“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:

PREPARATION OF VIRAL TRANSPORT MEDIUM

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:

  • 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)
  • Sterile, heat-inactivated fetal bovine serum (FBS)
  • Gentamicin sulfate (50mg/mL) (or similar antibiotic at an appropriate concentration to prevent bacterial contamination and growth)
  • Amphotericin B (250µg/mL) (Fungizone) (or similar antifungal at an appropriate concentration to prevent fungal contamination and growth)
  • Blood agar plate
  • 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:

The adverse effect of gentamicin on cell metabolism in three cultured mammary cell lines: “Are cell culture data skewed?”

“Many cells are cultured in media that contains an antibiotic to prevent bacterial contamination. Mycoplasma and other bacterial contamination is a serious problem for those involved in cell culture. Antibiotics in the media helps prevent this contamination and make life easier for the investigators; as performing cell culture experiments in antibiotic free media is difficult and requires vigorous sterile technique. 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 upregulated 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. These results showed that normal epithelial and breast cancer cells cultured in the media with gentamicin had increased HIF1a, aerobic glycolysis and DNA oxidative damage. If we use these unhealthy cells in the experiment, all data will be different, compared to cells grown in gentamicin free media. We have studied the detrimental effects of three antibiotics on mitochondrial function in the untransformed MCF-12A human mammary cell line and two human mammary cancer cell lines, MCF-7 and MB-MDA-231. 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.”

“We found in our studies that gentamicin in the culture media affected the morphology of the human mammary epithelial MCF-12A cell line. The cells were undifferentiated and scattered in the antibiotic media, but were differentiated and had a distinct ductal pattern in antibiotic free media (Fig 1). These morphological changes were not observed on the human breast cancer cell lines MCF-7 and MDA-MB-231. The addition of gentamicin to the cell culture media inhibited the mitochondrial membrane potential of both cancer cell lines MCF-7, MDA-MB-231, and also the normal MCF-12A cell line (Fig 2).

Gentamicin containing media also upregulated the gene expression of HIF-1a, glycolytic enzymes and glucose transporters in all three humans cell lines (Fig 3, S1 File). Gentamicin also increased lactate production of all three cell lines but was more pronounced and significant in the normal MCF-12A cell line. It was increased in the two cancer cell lines but was not significant (Table 2). Gentamicin induced mitochondrial superoxide in all three cell lines after 24 hours (Fig 4) and it was significant when compared to the cell lines incubated in antibiotic free media. Gentamicin also caused increased cell DNA oxidative damage. Cells cultured in media containing 0.05 mg/ml of gentamicin for 24 hours increased the concentration of 8-OHdG in the normal MCF-12A cell lines. It also increased it in the cancer cell lines but it was not significant (Table 3). These results suggest antibiotics may be more detrimental to normal cells. All of these results demonstrate that cell culture studies of cell metabolism should be done in antibiotic free media; as antibiotic media causes mitochondrial dysfunction resulting in inaccurate data. This suggests that many previous reports need to be re-evaluated.”

“We have demonstrated in this presentation that antibiotics cause mitochondrial dysfunction, and have emphasized this in previous publications [1920]. Different groups of antibiotics cause mitochondrial damage by different mechanisms, but it is absolute that antibiotics cause mitochondrial damage.”

“In summary, we believe that the main message and purpose of this communication has been accomplished and presented fairly. 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:

Imaging of human cells exposed to an antifungal antibiotic amphotericin B reveals the mechanisms associated with the drug toxicity and cell defence

“Amphotericin B is an antibiotic used in pharmacotherapy of life-threatening mycotic infections. Unfortunately, the applicability of this antibiotic is associated with highly toxic side effects. In order to understand molecular mechanisms underlying toxicity of amphotericin B to patients, two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of the drug and imaged with the application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. The results of the cell viability assays confirm high toxicity of amphotericin B towards human cells. The images recorded demonstrate effective binding of amphotericin B to biomembranes. Analysis of the images reveals the operation of a defence mechanism based upon the elimination of molecules of the drug from living cells via formation of small amphotericin B-containing lipid vesicles. The fact that exosomes formed are devoid of cholesterol, as concluded on the basis of the results of Raman analysis, suggests that sequestration of sterols from the lipid phase of biomembranes is not a sole mechanism responsible for the toxic side effects of amphotericin B.”

“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). Both CCD 841 CoTr and HT-29 cells were susceptible to AmB, but up to a concentration of 5 μg/ml the cytotoxic effect did not exceed 15% compared to the control (viability inhibition to 88.4 and 86.8% in CCD 841 CoTr and HT-29 cell cultures, respectively). At concentrations higher than 5 μg/ml the drastic fall of cells viability was noted and the effect towards normal cells (viability decreased to 3.6% compared to the control) was more severe than on cancer cells (inhibition to 41.8% of the control).”

“Assays of the viability of cells exposed to AmB, run prior to imaging, confirmed high toxicity of this antifungal antibiotic also for human cells. Interestingly, the resistance of normal cells to the toxicity of the drug has been determined significantly lower as compared to adenocarcinoma cells (Fig. 1).”

“The fact that despite the operation of such a defence mechanism, AmB is highly toxic to human cells, shows that molecules of the drug present in the plasma membranes affect the membrane physiological functionality. According to the results of the fluorescence lifetime and fluorescence spectral analyses, AmB incorporated into the cells appears in the form of small supramolecular structures. It is very likely that such structures promote uncontrolled ion flow through biomembranes, affecting electrostasis of living cells.”

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

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

ETHANOL:

Toxicity of Ethanol in Low Concentrations

“Results: 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. There were no significant differences in ethanol tolerance among the cell types.

Conclusion: 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:

Ethanol in cell culture: disinfectant or contaminant?

“Ethanol solution is used widely as a disinfectant in cell culture room at two usual percentages, which are 70% and 75%. The experimenter often sprays it on experimental tools, on gloves, lab coat, and over other necessary experiment items. 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. For instance, ethanol can induce neurodegenerative diseases [4], and it interferes with pathological mechanisms of diabetes [5].

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, and this proves the importance of reasonable use of its solution. In addition, it can dissolve many experimental compounds, and also pen ink used for writing notes on petri dishes, microplates, cryogenic tubes, etc. and this may cause much risk of cell contamination with undesirable factors. Hence the necessity of being careful when spraying this useful disinfectant, but also harmful product when undesirable if being in contact with cells when studying different diseases mechanisms, particularly those for which ethanol may play a pivotal role.”

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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