriate cell line is needed. This laboratory exercise will provide an
introduction to cell and viral culture, viral quantification, and observations of cell
and tissue damage resulting from certain viral pathogens.
Materials
Plaque assay cell plates
Dissection microscope
Compound microscope
Histology slides
Calculator
Procedure
Plaque assay and PFU’s
Obtain a cell plate that has been stained and, at the appropriate dilution (counts
should be in the range of 8 to 80 plaques per well) count the plaques for each
well for that dilution. Calculate the PFU’s/ml using the formulas given (second
page)
TCID50
Using the same cell plate as above, calculate the TCID50/ml infectious dose for
used to infect the plates using the formulas given (second page)
Cellular examination and impact
Observe the effect that virus can have at the cellular level on tissue and cell
culture slides. Note differences observed between uninfected control tissues and
cells vs. their infected counterparts.
All calculations for PFU’s and TCID50 should be recorded in your laboratory
notebook along with any notes or drawing that you find helpful in distinguishing
infected vs. no infected cell culture or histological tissues
Plaque Assay (PFU’s)
Calculating viral titer based on the plaque assay method
The viral titer is a quantitative measurement of the biological activity of a virus
and is expressed as plaque forming units (pfu) per ml. To calculate the viral titer,
count the number of well isolated plaques. Then use the following formula to
determine the titer (pfu/ml) of your viral stock.
Average # Plaques = PFU/ml
DxV
D = Dilution factor
V = Volume of diluted virus
added to the well
Example:
Wells observed 7 days after inoculation
with of 0.1ml viral solution
PFU’s/ml = 42 plaques observed plaques
(10-7 dilution factor)(0.1ml virus added)
TMTC
TMTC
TMTC
PFU’s/ml = 4.2 x109
42
5
0
Multiplicity of Infection:
Multiplicity of infection (m.o.i.) is the average number of virus particles which
infect a cell.
where:
m.o.i. = -ln P[0]
ln = natural log
P[0] = proportion of uninfected cells
In most virus infections, there are 3 classes of infected cells. Once you have P[0]
you can calculate the other classes:
Uninfected cells: P[0] = e-m
Cells infected with a single virus particle: P[1] = me-m
Cells infected with multiple virus particles: P[>1] = 1-(e-m(m+1))
Reed & Muench TCID50
The TCID50 method allows you to simply add up the total number of positive wells
from a plate and convert it to a titer that represents an endpoint (the tissue
culture infectious dose is 50% at this point). Use the following formula to perform
the calculation:
i. Proportionate Distance =
(% CPE at dilution above 50%) – (50%)
(% CPE at dilution above 50%) – (% CPE at dilution below 50%)
ii. -Log = dilution above 50% CPE ratio (i.e. 10-3 would be -3)
iii. ((PD)+(-log(dilution interval))
iv. TCID50 = 10(ii + iii)
This will give you the dilution of the original suspension that would be equal to
the TCID50. The reciprocal would give you the # of TCID50 in the original
suspension applied to the wells (usually 0.1 or 0.2 ml). To determine the titer per
ml, multiply by the reciprocal of the volume of the inoculum, then convert to log10.
Example:
Wells observed 7 days after
inoculation of 0.1ml of viral solution
PD = (66 – 50)
(66 – 33)
PD = .48
1
-Log dilution above 50% = 4 (from
10-4)
2
3
4
5
A
10-1
10-5
B
10-2
10-6
C
10-3
10-7
D
10-4
6
4(-Log)+ .48(PD)=4.48
4.48
TCID50 = 10
Control
/0.1ml infection dose
TCID50 = 105.48/ml viral titer
Dilution
10-1
10-2
10-3
10-4
10-5
10-6
10-7
Infected
3/3
3/3
3/3
2/3
1/3
0/3
0/3
% Infected
100
100
100
66
33
0
0