Half this lab's budget goes to buying serological pipettes They should invest in a vacuum pump and autoclave glass pipettes for aspirating media and PBS.
Yes, seems as if that is so, I'd be inclined to provide reusable glassware and a proper sterilization procedure and not use the single use plastic supplies - but the whole environmental cost/benefit from origin should be analysed when making that social decision (rather that basing that on the local business budget only).
Great video; however, 70 % alcohol does nor sterilise gloves or anything.It's a disinfectant agent. Sterilisation is achieved by autoclaving, dry heat, gamma radiation, ozone and some chemical products.
Very informative and pleased to witness a very competent operator. I trust the resulting materials are not for use in humans? I ask this because if so, the aseptic technique, procedures and the environment would require improvements. To follow up on Thyago's point regarding the use of 'sterilisation' he is right as the 70% ethanol sanitises the outer surfaces reducing bioburden and this cannot be referred to as sterilisation. I have never witnessed the supernatant being poured off the pellet of cells before, does the pellet always stay anchored or do you occasionally lose one?
Excellent video. I was wondering if the plastic pack containing the flasks was closed using tape or something similar, or kept opened until the next use? Thank you.
Great video. I was curious however why you pellet then resuspend the cells? I was under the impression that the trypsin is neutralized by the media and can simply be diluted with fresh media until the desired cell density is achieved? This surely removes an additional handling step as well as risking damaging the cells through vigorous pippetting?
This is a generic protocol and is meant as an introduction to the idea of cell culture and aseptic technique. This, as with other protocols may be adjusted to suit the requirements of the specific experiment undertaken/cells in use. However, It is common practice to centrifuge and resuspend cells after trypsinisation and this does not affect cell viability when done under appropriate conditions (e.g. normal levels of pipetting with a standard opening tip does not generate sufficient shear force to damage mammalian cells) You are correct that most cells are largely unaffected by simply leaving the trypsin on, diluted. The key point is that not performing the centrifugation step is really only appropriate where routine subculture is being undertaken. Centrifugation and resuspension is a critical step in a number of cases, e.g. Should the cells need to be completely “washed” of the media they are in, going into a short-term biological assay, the cell sample requires concentration to make a higher cell density. and so the decision for this training video was to include the centrifugation step.
