Basic techniques for sterile operation in microbial and cell culture

aseptic operation technology not only plays an important role in the research and application of
microbiology
, but is also widely used in many biotechnologies, such as genetically modified technology, monoclonal
antibody
technology, etc.aseptic room, microbial laboratory dedicated to a small room, outdoor set up a buffer room, buffer room door and sterile room door do not face the same direction, so as not to air flow into the clutter. Aseptic rooms and buffer rooms must be closed, sterile indoor floors, walls must be flat, not easy to hide dirt, easy to clean, indoor equipment for air
filtration
device. The work surface of the work station should be horizontal, the sterile chamber and buffer room are equipped with ultraviolet light (1 meter from the work surface), the staff should wear sterilized clothing and hats when entering the sterile room.ultra-clean table, the main function is to use the air stratification device to remove all kinds of small dust, including microorganisms on the work surface, through the electric device to make air through the high-efficiency filter tool into the work surface, so that the tabletop is always under the control of the flow of sterile air. In more difficult conditions, you can also use wooden sterile box instead of ultra-clean table (the front opens two holes, do not operate with push-and-pull door blocking, operation can be extended into the arms; the upper front is equipped with glass, easy to operate inside, the box is equipped with ultraviolet light, from the side door can be put into the appliance and bacteria, cell strains, etc.).microbial
culture in the microbial culture process, to maintain the purity of the culture, culture microorganisms, the important thing is to provide a good living environment for the required microorganisms, while preventing or inhibiting the growth of other microorganisms.(i)
Medium
1. Understanding the basic components of the medium and understanding why these basic components are needed in the medium from the perspective of the basic elements of the composition of the organism.
2. Use and type of culture base: liquid and solid two categories.
3. Different microorganisms often require different formulations of cultures, and although the formulations of the cultures vary, their basic components include water, carbon, nitrogen and inorganic salts.
4. Whether the medium is qualified (aseptic test): the medium in the
stration
box after insulation 1 to 2d sterile growth (liquid unchanged turbidity), indicating that the preparation of the medium is successful, otherwise it needs to be re-prepared.(ii) Aseptic technology1.The concept of aseptic technology
sterile operation refers generally to all methods to prevent clutter contamination in the operation of cultured microorganisms. In everyday living environments, where microorganisms exist everywhere, any unannored action can introduce a microorganism into a culture.
in a sterile environment and access to sterile materials, but also always maintain a sterile state in order to study or use the function of a particular known microorganism, otherwise the outside microorganisms are easy to mix in. The phenomenon of the mixing of irrelevant microorganisms from
is called
in microbiology.
pollution prevention is a key technology in microbiology: thorough sterilization and pollution prevention are two aspects of sterile technology.
In addition, to effectively avoid the operator's own microbial infection, but also to prevent the microorganisms studied, especially pathogenic microorganisms or genetically engineered microorganisms that do not otherwise exist in nature from our experimental containers to escape from the outside world (biosecurity). Aseptic operation is very important, whether it is reverse plate, flat line operation, or plate dilution coating method, its operation at every step needs to be "sterile", only skilled, standardized sterile operation, it is possible to successfully cultivate microorganisms.2. Concept of disinfection and sterilization
(1) Cultured cultured bacteria with petri dishes (need to be sterilized)
(2) glass rods, test tubes, flaming bottles and straws (needs to be sterilized)
(3) The hands of the experimental operator (need to be disinfected)
(4) inoculation rings, needles, coating sticks: burning sterilization (external flame).3. After sterilization of the reverse plate
(1), the medium should be cooled to 55 degrees C and the reverse plate operation should be carried out in a timely manner, if not in time, the medium needs to be placed in an electric heat tank at about 55 degrees C to prevent
Agarid
solidification (touching a conical bottle with a culture base by hand, feeling that the temperature of the conical bottle drops to just not hot, you can turn the pan); each 72mm petri dish requires a culture base of 12 to 15 ml.
(2) Why do you want the mouth of a conical bottle to pass through the flame?
: by burning and sterilization, to prevent microbial contamination of the culture base at the mouth of the bottle.
do you turn the plate upside down after the (3) plate condenses?
: After the plate condensation, the lid will condense water beads, the humidity of the solidified culture base surface is also relatively high, the plate will be upside down, not only can make the water on the culture base surface more volatile, but also to prevent the water beads on the lid of the dish from falling into the culture base, resulting in pollution.
(4) in the process of pouring the plate, if accidentally splashed the culture base between the lid and the bottom of the dish, this plate can also be used to culture microorganisms? Why?
: Microbes in the air may grow on a culture base between the lid and the bottom of the dish, so it is best not to culture microorganisms with this plate.4. Flat dashing
(1) Why burn the inoculation ring during the first step of operation and before each dash? Do I still need to burn the inoculation ring at the end of the dash operation? Why?
: The first step of the operation is to avoid the possible microbial contamination culture on the inoculation ring, each line before burning the inoculation ring is to kill the last line after the end of the inoculation ring residual bacteria, so that the next line, the bacteria on the inoculation ring directly from the end of the last dash, so that through the increase in the number of dashes, so that the number of bacteria at each dash gradually reduced, in order to get a single bacteria. Burning the inoculation ring after the dash can kill the bacteria left on the inoculation ring in time to prevent bacteria from polluting the environment and infecting the operator.
(2) after burning the inoculation ring, why wait for it to cool down before dashing?
: so as not to inoculation ring temperature is too high, kill bacteria.
(3) why do you always start at the end of the last dash when you're doing a second and subsequent dash operation?
: After the line, the number of bacteria at the end of the line is less than the beginning of the line, each time from the end of the last dash, can make the number of bacteria with the number of dashes gradually reduced, and eventually can be reproduced by a single bacteria from the fall.5. The coating plate
"sterile" from all details of the operation, and all operations of the coating plate should be carried out near the flame. For example, the distance between the alcohol lamp and the petri dish should be appropriate, the straw head should not touch any other object, the straw should be around the flame of the alcohol lamp, and so on.cell culture
in-body culture cells lack anti-infection ability, so preventing pollution is the key to the success or failure of cell culture. Even in well-equipped laboratories, if the experimenter is careless and the operating technology is not standardized, it can lead to pollution. Therefore, all operations must be as sterile as possible, and every work must be orderly and completely reliable. (Number in mind)(i) preparation before training
to develop a good experimental plan and operating procedures before starting the experiment. The calculation of the data should be done in advance. According to the experimental requirements, prepare all kinds of required equipment and items, inventory it correctly and place it in the operating place (culture room, ultra-clean table), and then start disinfection. This can avoid the start of the experiment, because the equipment is not all round-trip access to increase the chance of contamination.(ii) operation area disinfection
sterile culture room every day with 0.2% of the new Jieer tow wash the ground once (tow cloth to be dedicated), ultraviolet radiation disinfection 30-50min, ultra-clean work surface before each experiment with 70 alcohol scrubbing, and then ultraviolet disinfection 30min. Do not expose cultured cells and culture fluids to ultraviolet light at the same time when disinfecting the work surface, and do not place too many or overlapping supplies on the worktop when disinfecting, otherwise the blocking rays will reduce the disinfection effect. Experimental supplies, such as pipers, waste tanks, dirt boxes, test tube racks, etc. to use 70% alcohol wipe before being brought into the sterile operating table, while UV disinfection.(iii) Hand washing and dress is the same
in principle as surgery. Usually only do observation do not do culture operation, can wear cell culture indoor ultraviolet radiation 30min cleaning work clothes. When working with the ultra-clean table, wear long-sleeved cleaning overalls and disinfect hands with 70% alcohol before starting operation, as the entire forearm is to be extended into the box. If the hand touches potentially contaminated items during the experiment and enters and exits the culture room, wash your hands again with disinfectant. Entering the original training room requires thorough hand washing and wearing a mask and sterilizing clothing. (iv) Flame disinfection
first light an alcohol lamp when cultured in a sterile environment or doing other sterile work. All future operations, such as installing straw caps, opening or closing bottle openings, need to be carried out near the flame. But pay attention to: burned metal tweezers to be cooled to take tissue, so as not to cause tissue damage, after absorbing the nutrient liquid straw can no longer burn with flame, because the residual nutrients in the straw head can burn to form a carbon film, and then use will bring harmful substances into the nutrient solution; (v) Operation
turn on the sterile operation of the typhoon machine after 10 minutes, can start experimental operation, the action should be accurate and agile, but not too fast, the magnitude can not be too large, in order to prevent air flow, increase the chance of pollution. Sterile operation work area should be kept clean and spacious, necessary items, such as test tube racks, piper or straw head, etc. can be temporarily placed, other experimental supplies should be moved out in time after use, in order to promote gas circulation.
operation should be carried out in the sterile area in the center of the operator's station, not in the non-sterile area at the edge. Do not touch the disinfected utensils by hand, and if they have come into contact, replace them with flame-burning disinfection or take spare parts. In order to get convenient, the work surface supplies should have a reasonable layout, in principle, should be the right hand to use things on the right side, left hand supplies on the left, alcohol lamp in the center.
work should be kept sequential from start to finish, and tissues or cells should not be exposed to air prematurely until they have been treated. Similarly, do not open the bottle prematurely before use, and close the bottle immediately if it is no longer reused after use (opening upright can increase the chance of bactericide contamination). When absorbing nutrients, PBS, cell suspension and other liquids, straws should be used separately and should not be mixed to prevent enlargement of contamination or cross-contamination of cells.
not speak or cough towards the operating area during work to prevent saliva from contaminating the work surface by bringing bacteria or mynchons into the work surface. Hands or relatively dirty items can not pass above the open bottle mouth, do not operate the experimental container open directly above the open container, hold the cap by hand and hold the bottle body, tilt about 45 degrees angle to take, try not to put the cap mouth up on the countertop. Bottle mouth is the most easily contaminated, when liquid, such as straw, tip of the tip of the bottle, bottle outside, the straw should be replaced. Only one cell is treated at a time to avoid cross-contamination of the cells.
after the experiment, take the experimental items out of the workstation, if you need to continue the next experiment, wipe the sterile operating table with 70% alcohol, and then let the sterile operation of the typhoon machine after 10 minutes of operation before the next experimental operation. (vi) Safety and
staff should pay attention to their own safety, must wear laboratory clothes and gloves before conducting experiments. Special care should be taken for cell strains from human-origin or viral infections, and select an appropriate level of sterile operator's station (at least two levels). During operation, avoid the production of aerosols, be careful of toxic reagents, such as DMSO (dmethylene), and avoid sharp objects that hurt people. (7) regularly test the following items 1. CO
2
2 CO 2
pressure
2. 2. CO
2
concentration, temperature, and contamination of the water tray (sterile water for water on the
,
weekly); 3. Whether the air flow pressure in the sterile operator's station is normal or not, replace the UV lamp and HEPA filter film regularly, pre-filter (300 hours/pre-filter, 3000 hours/HEPA).