“SARS-COV-2” VTM Guidelines

Coronavirus test. Hand in gloves holds a test tube with a corona virus test label on blurred laboratory in the background. COVID-19 or SARS-CoV-2 test concept.

These are the guidelines for Viral Transport Media for “SARS-COV-2” put forth by the CDC. Note that this was put out there to provide a standard to follow as an alternative to commercially available products which means that the VTM is not standardized across all laboratories. They even allow alterations as long as it is documented:

Purpose:

“TO PROVIDE A STANDARD OPERATING PROCEDURE (SOP) for producing viral transport medium (VTM) for specimens for viral culture or other means of viral detection.

This SOP PROVIDES AN ALTERNATIVE TO COMMERCIALLY AVAILABLE PRODUCTS. THERE ARE MANY ACCEPTABLE FORMULATIONS OF THIS MEDIUM THAT MAY BE SUITABLE FOR THE UNIQUE CONDITIONS OF INDIVIDUAL LABORATORIES. The specific needs of the shipping and receiving laboratories should be considered when choosing a VTM formulation.”

Responsibility:

“It is the responsibility of personnel preparing VTM for CDC’s COVID-19 outbreak response to follow this SOP accurately. IF THERE ARE VARIATIONS from this SOP, THE VARIATIONS SHOULD BE DOCUMENTED, AND DATA GENERATED TO DEMONSTRATE EQUIVALENT PERFORMANCE OF THE VTM.”

Here is the VTM formula recommended by the CDC:

Reagents:

“6.11 Sterile Hanks Balanced Salt Solution (HBSS) 1X with calcium and magnesium ions, no phenol red, 500mL bottle (or HBSS containing phenol red as a pH indicator)

6.12 Sterile, heat-inactivated FETAL BOVINE SERUM (FBS)

6.13 GENTAMICIN SULFATE (50mg/mL) (OR SIMILAR ANTIBIOTIC at an appropriate concentration to prevent bacterial contamination and growth)

6.14 AMPHOTERICIN B (250µg/mL) (Fungizone) (or similar antifungal at an
appropriate concentration to prevent fungal contamination and growth)

6.15 Blood agar plate

6.16 Disinfectant, such as 70% ETHANOL”

https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.cdc.gov/coronavirus/2019-ncov/downloads/Viral-Transport-Medium.pdf&ved=2ahUKEwigj-r3x_vuAhUMV80KHYMbAFAQFjAIegQIDxAC&usg=AOvVaw3pTsWq8rGHibhocLI1EVl0

A quick note on the use of Gentamicin and Amphotericin B. THEY ARE TOXIC TO CELLS:

GENTAMICIN:

“THERE ARE MANY REPORTS OF ANTIBIOTICS CAUSING MITOCHONDRIAL DAMAGE. In this study, we tested the effect of gentamicin in culture media on human mammary epithelial MCF-12A and breast cancer MCF-7 and MDA-MB-231 cell lines by real time PCR, immunofluorescent microscopy, lactate assay, DNA damage assay. WE FOUND THAT THE ADDITION OF GENTAMICIN IN MEDIA UNREGULATED THE GENE EXPRESSION OF HYPOXIA INDUCER FACTOR 1 ALPHA (HIF1a), GLYCOLYTIC ENZYMES AND GLUCOSE TRANSPORTERS, compared to the cells cultured in gentamicin free media. GENTAMICIN ALSO INCREASED THE LACTATE PRODUCTION AND INHIBITED MITOCHONDRIAL MEMBRANE POTENTIAL OF THE CELL LINES. Furthermore, THE ANTIBIOTICS IN MEDIA INDUCED MITOCHONDRIAL REACTIVE OXYGEN SPECIES CAUSING DNA DAMAGE. We found an increase of 8-hydroxy-2’-deoxyguanosine a product of DNA oxidative damage in the media of MCF-12A, MCF-7 and MDA-MB-231 cell lines.”

“THE METABOLIC CHANGES IN ALL CELL LINES WERE DRAMATICALLY DIFFERENT BETWEEN THOSE IN ANTIBIOTIC FREE MEDIA VERSUS ANTIBIOTIC CONTAINING MEDIA. There was a MARKED DIFFERENCE IN GENE EXPRESSION OF GLYCOLYTIC ENZYMES, REACTIVE OXYGEN SPECIES PRODUCTION AND EFFECTS ON MEMBRANE POTENTIAL. Ironically, our first studies were done in media containing gentamicin, and repeated studies were done in gentamicin free media. THE RESULTS WERE VERY DIFFERENT. The purpose of this report is to EMPHASIZE THAT METABOLIC CELL CULTURE DATA MAY BE INACCURATE BECAUSE EXPERIMENTS WERE PERFORMED IN CELL CULTURE MEDIA CONTAINING ANTIBIOTICS.”

“Based on our study of the effect of gentamicin in three mammary cell lines, WE ARE CONVINCED THAT ANTIBIOTICS DO CAUSE MITOCHONDRIAL DYSFUNCTION, AND THIS IS TRUE FOR BACTERICIDAL AND BACTERIOSTATIC ANTIBIOTICS REGARDLESS OF PARADOXICAL REPORTS IN THE LITERATURE. We have emphasized the importance of CELL CULTURE STUDIES BEING DONE IN ANTIBIOTIC FREE CULTURE MEDIA; especially when studying cellular metabolism. We have stressed that MANY REPORTS MAY HAVE INACCURATE DATA, as the study was done in antibiotic containing cell culture media. When studying mitochondrial function, we must remember that mitochondria are evolutionary bacteria; AND ANTIBIOTICS HAVE DAMAGING EFFECTS ON THEM AND BACTERIA.”

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214586

AMPHOTERICIN B:

“The results of the cell viability assays CONFIRM HIGH TOXICITY OF AMPHOTERICIN B TOWARDS HUMAN CELLS.”

“According to a current knowledge, biomembranes of human and fungi cells are a primary target of the drug and both the therapeutic and toxic side effects of AmB are BASED UPON IMPAIRING OF PHYSIOLOGICAL PROCESSES TAKING PLACE IN MEMBRANES.”

“Two human cell lines, CCD 841 CoTr and HT-29, were cultured in the presence of AmB in a concentration range of 0.05 to 25 μg/ml in the growth medium. AS EXPECTED, HIGHER CONCENTRATIONS OF THE ANTIBIOTIC ARE TOXIC TO HUMAN CELLS (above 5 μg/ml, see Fig. 1).”

https://www.nature.com/articles/s41598-018-32301-9

Oh yeah…we can’t forget the 70% Ethanol.

ETHANOL:

“ALL CELLS WERE KILLED BY A 15-s EXPOSURE TO 30–40% ETHANOL WHILE A CONCENTRATION AS LOW AS 15–20% GAVE A TOTAL RESPONSE AFTER 5–10-min EXPOSURES. After a one-hour exposure of F9 carcinoma cells and hepatocytes, a total or nearly total response was achieved with 10% ethanol. THE CYTOTOXIC EFFECT WAS THUS DEPENDENT BOTH ON THE EXPOSURE TIME AND ON THE CONCENTRATION OF ETHANOL .”

“ETHANOL SEEMED TO KILL CELLS IN THE CELL CULTURE EFFECTIVELY IN MUCH LOWER CONCENTRATIONS than those currently used in tumour ablation.”

https://www.tandfonline.com/doi/abs/10.3109/02841859609175470

And this is primarily about Ethanol being a CONTAMINANT when sprayed as a disinfectant in laboratories:

“Here we want to put a spotlight on the importance of being careful on the quantity sprayed and where to spray it, and particularly to avoid its contact with experimental cells, SINCE THIS WILL LEAD TO RADICAL INFLUENCE ON CELLS PATHOPHYSIOLOGICAL CONDITION.”

“In conclusion, ethanol is largely utilized as antiseptic in cell experiment environment, and at the same time IT HAS A HUGE NUMBER OF POSSIBLE IMPLICATIONS IN DIFFERENT CELLULAR MECHANISMS”

https://medcraveonline.com/JMEN/ethanol-in-cell-culture-disinfectant-or-contaminant.html

REMEMBER: samples from sick patients are immediately placed in Viral Transport Media.

Why are these obviously toxic Antibiotics (along with the damaging effects of Fetal Bovine Serum and the potential contamination by Ethanol) added to the samples before testing, culturing, sequencing, etc.? Why is it assumed they will have no effect on the sample when the evidence clearly shows otherwise?

There is nothing pure about the cell culture process even from the very first step.

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