I have prepared a TCID50 assay, but I have doubts regarding how to consider a positive cytopathic effect. Do I need to take into account just those wells where the cytopathic effect appears in more than 50% of inoculated tissue culture cells? Or otherwise all the cultures showing cytopathic effects regardless of the percentage?
To calculate the TCID50 Reed and Muench method will be suitable. To calculate the number of infected wells just above 50% and just below 50% and use the equation to get the TCID50.
Hi Jaime! Please find the attached file. It helped me to understand the Red-Müench method. I compared it with Spearman-Kärber and Kärber methodsand the results were the same. Good luck.
Hi, I want add that the Reed-Muenchen method is to evaluate an endpoint where 50% of the cell cultures are infected when the Karber method (that I may use in your case) is used to directely estimate the TCID50.
Hi Jaime,
Our protocol for determining titer of Adenovirus by TCID50 is as follows. Hope this helps.
Limiting Dilution/ TCID50 Assay ( 293 cells)
This assay reports titer in terms of TCID50 units per ml, where TCID50 stands for “tissue-culture infectious dose.” One TCID50 unit per ml is essentially an approximation of 1 pfu per ml, but since plaques are not being scored, the term “pfu” is not accurate. The method is a bit less quantitative than a plaque assay, but is relatively foolproof and is thus a consistent method for measuring adenovirus vector titers.
1. Adenovirus stocks generally have titers of 10^8 - 10^10 pfu/ml. Based on this range, prepare 3 ml of a 10-6 dilution of the adenovirus stock (i.e. the most concentrated dilution that will be added to the 1st column of 293 cells in a 96-well plate).
2. Make 11 serial 1/3rd -log dilutions (1390 : 3000 ul).
3. Seed a 96-well plate with 2.5 x 10^3 293 cells per well
(volume = 100ul per well; medium = complete DMEM, 5%FBS, no methylcellulose).
4. Transfer 50 ul of viral dilution to each well. This is most easily done with a reagent reservoir and multichannel pipettor. Transfer 50 ul of the 12th viral dilution (dilution factor = 2 x 10^-10) into the 12th column on the plate, discard the excess from reagent reservoir, and then transfer 50 ul of the 11th viral dilution into the 11th column on the plate, etc.
5. On days 7 and 14 after inoculation, score each well as either positive or negative for cytopathic effect (this should reach completion by 10 days after inoculation).
6. The titer of the viral stock is calculated as TCID50 /ml = 20 x dilution at which CPE develops in only ½ of replicate cultures. The multiplication factor of 20 reflects the fact that only 1/20th of a ml is added to each culture of 293 cells.
If you are finding wells that are difficult to determine CPE, I recommend you do a duplicate transfer of media from the entire plate to fresh cells at the point where you start to see the CPE. You'll quickly get an easily interpreted CPE in the second round viral amplification and the TCID50 will be much easier to estimate.
Dear colleagues!
I have published a very simple formula to calculate TCID50. The full paper can be downloaded from the following PubMed/Research gate links
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861875/
https://www.researchgate.net/publication/298789586_Determination_of_50_endpoint_titer_using_a_simple_formula?ev=prf_pub
Article Determination of 50% endpoint titer using a simple formula
Hi Muthannan Andavar Ramakrishnan,
Is the formula also applicable with cells? Can you show me the formula if it is. Thank you very much.
Hi Lei,
The same formula can be used for any endpoint including TCID50. Yo can substitute wells showing CPE and wells not showing CPE in place of animals died and survived.
You can try your value with this formula and let me know your answers.
Thanks
Rama
Muthannan Andavar Ramakrishnan
I used your formula in cell culture infected with HSV type 1 and it gave the same results as Reed and Muench method
Many thanks.
Hi Dr. Ramakrishnan
How can I calculate tcid50 formula while making dilution as 1:2 fold serial ( not 10 fold)???
Thanks in advance
Dear Dr Hossain,
Please apply 0.301in dilution factor. This is log value of 2.
Thanks
Rama
Hi,
please suggest me how to calculate TCID50 of HIV pseudovirus? what does 200 TCID indicates?
Please suggest me that how to calculate virus titre by TCID50 per ml for 96 well plate?
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