In this document the various end point dilution methods like ID50, LD50, TCID50, EID50 were detailly explained.

1. End point dilution methods:
Endpoint dilution methods are laboratory techniques used to determine the
infectivity rate of an infectious agent, such as a virus or a bacterium, in a
sample. These methods involve serially diluting the sample and then
determining the highest dilution at which the infectious agent is still capable of
causing infection or producing a specific observable effect.
Types:
 ID50
 LD50
 EID50
 TCID50
LD50
LD50 stands for lethal dose 50. It’s a dilution method used to determine the lethal
dose of a substance by diluting it and testing its effects on test animals. It refers
to a dose of a substance that expected to be lethal to 50% of test subjects.
Procedure:
1. Selection of Test Animals: Choose appropriate animal species for the study, often
rodents such as mice or rats. Consider factors such as size, availability, and similarity
to humans in terms of metabolism and physiology.
2. Grouping and Randomization: Divide the animals into groups, usually consisting of
10 or more animals per group. Randomly assign animals to different groups to
minimize bias.
3. Dose Range Determination: Determine a range of doses to be tested. Start with a
relatively low dose and increase it in a stepwise manner to higher doses. The range of
the doses expected to cause neither minimal effects nor cause severe toxicity and
death.
4. Dose injection: Inject the test substance to each group of animals according to the
chosen dosing route, which can be oral, intravenous, intraperitoneal, etc. The
substance may be injected as a solution, suspension, or solid form, depending on its
characteristics.
5. Observation and Recording: Observe the animals closely for a predetermined
period, typically 14 days, noting any signs of toxicity, illness, or death. Record
observations such as changes in behaviour, appearance, food and water intake, body
weight, and mortality.
6. Data Analysis: Calculate the LD50 value using statistical methods. The LD50 is the
dose at which 50% of the animals in a group die.

2. ID50
It measures the minimum size of a population of infectious agents required to
start an infection with 50% probability. (or) Infectious dose of a pathogen
required to infect 50% of the total exposed population.
Procedure:
1. Selection of Test Animals: Choose appropriate animal species for the
study, often rodents such as mice or rats. Consider factors such as size,
availability, and similarity to humans in terms of susceptibility to the
infectious agent.
2. Grouping and Randomization: Divide the animals into groups, usually
consisting of 10 or more animals per group. Randomly assign animals to
different groups to minimize bias.
3. Dose Range Determination: Determine a range of doses to be tested.
Start with a relatively low dose and increase it in a stepwise manner to
higher doses. The range should include doses expected to cause no or
minimal effects as well as doses likely to cause infection in a significant
proportion of animals.
4. Dose Administration: Administer the infectious agent to each group of
animals according to the chosen route of infection. This could involve
intranasal, intravenous, intraperitoneal, oral, or other relevant routes of
exposure. Prepare the infectious agent in an appropriate form, such as a
suspension or solution, to ensure uniform and controlled delivery.
5. Observation and Recording: Observe the animals closely for a
predetermined period, typically monitoring for signs of infection or
illness. Record observations such as changes in behaviour, appearance,
body weight, survival, and any other relevant parameters.
6. Data Analysis: Calculate the ID50 value using statistical methods. The
ID50 is the dose at which 50% of the animals in a group become infected.
It is usually determined by interpolation between the highest dose that
results in less than 50% infection and the lowest dose that results in more
than 50% infection.

3. EID50
Egg infective dose 50 is a test that is used to determine the amount or quantity
of a viral sample that will infect 50% of inoculated eggs.
Procedure:
1. Preparation of Embryonated Eggs: Obtain specific-pathogen-free
(SPF) embryonated eggs from a reliable source. The eggs are typically
chicken eggs that have been fertilized and incubated for a specific period
to develop embryos. Check the health and viability of the eggs before use.
2. Grouping and Randomization: Divide the embryonated eggs into
groups, typically consisting of 10 or more eggs per group. Randomly
assign eggs to different groups to minimize bias.
3. Dilution Series: Prepare a series of serial dilutions of the virus or
pathogen to be tested. The dilutions should cover a range of
concentrations, including dilutions that are expected to result in no
infection and those likely to result in infection in a significant proportion
of the eggs.
4. Inoculation of Eggs: Inoculate each group of embryonated eggs with a
specific dilution of the virus or pathogen. The inoculation is typically
performed by injecting the virus or pathogen into the egg through the
shell or a small hole made in the shell using a fine needle. Care must be
taken to avoid contamination during the inoculation process.
5. Incubation: Incubate the inoculated eggs under controlled conditions,
typically at a specific temperature and humidity suitable for the
development of the embryos and virus replication. The incubation period
depends on the specific virus or pathogen being studied and is usually
predetermined based on previous knowledge or established protocols.
6. Observation and Recording: Monitor the eggs for signs of infection or
disease over the incubation period. Observe characteristics such as

4. embryonic death, growth retardation, deformities, or other visible signs of
infection. Record the number of infected and uninfected eggs in each
group.
7. Data Analysis: Calculate the EID50 value using statistical methods. The EID50 is the
dilution at which 50% of the eggs in a group are infected. It is typically determined by
interpolation between the highest dilution resulting in less than 50% infection and the
lowest dilution resulting in more than 50% infection.
TCID50
50% Tissue Culture Infectious Dose (TCID50) assays are virus titration
experiments which can be used to quantify virus infective rate by measuring the
cytopathic effects of a virus on an inoculated host cell culture. TCID50 assays are an
important tool for the evaluation of anti-viral drugs, as they enable the investigation of
the effect of drugs on the life cycle of a virus.
PRINCIPLE
The 50% Tissue Culture Infectious Dose (TCID50) used to quantify and to assess
the infectivity of a virus in cells. Compared to the plaque assays, TCID50 assays offer
the advantage that even viruses that do not form plaques or infect cell monolayers can
be quantified.
In TCID50 assays, varying virus dilutions are added as an endpoint dilution to host cell
populations with the same number of cells and incubated until a cytopathic effect can

5. be seen. Here, the TCID50 value represents the amount of virus dilution required to
induce cytopathic effects in 50% of the inoculated cell culture after a defined period.
TCID50 assays assess this threshold either by visually counting the number of
affected wells or by using cell viability assays as readout. The TCID50 value is
determined when the cytopathic effect or cell viability assay read-out appear the same
for a dilution in 3 separate readings. An example of the application of cell
viability/toxicity assays for the evaluation of viral cytopathic effects can be found in
the Viral cytopathic effects measured in a drug discovery screen.
TCID50 calculation
The results of 50% Tissue Culture Infectious Dose (TCID50) assays can be
analysed by different calculations. Several mathematical approaches have been
developed for this purpose, including the Reed-Muench , Spearman-Kärber or Weil
method. The formula after Reed-Muench is depicted as an example below.
Where I is the interpolated value of the 50% endpoint and h is the dilution factor.
TCID50 assays in microplate format
50% Tissue Culture Infectious Dose (TCID50) assays can be performed with a
variety of readouts in comparison to plaque assays which relies on plaque formation.
While traditionally, the wells which show a cytopathic effect are counted on a
microscope or microplate readers offer automated and sensitive approaches for the
qualitative measurement of TCID50 assays using absorbance, fluorescence,
or luminescence readouts. Most often, cell viability assays such as MTT are used for
this purpose, where the decrease in cell viability are monitored as indicator of cytopathic
effects due to virus replication. Another way to assess the infectivity of a virus in a cell
culture is by immunostaining for a viral antigen. Immunofluorescence can also be used
as readout of TCID50 assays. Since the immunofluorescence staining of viral antigens
offers overall a higher sensitivity than cell viability experiments, even infections that
do not induce a strong cytopathic effect in cells can be detected.

6. PROCEDURE:
1. Cell Culture:
 Prepare the appropriate cell culture medium and supplements for the cells you
will be using. Common cell lines used for TCID50 assays include Vero cells
or MDCK cells, depending on the virus being tested.
 Seed the cells in appropriate culture vessels (e.g., multiwell plates or T-25
flasks) and incubate them until they reach the desired confluency (usually 80-
90% confluency).
2. Virus Sample Dilution:
 Prepare serial dilutions of the virus sample using a virus diluent or culture
medium. The dilution series should typically start with a high concentration
and gradually decrease.
 For example, you can start with a 1:10 dilution, then perform further 1:10
dilutions (e.g., 1:100, 1:1000, etc.) to create a range of dilutions.
3. Inoculation of Cells:
 Remove the culture medium from the cell culture vessels and add the diluted
virus samples to the cells.
 Include positive and negative controls (e.g., virus-free diluent) along with the
virus samples.
 Incubate the cells at an appropriate temperature and CO2 concentration for the
virus being tested. This is usually 37°C with 5% CO2, but it can vary
depending on the virus.
4. Observation and Scoring:
 After an appropriate incubation period (e.g., 3-7 days), examine the cells for
the presence of cytopathic effects (CPE) caused by the virus.
 CPE may include cell rounding, detachment, syncytium formation, or other
characteristic changes depending on the virus.
 Score each well or dilution based on the presence or absence of CPE and
record the results.
5. Calculation of TCID50:
 Use the results from the scoring to calculate the TCID50 titer of the virus
sample using statistical methods, such as the Spearman-Karber method or the
Reed-Muench method.
 These methods involve determining the dilution(s) that produce a 50% CPE
response and applying mathematical formulas to calculate the TCID50 titer.

7. 6. Data Analysis and Interpretation:
 Analyze and interpret the TCID50 titer results obtained from the assay.
 The TCID50 titer represents the reciprocal of the highest dilution that
produces a 50% CPE response. A lower TCID50 titer indicates a higher viral
concentration in the original sample.