A series of hydrogen ion buffers covering the pKa range 6.15-8.60. Most of these buffers are amino acids. The synthetic buffer 4-2hydroxyethyl-1-piperazineethanesulfonic acid, HEPES, has gained widespread use recently.
The preservation of animal cells in liquid nitrogen, which may be seen under biology microscopes, is now a routine practice. Damage associated with freezing has been prevented with the use of glycerol. Although some animal cells can be stored for 5 years in a dry ice chest, the percentage of viable cells that be recovered is very low. Cryoprotective agents, glycerol and dimethyl sulfoxide appear to be almost equally effective in preserving many cell lines. Glycerol is best for certain lines while dimethyl sulfoxide is also best for others.
During the harvesting of cells, fresh trypsin or a trypsin-Versene solution is used in dislodging the cells from the surfaces of the culture vessels with the aid of biology microscopes. The culture medium is removed and 5 to 10 ml of the trypsinizing solution is added to each flask. The culture is then allowed to stand at room temperature for 5 to 10 minutes or until the cell sheet begins to slough off. At this time, an equal amount of fresh culture medium is added to each flask to inhibit further tryptic action, and the crude suspension is gently aspirated to break up the large clumps of cells. The resulting suspensions of single cells and small aggregates of cells are then collected and dispensed into centrifuge tubes and centrifuged at approximately 200-300 g for 20 to 25 minutes at room temperature.
In cases where the tissue culture is contaminated with bacteria or fungi, which may be identified with the use of biology microscopes, it is recommended that infected cell cultures be autoclaved at once and to start over again with a frozen stock of clean cells stored previously in liquid nitrogen. Microbial contamination of cell culture media can be applied to infected cell cultures because they would also eliminate the cells.
Many investigators use antibiotics to prevent infection or to free cell cultures of contamination, but it seems like a futile exercise. The recommended procedure is to test the sensitivity of the contaminant to a battery of antibiotics to identify those most lethal for the contaminant, then, determine the minimal toxic dose of the most effective ones for the cell culture under study. Since elimination of bacterial and fungal contamination is so unsatisfactory and detection is quite laborious, it is recommended that emphasis be placed on prevention.
