In vitro cultured cells are severely polluted, and sometimes they are difficult to recover.
Therefore, prevention is the key to prevent pollution.
Only when preventive measures are carried through the whole cell culture can the possibility of contamination be minimized.
General prevention can be taken from the following aspects:
(1) add antibiotics
Various antibiotics nature, have different effects on microorganisms, combined use of combination than effect is good, the prophylactic use of better than used after pollution (but so far there is no against mycoplasma effects of antibiotics).
But the repeated use of antibiotics can make microbial resistance, and also have certain influence to the cell itself, so to avoid induced resistant bacteria, antibiotics should be replaced periodically training system of, or as far as possible need not antibiotic treatment.
(2) start with sterilization and sterilization of articles and supplies
The cleaning and disinfection of the products used in cell culture should be thorough. The bacteria should be carefully sterilized and confirmed asepsis after a week of sampling.
At the same time, in the aseptic filtration operation, as the filtration volume increases, the possibility of membrane damage increases, so the final filtered liquid should be selected for testing.
Regularly disinfect carbon dioxide incubator.
Specific operation: after the 75% alcohol wipe the incubator, use the removable uv lamp at least 30min, add high pressure sterilization superpurity water in the incubator tank to keep the humidity.
It can also be prepared with 300ml saturated sulphate and 3L sterilized super pure water mixed with copper sulphate solution into the tank.
(3) start with the operator
A. Wash your hands thoroughly before entering the sterile room and wear the segregating clothes as required.
Before starting the operation, use 75% alcohol cotton ball to wipe hands, wipe the mouth of the bottle and cauterize the mouth.
B. The operator should be light, and the installation of the straw cap, opening or sealing of the bottle should be done in the vicinity of the fire and by burning.
But be aware that metal instruments cannot be burned in flames for long periods of time to prevent annealing.
The burnt equipment must be cooled before it can be used;
Has smoked culture can no longer use the flame burning of straw, due to the residual in the straw composition of the culture medium can produce harmful substances such as protein after burning, straws and took it to a culture;
Stopper, rubber nipple and plastic cell culture supplies a flame is also cannot too long, lest produce poisonous gas, burning harm culture cell, and plastic deformation effects also use cell culture products.
C. It is not advisable to open the bottle before using the culture medium, and the culture medium after the bottle should be kept oblique to avoid the pollution of the falling bacteria.
The culture medium that is no longer used should be sealed off immediately, and the cultured cells should not be exposed to the air prematurely before processing.
D. Try not to talk as much as possible, coughing to prevent contamination from saliva and exhaled airflow.
E. learn culture, cell suspension, should charge is special, once found the suction mouth contact with their hands and other pollution items should be abandoned, in order to prevent the pollution to expand or cause cross contamination between cultures.
F. After the operation, you should tidy up the countertop and wipe the table with the sponge soaked in the disinfectant.
(4) prevent cross-contamination of cells
All from elsewhere or cell lines should be built by early reserves, have a plenty of cryopreserved once suspected cross-contamination, can be used as cell genetics appraisal, if discover the original cell genetic change, can be used in early recovery of cryopreserved cells.
The transmission of important cell lines (strains) should be carried out independently by two people.
(5) sterilization of sterile room
A. New jer extinguishes the thorough cleaning of the sterile room.
Before use, it should be diluted.
New jie er die compared with alcohol, the biggest advantage is cheap, because the new just 5 yuan jie, destroyed 500 ml, and can be diluted 50 times with, and the price of the same amount of alcohol may be a new clean out dozens of times.
B. formaldehyde fumigation method: formaldehyde is a broad-spectrum sterilizing agent, and its aqueous solution and gas have the effect of killing bacteria, spores and fungi.
Formaldehyde is cheap, fumigation and sterilization do not damage clothes, furniture, leather, rubber and so on.
The formaldehyde solution that is sold in the city commonly contains 37-40% formaldehyde.