This is so brilliant! Why has no one ever thought of this before?! Kudos.
Just one thing …. Please, please don’t say that you want to be a ‘thought leader’. That’s what the ‘controllers’ call their people; you hear it all the time.
I will certainly be supporting your project. I decided a couple of months ago that I would only be financially supporting people involved in revealing the virus scam - I believe that it is that important.
Hi Susan, Thank you for your candid and enthusiastic reply. Your support and donations mean a lot!
As I said the words "thought leader" they sounded a bit off in my head... maybe it was subliminally because I had heard one of "them" say it before... Noted and sorry to have pinged the radar 😂.
One scientist from America admitted that they get sars-cov-2 construct from PCR-negative nasopharyngeal sample BUT with poor alignment. :)) As well as the Chinese. However, without documented step-by-step controls, there is a lot of room for speculation. Hopefully people around the world will support this project with step-by-step documented controls from Jamie and co. This is very optimistic, especially since it is open source, free and thoroughly documented, and anyone can inform family members, friends, Research Integrity Teams that virologists and their collaborators are intentional or unitentional fraudsters in science because they misinterpret/fabricate parts of healthy human and animal bodies as alleged viruses.
Off topic - be careful when you suggest bitcoin is outside the system. It may well be an intelligence operation to test digital currency before implementing the state version, or the state just adopting bitcoin.
Regarding the counting of cells. Though automated counters are easier and more accurate, the counting process itself is an age old technique we did by hand using hemocytometers, in which a dye (Trypan Blue) is mixed with cells- cells with intact membranes (living cells) exclude the dye and thus appear white under the microscope, while cells with compromised membranes (a compromised membrane is a sign of impending cell death) allow the Trypan Blue to enter the cell, and thus the cells appear blue under the microscope. The fancy machine merely counts white and blue cells, and calculates the percentage of white cells over the total number of cells.
What occurred to me during the discussion is that cells with a compromised membrane will fall apart within a day or so, breaking into tiny pieces, and those cells would not be counted at all during the Trypan Blue counting process. So counts must be done at least daily to get accurate numbers.
Alongside those, another way of ensuring valid counts, would be to also count samples which had not been subjected to low serum concentrations or whatever stress conditions are being used. Those counts would give you an idea of how many total cells one might expect to have at a given timepoint, and can be used to compare the count totals from the stressed culture samples.
That said, it's likely that the doubling rate of the stressed culture samples will slow so that fewer cells would result even if all cells were accounted for.
I agree with critics who say the cultures should not be allowed to grow to confluency. When we designed our experiments, we always predetermined how many cells needed to be seeded so that at the end of the experiment, the cells would not have reached confluence. One way of doing that is to determine how many cells are in a given vessel (well, flask, dish) when confluent, and then based on the doubling time of the particular cell line (my guess is HEK293T cells probably have a doubling time of 15 hours or so) one can back calculate the number of starting cells depending on the duration of the experiment.
One other thing. The term viral titer is a concentration. Historically, the unit used for virus stocks was pfu/ml (plaque forming units/milliliter) with one plaque forming unit supposedly representing one virus capable of infecting a cell. And regarding infections of cultures, the term MOI (multiplicity of infection) is used, with an MOI of 10, for example, meaning that 10 supposed viruses were added per each cell in a culture. So a sample which receives no virus stock has an MOI of 0 (which typically serves as the negative control.) One thing I noticed looking at the virology protocols was that they often did not have an MOI=0 control, so that even in an experiment in which they varied the amount of virus stock added, every well got some of that virus stock.
Anyway, sorry for such a long comment, but I personally find your work both extremely important and fascinating, and the more you explain the nuts and bolts of these experiments (which is really not that complicated,) the quicker the general public will understand what is being hidden behind their scientific jargon.
Great comment, never knew about the method of counting cells, could you possibly clarify my laymans understanding of the Plaque Assay? Which is this; cell culture supernatant (thought to contain "viruses"), is poured onto a "lawn" of bacterial culture, clear spots on the "lawn" are referred to as "plaques", these "plaques" are considered evidence of a "virus". Dilated supernatant is poured onto a separate "lawn" & the difference in "plaques" is used as a means to calculate the amount of "virus" in the supernatant.
You've got the general principle correct. And, yes, the object of the plaque assay is to determine the number of virus present in a given viral stock solution.
A plaque is sometimes also called a "zone of clearing" which is a small area in a "lawn" of confluent cells (plaque size is typically 1-2mm in diameter) where the cells have lysed or broken apart as a result of having been infected by a lytic virus (we're taught that some viruses infect cells but do not kill the cell (lysogenic?) while other viruses are lytic and burst the cells at the end of the lytic cycle.) A single discrete plaque supposedly represents the active infection process of a single virus.
Anyway, a typical high titer virus stock would be about 500 million viruses (pfu) per milliliter (which makes me wonder why nobody has successfully imaged such stocks) and are stored at refrigerator temperatures for years (they tell us that the titer of a high titer stock decreases about 10% per year when stored in the fridge.)
So, in order to get single discrete plaques, each the result of a single virus, the high titer stock must be diluted (we ultimately want to cover the range of 0 to 1000 viruses in a given sample addition- probably anywhere from 1 to 200 discrete plaques can be distinguished on the "lawn" depending on the size of the growth container used. So, typically, a 10-fold serial dilution is done of the high titer stock (50 million pfu/ml, 5 million pfu/ml, 500 thousand pfu/ml, etc. down to what one might expect would be 0 pfu/ml.)
A small volume of each dilution is then combined with the cells (which are suspended in nutrient medium) and the mixture added to the growth container. A minimum of 3 replicates is done for each dilution. Cells are then grown several days and examined for the presence of plaques. Plaques are counted and used to calculate the original titer or concentration.
I did a bunch of these as part of different projects in 3 different labs, and I found this technique to be one of the most difficult to obtain satisfactory consistent results, so much so that I spent quite a bit of time trying to find alternative methods for quantitating my viral stocks. I never question the virus hypothesis, so looking back on it many years later, it's difficult to interpret what might have been going on.
I should also say that the purpose of my work was to use the virus to express protein, but the expression system I used (baculovirus) was a DNA plasmid based system in which the supposed genome of the baculovirus was carried in a giant 80 kilobase (kb) plasmid. But the system worked as advertised, and I expressed and purified protein from my cell cultures which had been "infected" with the DNA plasmid.
But, that's not to say anything about the work that Jamie's done. This virology technique of starving cells of fetal bovine serum and then claiming cells are dying from a virus is a complete sham. I've adapted cells to serum free media and removing that much serum (dropping the concentration from 10% to 2% or 1%) will cause massive cell death every time.
And the Fan Wu SARS-CoV-2 "genome" is a total scam also. I did the RNAseq work in my final 3 years, including all the in silico alignment software work, and the Fan Wu paper does not even qualify as a valid scientfic experiment.
The same goes for the fake PCR test. I developed those assays for years using the identical technology, and it was the "PCR test," both its design and implementation, that first alerted me that we had a fake pandemic. In fact, it was my refusal to stick something up my nose to run an unvalidated assay to detect a virus that had never been scientfically proven to exist that ended by employment.
Thankyou for that informative reply (now C&P'd into my "notes"), plaque assays have been bugging me since polio "virus" was supposedly found in sewage.
Huh PCR eh? Even the instructions state; "RT-PCR is not able to distinguish whether infectious virus is present."
I would agree that PCR is not "the end of the road"... I.e they kick the can further down the road to Whole Genome Sequencing to "confirm". I think it is important to falsify this area in and of itself as it was used as the main tool to perpetuate the fake pandemic...(and will continue to be used unless falsified properly)
Agree with falsifying the W.G.S. to me the genomic sequencing is "mad professor" stuff, i.e reads, contigs & assembling, Rasnick summed it up thus; "An ‘in silico’ sequence is a computer-generated, theoretical genome sequence of a theoretical virus, which is then declared to be the cause of the subject’s symptoms. That is not science. It is technology passing itself off as science."
Even the extraction of nucleic acid with its chemical lysing, elution buffers, centrifugation etc. is, as someone (Hillman?) wrote akin to using a hammer to find out how a watch works, as can be seen here;
This is so brilliant! Why has no one ever thought of this before?! Kudos.
Just one thing …. Please, please don’t say that you want to be a ‘thought leader’. That’s what the ‘controllers’ call their people; you hear it all the time.
I will certainly be supporting your project. I decided a couple of months ago that I would only be financially supporting people involved in revealing the virus scam - I believe that it is that important.
Bon courage!
Hi Susan, Thank you for your candid and enthusiastic reply. Your support and donations mean a lot!
As I said the words "thought leader" they sounded a bit off in my head... maybe it was subliminally because I had heard one of "them" say it before... Noted and sorry to have pinged the radar 😂.
Stay in touch.
Cheers
J
One scientist from America admitted that they get sars-cov-2 construct from PCR-negative nasopharyngeal sample BUT with poor alignment. :)) As well as the Chinese. However, without documented step-by-step controls, there is a lot of room for speculation. Hopefully people around the world will support this project with step-by-step documented controls from Jamie and co. This is very optimistic, especially since it is open source, free and thoroughly documented, and anyone can inform family members, friends, Research Integrity Teams that virologists and their collaborators are intentional or unitentional fraudsters in science because they misinterpret/fabricate parts of healthy human and animal bodies as alleged viruses.
Regards from Serbia
Thank you for your kind words and support. They mean a lot. J
Great interview.
Off topic - be careful when you suggest bitcoin is outside the system. It may well be an intelligence operation to test digital currency before implementing the state version, or the state just adopting bitcoin.
Not asserting that this is definitely the case,
Maybe… I was quite surprised to see Bitcoin dip in value in line with the stock market when opening this week… A bit odd.
Nice presentation.
Regarding the counting of cells. Though automated counters are easier and more accurate, the counting process itself is an age old technique we did by hand using hemocytometers, in which a dye (Trypan Blue) is mixed with cells- cells with intact membranes (living cells) exclude the dye and thus appear white under the microscope, while cells with compromised membranes (a compromised membrane is a sign of impending cell death) allow the Trypan Blue to enter the cell, and thus the cells appear blue under the microscope. The fancy machine merely counts white and blue cells, and calculates the percentage of white cells over the total number of cells.
What occurred to me during the discussion is that cells with a compromised membrane will fall apart within a day or so, breaking into tiny pieces, and those cells would not be counted at all during the Trypan Blue counting process. So counts must be done at least daily to get accurate numbers.
Alongside those, another way of ensuring valid counts, would be to also count samples which had not been subjected to low serum concentrations or whatever stress conditions are being used. Those counts would give you an idea of how many total cells one might expect to have at a given timepoint, and can be used to compare the count totals from the stressed culture samples.
That said, it's likely that the doubling rate of the stressed culture samples will slow so that fewer cells would result even if all cells were accounted for.
I agree with critics who say the cultures should not be allowed to grow to confluency. When we designed our experiments, we always predetermined how many cells needed to be seeded so that at the end of the experiment, the cells would not have reached confluence. One way of doing that is to determine how many cells are in a given vessel (well, flask, dish) when confluent, and then based on the doubling time of the particular cell line (my guess is HEK293T cells probably have a doubling time of 15 hours or so) one can back calculate the number of starting cells depending on the duration of the experiment.
One other thing. The term viral titer is a concentration. Historically, the unit used for virus stocks was pfu/ml (plaque forming units/milliliter) with one plaque forming unit supposedly representing one virus capable of infecting a cell. And regarding infections of cultures, the term MOI (multiplicity of infection) is used, with an MOI of 10, for example, meaning that 10 supposed viruses were added per each cell in a culture. So a sample which receives no virus stock has an MOI of 0 (which typically serves as the negative control.) One thing I noticed looking at the virology protocols was that they often did not have an MOI=0 control, so that even in an experiment in which they varied the amount of virus stock added, every well got some of that virus stock.
Anyway, sorry for such a long comment, but I personally find your work both extremely important and fascinating, and the more you explain the nuts and bolts of these experiments (which is really not that complicated,) the quicker the general public will understand what is being hidden behind their scientific jargon.
Great comment, never knew about the method of counting cells, could you possibly clarify my laymans understanding of the Plaque Assay? Which is this; cell culture supernatant (thought to contain "viruses"), is poured onto a "lawn" of bacterial culture, clear spots on the "lawn" are referred to as "plaques", these "plaques" are considered evidence of a "virus". Dilated supernatant is poured onto a separate "lawn" & the difference in "plaques" is used as a means to calculate the amount of "virus" in the supernatant.
Cheers in advance.
You've got the general principle correct. And, yes, the object of the plaque assay is to determine the number of virus present in a given viral stock solution.
A plaque is sometimes also called a "zone of clearing" which is a small area in a "lawn" of confluent cells (plaque size is typically 1-2mm in diameter) where the cells have lysed or broken apart as a result of having been infected by a lytic virus (we're taught that some viruses infect cells but do not kill the cell (lysogenic?) while other viruses are lytic and burst the cells at the end of the lytic cycle.) A single discrete plaque supposedly represents the active infection process of a single virus.
Anyway, a typical high titer virus stock would be about 500 million viruses (pfu) per milliliter (which makes me wonder why nobody has successfully imaged such stocks) and are stored at refrigerator temperatures for years (they tell us that the titer of a high titer stock decreases about 10% per year when stored in the fridge.)
So, in order to get single discrete plaques, each the result of a single virus, the high titer stock must be diluted (we ultimately want to cover the range of 0 to 1000 viruses in a given sample addition- probably anywhere from 1 to 200 discrete plaques can be distinguished on the "lawn" depending on the size of the growth container used. So, typically, a 10-fold serial dilution is done of the high titer stock (50 million pfu/ml, 5 million pfu/ml, 500 thousand pfu/ml, etc. down to what one might expect would be 0 pfu/ml.)
A small volume of each dilution is then combined with the cells (which are suspended in nutrient medium) and the mixture added to the growth container. A minimum of 3 replicates is done for each dilution. Cells are then grown several days and examined for the presence of plaques. Plaques are counted and used to calculate the original titer or concentration.
I did a bunch of these as part of different projects in 3 different labs, and I found this technique to be one of the most difficult to obtain satisfactory consistent results, so much so that I spent quite a bit of time trying to find alternative methods for quantitating my viral stocks. I never question the virus hypothesis, so looking back on it many years later, it's difficult to interpret what might have been going on.
I should also say that the purpose of my work was to use the virus to express protein, but the expression system I used (baculovirus) was a DNA plasmid based system in which the supposed genome of the baculovirus was carried in a giant 80 kilobase (kb) plasmid. But the system worked as advertised, and I expressed and purified protein from my cell cultures which had been "infected" with the DNA plasmid.
But, that's not to say anything about the work that Jamie's done. This virology technique of starving cells of fetal bovine serum and then claiming cells are dying from a virus is a complete sham. I've adapted cells to serum free media and removing that much serum (dropping the concentration from 10% to 2% or 1%) will cause massive cell death every time.
And the Fan Wu SARS-CoV-2 "genome" is a total scam also. I did the RNAseq work in my final 3 years, including all the in silico alignment software work, and the Fan Wu paper does not even qualify as a valid scientfic experiment.
The same goes for the fake PCR test. I developed those assays for years using the identical technology, and it was the "PCR test," both its design and implementation, that first alerted me that we had a fake pandemic. In fact, it was my refusal to stick something up my nose to run an unvalidated assay to detect a virus that had never been scientfically proven to exist that ended by employment.
Hi David,
Sorry to read that you were ousted from your previous employer for not taking a PCR test. Are you currently working in industry?
Did you do RT-PCR tests in industry? What leads you to believe that it is fake in its design and implementation?
Thankyou for that informative reply (now C&P'd into my "notes"), plaque assays have been bugging me since polio "virus" was supposedly found in sewage.
Huh PCR eh? Even the instructions state; "RT-PCR is not able to distinguish whether infectious virus is present."
I would agree that PCR is not "the end of the road"... I.e they kick the can further down the road to Whole Genome Sequencing to "confirm". I think it is important to falsify this area in and of itself as it was used as the main tool to perpetuate the fake pandemic...(and will continue to be used unless falsified properly)
Showing PCR's lack of diagnostic capabilities is indeed very important, this can be demonstrated quite easily to Mr/Ms Average by asking them to "google" the following key words; "understanding cycle threshold in RT PCR". Result, following link; https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/926410/Understanding_Cycle_Threshold__Ct__in_SARS-CoV-2_RT-PCR_.pdf#:~:text=Cycle%20threshold%20%28Ct%29%20is%20a%20semi-quantitative%20value%20that,much%20viral%20genet First point out this is an official Government document, then ask them to scroll down to page 6, where it states, in bold; "RT-PCR is not able to distinguish whether infectious virus is present."
Agree with falsifying the W.G.S. to me the genomic sequencing is "mad professor" stuff, i.e reads, contigs & assembling, Rasnick summed it up thus; "An ‘in silico’ sequence is a computer-generated, theoretical genome sequence of a theoretical virus, which is then declared to be the cause of the subject’s symptoms. That is not science. It is technology passing itself off as science."
Even the extraction of nucleic acid with its chemical lysing, elution buffers, centrifugation etc. is, as someone (Hillman?) wrote akin to using a hammer to find out how a watch works, as can be seen here;
https://www.integra-biosciences.com/united-kingdom/en/blog/article/comprehensive-introduction-dna-extraction?utm_campaign=Blog%20US%20%E2%80%93%20EN&utm_source=bing&utm_medium=cpc&utm_content=Blog%C2%A01%3A%20Nucleic%20Acid%20Extraction&utm_term=dna%20extraction%20methods&msclkid=66e05a5be5861051e9de3c36b48d6f13