Comments on: Measurement of viruses by end-point dilution assay

By: Anthony

Anthony — Wed, 01 Feb 2012 21:26:00 +0000

“The 50% end point, which falls between the fifth and sixth dilutions, is calculated to be 10-6.5.” (from the last paragraph)

isn’t it 6th and 7th dilution?

By: Nisha D

Nisha D — Mon, 09 Jan 2012 21:07:00 +0000

Hi Dr. Racaniello,

I was reading a paper where they inactivated A/PC using gamma radiation. When they immunized mice in their study they claim that they used doses of 3.2 x 10^6 PFU equivalents. Is the term “PFU equivalent” widely used and what does it actually mean? Would you be able to explain the process of quantifying inactivated virus? Is there a way to use something like a TCID50 equivalent?

Thanks for your help and time!

Nisha

By: Rama

Rama — Fri, 09 Dec 2011 10:19:00 +0000

Hi friends!
Read the following. Really helpful in understanding TCID50. Further, I send my new formula for the publication and will upload after acceptance.
http://whqlibdoc.who.int/monograph/WHO_MONO_23_(3ed)_appendices.pdf

M. A. Ramakrishnan, Senior Scientist, Indian Veterinary Research Institute, Mukteswar, India
maramakrishnan@gmail:disqus .com

By: Sgalattas

Sgalattas — Fri, 07 Oct 2011 19:14:00 +0000

What is the equation 4 TCID50 ? How can i explain it to my students ?

By: Shahzaduaar772

Shahzaduaar772 — Thu, 08 Sep 2011 17:49:00 +0000

hello
i am here in pakistan a phd student. i need original paper of reed and Reed L.J. & Muench M. (1938) A simple method of estimatingfifty percent end points. American Journal of Hygiene
Reed L.J. & Muench M. (1938) A simple method of estimating
fifty percent end points. American Journal of Hygiene
27, 493–497. for calculation of LD 50.493–497. for calculation of LD 50.

KIND REGADS

By: Kamola

Kamola — Fri, 02 Sep 2011 14:45:00 +0000

Hello,
I am making TCID 50, what does it mean, make serial dilutions from virus stock, is it virus supernatant from third amplification step, or purified virus recovered from supernatant by ultracentrifugation. Purified virus is stored in storage medium containing glycerol, can this somehow affect the assay. the protocol i’m using requires me to take 200ul of virus (not sure, the supernatant or the purified virus), if its purified virus, its too much, my cells will die in 2 days, can i some how minimize, the volume?
Thanks

By: Thuyniheje

Thuyniheje — Fri, 02 Sep 2011 12:33:00 +0000

I am in Viet Nam. I know the Reed and Muench. If you want to know it, I can give to you. Contact me: thuyniheje@yahoo.com.

By: Phuongthuy

Phuongthuy — Fri, 02 Sep 2011 12:27:00 +0000

Thank you very much. But I think the medium with serum that add after remove virus and incubate is very imfortant. Can you tell me which is medium? MEM or other? Thank you.

By: aditya asopa

aditya asopa — Thu, 25 Aug 2011 17:00:00 +0000

great job. Would you please tell us about Karber’s method.?

By: profvrr

profvrr — Fri, 29 Jul 2011 20:30:00 +0000

My colleague Saul Silverstein, who worked on varicella virus sent me the following: To titrate virus dilute in PBS containing 2% calf serum and overlay a cell monolayer with a minimal volume of liquid (100ul for a 3.5 cm dish), incubate at 37 and gently tap the sides of the dish even 15 min for 1 hr. After 1hr remove virus and add 3mls of medium with serum and incubate. You should see plaques developing in a few days. You can add 0.1% pooled human gamma globulin to reduce plaque spread. Remove medium, fix with methanol and stain with crystal violet.

By: phuongthuy

phuongthuy — Fri, 29 Jul 2011 12:48:00 +0000

Dear sir.
Im VietNamese. I am studying about Varicella virus. I want to determine the titer of virus in vaccine but
I face a problem when choose the media diluent virus and the media for overlay. Can you help me? My email:thuyniheje@yahoo.com. Thank you.

By: Sonal Mundhra

Sonal Mundhra — Wed, 13 Jul 2011 04:34:00 +0000

Thanks a lot sir for the paper.

By: shri

shri — Tue, 12 Jul 2011 11:57:00 +0000

dear sir can you please mail me the same artical i dont have online access.
dav0082@gmail.com

By: Sonal Mundhra

Sonal Mundhra — Tue, 12 Jul 2011 07:08:00 +0000

Vincent sir,
I am Ph.D student working on virus. Kindly end me the Reed and Muench original paper on end point dilution assay for virus titer. My email id is sonal.mundhra@gmail.com
Thank you.

By: Senvetpath

Senvetpath — Sun, 03 Jul 2011 06:39:00 +0000

hello rama please send your formula, my mail id is senvetpath@gmail.com

By: M Denise

M Denise — Wed, 01 Jun 2011 15:05:00 +0000

Hi,
I work in an animal disease lab, where we use TCID50 to determine virus use in VN/SN testing. For the past few months there has been some differing opinions on the best way to measure the TCID50 units. After receiving information from Dr. Rama, everyone involved is in agreement that his formula is the most accurate and less complicated way to measure. I am very grateful to Dr. Rama for emailing his formula to me. Thanks Again, Dr. Rama
M Denise

By: profvrr

profvrr — Wed, 13 Apr 2011 18:53:00 +0000

Not sure what you are asking – please email virology@virology.ws and we’ll figure it out.

By: KC vet

KC vet — Sun, 10 Apr 2011 11:14:00 +0000

Hi,
I’m a PhD student struggling to understand the whole purpose of the TCID50. I have calculated using the Reed and Muench method that my virus’ TCID50 is 5.1E-7. In the experiment I did a series of 10 fold dilutions, set up by adding 0.3ml virus to 2.7ml medium etc etc. I then put 100microlitres of each dilution into wells containing 100microlitres of cells in medium. I did twelve wells per dilution. So how do I know what my TCID50/ml is? Or is the value I calculated already per ml? And what units is it expressed in i.e what does it actually mean?! I have previously calculated the titre of the virus using plaque assay, how do the two relate? I am succeeding in getting myself more and more confused the more I read, so any help much appreciated! Thanks, KC

By: Ramakrishnan

Ramakrishnan — Wed, 16 Mar 2011 08:43:00 +0000

Please send your email address to maramakrishnan@gmail.com

By: Ramakrishnan

Ramakrishnan — Wed, 16 Mar 2011 08:41:00 +0000

please send your email address to maramakrishnan@gmail.com

By: elisha

elisha — Tue, 22 Feb 2011 12:13:00 +0000

Dear profvrr
i will apreciate if you send me a copy of Reed and Muench paper

elisha kutto
yonkutt@yahoo.com

By: elisha

elisha — Tue, 22 Feb 2011 12:03:00 +0000

I am an MSc student of clinical pathology from Kenya.I would be very gratefull if you send me the paper by Reed and Muench.Thank you

By: elisha

elisha — Mon, 21 Feb 2011 08:50:00 +0000

Hello
please send me you formulae

By: elisha

elisha — Mon, 21 Feb 2011 08:47:00 +0000

your comment seems can solve my problem which has been an headage for me. please clarify for me. what is the difference between the 50% end point titre and the ld50.how can i calculate the number of organisms (cfu) per ld50?. must i know the concentration of the stock solution to determine the ld50?
Thank you,elisha yonkutt%yahoo.com

By: Elisha Kutto

Elisha Kutto — Mon, 21 Feb 2011 08:32:00 +0000

please send me your formulae
Elisha kutto
yonkutt@yahoo.com

By: XMRV infection of Rhesus macaques

XMRV infection of Rhesus macaques — Thu, 17 Feb 2011 20:20:54 +0000

[...] that they were not previously infected. Animals were inoculated intravenously with 3.6 million TCID50 of purified XMRV – a good amount of virus, to ensure infection. The virus used, VP62, was [...]

By: Terrasse Rachel

Terrasse Rachel — Tue, 08 Feb 2011 14:37:00 +0000

Hello,

Could you please send me your formula?

Best regards

By: Terrasse Rachel

Terrasse Rachel — Tue, 08 Feb 2011 14:33:00 +0000

Dear Dr,

I’m a PhD student and I would be interested in having a copy of this paper, could you send it to me please?

Thanks a lot.

By: profvrr

profvrr — Mon, 31 Jan 2011 01:06:00 +0000

Yes, you can flick the medium off of the 96 well plates – use a broad
tray to receive the liquid. You can flick the plate with your wrist,
or tap it – try it on some blank plates to get a feel for what works
for you. We do this all the time when changing medium in 96 well
plates – as you say, it takes too long to remove the medium by
aspiration.

By: Victor

Victor — Mon, 31 Jan 2011 00:02:00 +0000

Hi Vincent,
I have a technical question about the TCID50 method. I have to assay lots of samples from infected mice and I set them up in quadruplicate so I usually have a lot of 96well plates to work with(20-30 at a time). After absorbing virus on MDCKs for 24 hrs I replace the media with VGM by aspiration. Aspirating the media off is very time consuming and uses up a lot of tips. I was wondering if I could save time by “flicking” the media off of my plates into a container instead of aspirating. Any tips for saving time would be helpful.

By: Prashant

Prashant — Fri, 03 Dec 2010 11:40:00 +0000

Respected sir,
I would like to know weather TCID50 can be used to evaluate antiviral activity of compounds.

regards
Prashant,PhD

By: Cyc10

Cyc10 — Wed, 20 Oct 2010 20:34:00 +0000

Hi Dr. Racaniello,
I am also looking for the Reed and Muench, I would very much appreciate if I can obtain the paper from you. Thanks a lot.
cyc

By: Thietmo

Thietmo — Mon, 27 Sep 2010 08:25:15 +0000

Dear Vincent,

I am a master student in Biotechnology from Vietnam. I am looking for the paper by Reed and Muench on the internet and our school library, however, I can not find it. May I have the paper from you? Appreciate for your help.

By: DIPAKSINH BHATI

DIPAKSINH BHATI — Sun, 12 Sep 2010 14:12:32 +0000

PLS SEND ME UR CALCULATION FORMULA

By: Bmschen

Bmschen — Wed, 23 Jun 2010 10:34:53 +0000

I am interested to have a read of this paper. May I have a copy from you? Appreciate.

By: profvrr

profvrr — Mon, 21 Jun 2010 19:35:57 +0000

Yes, it's a good idea to try to disrupt IFN production. You could try
another cell line with an IFN defect (such as VERO, which don't
produce IFN, or cells lacking the type I IFN receptor), or you could
alter the LLC-MK2 so they are defective in the IFN response.

By: Murine

Murine — Mon, 21 Jun 2010 10:35:42 +0000

Thanks Dr Vinc, Your advise was quite useful in enabling me to select samples containing only single virus. Recently I tried to determine the replication kinetics of many different strains of the same virus. I found out that the replication of the viruses was quite low and eventually became undetecable by qPCR after 04 passages on LLC-MK2 with inapparent CPE. What would you advise in case that I try antagonizing the IFN, can it lead to any improvement in the replication?

By: profvrr

profvrr — Tue, 08 Jun 2010 01:26:43 +0000

I would try either a different cell line, hoping that it will only
propagate your virus, or try diluting the virus before infecting
LLC-MK2 so that only a few virions are used to infect, thereby
favoring infection with one or the other virus. If your virus is in
the majority this should work, especially if done a few times.

By: FALEYE TEMITOPE .O .C

FALEYE TEMITOPE .O .C — Mon, 31 May 2010 20:49:37 +0000

DMc,
IT'S OBVIOUS FROM YOUR COMMENT THAT YOU'VE DIGESTED THE CLASSIC OF REED AND MUENCH. PLEASE, CAN YOU SEND ME A COPY?
faleyetope@yahoo.com
ps, I'm a graduate student of virology in Nigeria. The logic behind the calculation still eludes me. Thanks.

By: Emily T. S. Wu

Emily T. S. Wu — Tue, 25 May 2010 19:27:11 +0000

Dear Vincent,
I am a master student in Biotechnology from Taiwan. I am looking for the paper by Reed and Muench on the internet and our school library, however, I can not find it. May I have the paper from you? Appreciate for your help.

By: murine

murine — Mon, 05 Apr 2010 15:49:04 +0000

Dear Dr Vincent,
My study virus disappears in the process of passaging (after 3 passages) and is replaced by another unknown CPE producing virus on LLC-MK2. I cannot understand that how do I identify this unknown virus and select my original virus. Since i need to prepare the viral stock for my original virus. The approval of cloning based detection methods would take around 2-3 months. In passage 2 original virus is there and in the passge 3 it disappears. Perhaps the exclusion principal is involved here. SHould i try even more cell lines or the available antibodies. Any thoughts or golden piece of advice? Yoroshiku one gai shimas.

By: murine

murine — Mon, 05 Apr 2010 08:49:04 +0000

By: wang

wang — Fri, 26 Mar 2010 04:43:41 +0000

Dear profvrr,
can you send me a copy, i am interested in Reed an Muench method,because it is too early ,i can not search it in our library. thank you !

By: dmcilroy

dmcilroy — Mon, 22 Mar 2010 19:23:40 +0000

OK, after reading the classic paper, I think I have a (slightly) better understanding of why using cumulative numbers of dead mice/infected wells and cumulative alive mice/non-infected wells works. In the actual paper, the example they gave was protection of mice from infection by transfer of different dilutions of immune serum. However, to make this relevant to viral titres in cell culture, I will try to explain how I understood the paper as if the data were referring to a TCID50 determination .

I found the figure much clearer than the text in explaining the use of cumulative numbers. If you plot the cumulative number of infected wells going from the most dilute to the least dilute, then you get a curve that goes down as the inoculum gets more dilute. Conversely, the curve for cumulative number of non-infected wells goes up as the inoculum gets more dilute. Where the two curves cross is the dilution that gives you the TCID50.

However, they didn't use these two curves to calculate the TCID50. This was done using the Reed+Muench formula on the sigmoid curve of cumulative or pooled % of infected wells at each dilution. I guess that finding where you get 50% infected wells on this curve is identical to finding where the number of infected/non-infected wells is the same.

OK, so now I understand why it's a valid approach. But what is the utility? Well if I understood correctly, using cumulative numbers is supposed to be more statistically robust, since the number of animals/cell culture wells analyzed at each dilution is quite low (typically 6 to 8). If this is correct, then the variance of a TCID50 measure using the Reed-Muench method correctly (i.e. with cumulative numbers) should be lower than if the Reed-Muench formula is applied directly to the observed proportion of infected wells at each dilution.

It occurs to me that I could test this quite soon, as I will be getting a bunch of write-ups from students who did a TCID50 in the lab this semester, so I have a data set with several determinations on the same virus stock.

DMc

By: dmcilroy

dmcilroy — Sat, 20 Mar 2010 13:11:05 +0000

No, I haven't. I think about the only paper I have ever read from that period was the Ellis and Delbruck paper on the single-step growth curve. (Even with that one, though, the credit goes to Alan Cann for putting it on the CD that came with his text book). If you could send me a copy, I would be much obliged. I'll even write back here to let you know if I understood it better this time.

DMc

By: Rama

Rama — Fri, 19 Mar 2010 19:18:55 +0000

Do you mean LD50? Then Yes I have

By: profvrr

profvrr — Fri, 19 Mar 2010 19:00:17 +0000

Have you read the 19389 paper by Reed and Muench? The logic is best
explained there. If you have not, I can send you a copy of the paper.

By: dmcilroy

dmcilroy — Fri, 19 Mar 2010 18:21:08 +0000

Actually, I have never understood the utility, or even the validity, of pooling the results at each dilution.
In the example shown above, why is the mortality ratio of 27% at 1x10-7 , calculated from the cumulative data considered to be more valid than the actual observed mortality ratio at that dilution (3/8 or 37.5%) ?

I have read (and even tried to understand) textbook explanations of this, and I have not found the explanations very clear.

DMc

By: khemmie

khemmie — Thu, 18 Mar 2010 06:18:16 +0000

Do you have formula in solving thr LT50?

By: khemmie

khemmie — Thu, 18 Mar 2010 06:16:20 +0000

How about determining the LT50?