In vitro culture
cellWhen it is seriously polluted, sometimes it is difficult to recover even if you try every means. Therefore, prevention is the key to preventing pollution. The precautionary measures can be used throughout the entire cell culture to minimize the possibility of contamination. General prevention can be started from the following aspects:
1
, from the sterilization of articles and supplies
The items used for cell culture should be thoroughly cleaned and disinfected. All kinds of solutions should be carefully sterilized and sterilized before use. At the same time, in the sterile filtration operation, as the filtration volume increases, the possibility of damage to the membrane increases, so the final filtered liquid should be selected for testing. The carbon dioxide incubator is disinfected regularly. Specific operations:
75%
After the alcohol is swabbed, use a removable UV lamp to at least disinfect
30min
Add hyperbarted ultrapure water to maintain humidity in the incubator tank. Can also be formulated
300ml
Saturated copper sulfate plus
3L
The sterilized ultrapure water is mixed into a copper sulfate solution and added to the water tank.
2
Add antibiotics
The nature of various antibiotics is different, and the effects on various microorganisms are also different. The combined application is better than the single use, and the preventive application is better than the use after pollution. However, repeated use of antibiotics can cause microbial resistance and affect the cells themselves. Therefore, in order to avoid the induction of drug-resistant bacteria, the antibiotics in the culture system should be replaced regularly, or as far as possible without antibiotics.
3
Starting from the operator
(
1
Wash hands thoroughly before entering the sterile room and wear gowns as required. Use before starting operation
75%
Alcohol cotton balls rub the hands, wipe the mouth and burn the mouth of the bottle.
(
2
The operator should be light, and the operation of installing the straw cap, opening or closing the bottle mouth should be carried out in the vicinity of the flame and cauterized. However, it should be noted that metal instruments cannot be cauterized in the flame for a long time to prevent annealing; the burnt instruments should be cooled before they can be used; the straws that have absorbed the culture solution can no longer be burned with flame, because the composition of the culture solution remaining in the straws If the protein is scorched, it will produce harmful substances. When the straw is reused, it will be brought into the culture solution. The rubber culture of the rubber stopper, rubber nipple and plastic can not be too long to avoid the burning of toxic gas. Hazardous culture cells, while plastic cell culture supplies can also be used for deformation effects.
(
3
Try not to talk when you are operating, coughing to prevent contamination from the sputum and exhaled airflow.
(
4
It is not advisable to open the bottle too early before using the culture solution. The culture solution after opening the bottle should be kept oblique and avoid standing upright to prevent the contamination of the falling bacteria. The culture medium that is no longer used should immediately close the mouth of the bottle, and the cultured cells should not be exposed to the air prematurely before treatment.
(
5
After finishing the operation, the work surface should be arranged and the table top should be wiped with gauze soaked in disinfectant water.
(
6
)
When taking up the culture solution or cell suspension, it should be used exclusively. Once the pipette mouth is found to be in contact with hands and other contaminated items, it should be discarded to prevent the pollution from expanding or causing cross-contamination between the cultures.
4
To prevent cell cross-contamination
All cell lines that have been transferred from elsewhere or built by themselves must have sufficient frozen storage in the early stage. Once cross-contamination is suspected, cytogenetic identification can be done, such as the discovery of the original cellular genetic material. Changes can be used to resuscitate early frozen cells. The passage of important cell lines should be carried out independently by two people.
5
Thorough disinfection of sterile rooms
1
)
The new cleansing thoroughly cleans the sterile room thoroughly. It should be diluted and used immediately before use. Compared with alcohol, the biggest advantage of chlorpheniramine is that it is cheaper because it is new.
500ml
Just
5
Yuan, and can be diluted
50
Use more, and the same amount of alcohol may be dozens of times more than the new clean.
2
Formaldehyde fumigation method: Formaldehyde is a broad-spectrum sterilizing agent, and its aqueous solution and gas have a killing effect on various bacteria, spores and fungi. Formaldehyde is cheap, and it does not damage clothes, furniture, leather, rubber, etc. during fumigation. Commercially available aqueous formaldehyde solution generally contains
37
-
40%
Formaldehyde.
Its main usage is:
a
Fumigation: the amount of formaldehyde per cubic meter of space is
10
~
15
ML, fumigation and disinfection should be closed at least closed doors and windows
4h
the above. Potassium permanganate
40%
formaldehyde(
1
:
1
After mixing, put it into a development container and immediately see white formaldehyde fumes. Can be placed after disinfection
25%
The ammonia water evaporates to neutralize the irritating odor. The amount of ammonia water is half of the aqueous formaldehyde solution used, and the time is
30
minute. Formaldehyde gas is very toxic, so be sure to wear a gas mask when handling.
b
) Natural volatilization:
Put a larger amount of aqueous formaldehyde solution into an open container, and keep the indoor temperature at
18
Above Celsius, while spraying water indoors to maintain relative humidity
70%
Above
16
Hours can achieve indoor disinfection purposes
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