Fetal Bovine Serum (FBS) is a vital component used in eukaryotic cell cultures (Arora, 2013). Outside cell culturing, FBS has a myriad of application that ranges from its usage in research, control, and manufacture of human and animal vaccines to facilitating biotechnological processes of drug development. In the context of this laboratory experiment, FBS is utilized because of its low gamma globulin and growth factors. Also, FBS contain significantly low levels of complements as opposed to those derived from newborns or adults. The low composition of complements is significant in the sense that high complement concertation as noted in adult and newborn serums tend to interfere with immunoassays and cause lysing of the cells in the culture (Chwalek, Partlow, & Kaplan, 2016). Holistically, the functions of FBS in cell culture is to deliver growth factors, nutrients, and to facilitate the attachment of proteins to the cells in the culture. Also, FBS prevents apoptosis.
Dulbecco's Modified Eagle's medium is a serum-free formulation that is primarily used in the preparation of a culture. Serum-free DMEM is particularly distinctive from other culture media because its amino acid and vitamin concertation are four times more than the composition in Eagle’s Minimal Essential Medium (Mori et al., 2015). DMEM is usually used alongside sodium pyruvate and high glucose level. DMEM is widely used in culturing mouse and chicken cells. Serum-free DMEM is also used in the formation of viral plague and contact inhibition experiments.
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Phenol red exists typically as a red crystal with a solubility of 0.77 grams per liter and 2,9g/l in ethanol and water respectively. A solution of phenol red is utilized in the cell culture as an indicator that accurately establishes the pH of the media (Zamani et al. 2015). The color of phenol as an indicator exhibits a gradual transition from yellow at a pH of 6.8 to a bright pink at a pH that exceeds 8.2. Under a very low pH, the phenol crystals become a zwitterion, a molecule with has two or more functional groups. At least one of the functional groups is positively charged while the other is negative, and the total charge is zero (Phelan & May, 2015). To be precise, the negative charge is carried by the sulphate group while the ketone group contains the extra proton that contributes the positive charge. Ultimately, an increase in pH results in the loss of a proton from the ketone group transforming the colour of the phenol indicator from orange-red to yellow (Herman, & Pauwels, 2015). Further loss of a proton by the phenol hydroxyl group as a result of the increase in pH changes the color from yellow to red. The change is the colour of phenol indicator is as depicted in the figure below. In the context of the experiment, the medium turned red denoting that it was not contaminated (Freshney, 2015). The contamination of the media added a hydroxyl group hence reducing the pH (Hemeda, Giebel, & Wagner, 2014). As a result, the color of phenol indicator turns to yellow.
Aseptic technique is typically any practices and procedures utilized in a laboratory setting aimed at preventing contamination from pathogens. Ideally, aseptic techniques encompass implementation of the strictest rules designed to diminish the risk of infection. Aseptic techniques are vastly invoked in surgery rooms, outpatient care centres, and medical and science laboratories. In the experiment, the sample was kept free from contamination by avoiding putting the tip of the pipette in contact with the hand or any surface. The pipette was returned into its sterile package when not in use.
Principally, glass and electric pipettes are used in the cell culture because they are easy to sterilize and guarantee accurate measurement of the compound to be introduced to the culture. The petri dish is kept partially open to allow equal distribution of the conditions in the incubator on the sample. The 5% carbon dioxide concentration is maintained in the incubator to keep the pH constant and match physiological conditions needed by the cells. Moreover, 37°C is an optimal temperature required to match physiological conditions.
References
Arora. M. (2013). Cell culture media: A review. University of Pittsburgh Medical Center . Retrieved from https://www.labome.com/method/Cell-Culture-Media-A-Review.html .
Chwalek, K., Partlow, B., & Kaplan, D. L. (2016). Phenol red-silk tyrosine cross-linked hydrogels.
Freshney, R. I. (2015). Culture of animal cells: a manual of basic technique and specialized applications . John Wiley & Sons.
Hemeda, H., Giebel, B., & Wagner, W. (2014). Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells. Cytotherapy , 16 (2), 170-180.
Herman, P., & Pauwels, K. (2015). Biosafety Recommendations on the Handling of Animal Cell Cultures. In Animal Cell Culture (pp. 689-716). Springer, Cham.
Mori, S., Araki, M., Ishii, S., Hirane, M., Fukushima, K., Tomimatsu, A., ... & Tsujiuchi, T. (2015). LPA signaling through LPA receptors regulates cellular functions of endothelial cells treated with anticancer drugs. Molecular and cellular biochemistry , 408 (1-2), 147-154.
Phelan, K., & May, K. M. (2015). Basic techniques in mammalian cell tissue culture. Current protocols in cell biology , 66 (1), 1-1.
Zamani, M., Sadeghizadeh, M., Behmanesh, M., & Najafi, F. (2015). Dendrosomal curcumin increases expression of the long non-coding RNA gene MEG3 via up-regulation of epi-miRs in hepatocellular cancer. Phytomedicine , 22 (10), 961-967.
