Clean and wipe the work area, and then place bottles, pipettes, etc. in the work area. Then, start the preparatory work of preparing the culture medium and other reagents), and after the preparatory work is completed, carry out the culture work. Finally, the bench is organized and the work surface is wiped with 70% ethanol.
Operation method
Ultra-clean table aseptic operation method Move Laboratory materials Caveat For operation in vertical benches, do not operate immediately above open dishes.For operation in horizontal benches, do not operate behind open containers. For more product details, please visit Aladdin Scientific website.
Sterile items
1. Eagle's 1 X MEM: 100 ml of various sizes of corked glass pipettes in square pipette tubes 1 ml, 5 ml, 10 ml, 25 ml, or individually wrapped plastic pipettes, prepared with Hank's solution containing HCO3.
2. Culture flasks (pre-weighed if used for lab work1) 25cm2
Non-sterile items
Pipette or ear bulb; 70% ethanol spray bottle; lint-free cotton swabs; absorbent paper towels; pipette tubes with water and disinfectant, scissors, ethanol-resistant markers, notebooks, pens, lab guides, etc.
Procedure
1. Wipe the work surface and the inside of the clean table with a cotton swab or paper towel moistened with 70% ethanol. This includes the inside of the front panel.
2. Remove culture media from the freezer, water tank, or refrigerator. Thaw. Scrub bottles with 70% ethyl alcohol and place immediately needed items on the clean table.
3. Select pipettes and place them in an accessible position on the side of the work surface (Fig. 6.2).
(a) In the case of glass pipettes, open the pipette box and place the lid on it or on one side, with the open end downwards.
(b) In the case of plastic straws, remove the outer packaging, sort the straws by size and type, and place the wrapped straws on a shelf or in a box.
4. Select the other glassware, instruments and equipment you need. Put them on a nearby cart or workbench.
5. Loosen (but do not remove) the lids of all the bottles to be used.
6. Remove the lids from the test or culture bottles to be pipetted and place them, mouth side up, on one side of the clean table behind the reagent bottles}.
Place the bottle to be pipetted, mouth side up, on the single side of the clean table, behind the reagent bottles, so that no hands can pass over the top of the bottle. If only one bottle is uncapped at a given time, hold the cap in the crook of the little finger and palm (Figure 6.6) and, when pipetting is complete, place it back on the original bottle.
7. Selection of pipette.
(a) In the case of a glass pipette, remove the pipette from the box. Keep the pipette in the box parallel to the other pipettes and touch them as little as possible, especially the head of the pipette; if the pipette you are holding touches the end of another pipette still in the box, you cannot use it again; if the pipette you are holding touches the end of another pipette still in the box, you cannot use it again. If the pipette is held in contact with the ends of other pipettes still in the box, it must not be used; insert the pipette into the pipette with the straw pointing outwards towards you, holding it above the scale so that the part of the pipette that goes into the reagent bottle or culture flask does not become contaminated (Fig. 6.9).
Safety tip: Do not use too much force when inserting the pipette into the lancet or pipette. The pipette may break (Figure 7.1.7.5.3).
8. When inserting the pipette into the lancet or pipette, keep the pipette at an appropriate angle. Always keep the pipette positioned so that the tip does not touch the outside of the reagent bottle or the inside surface of the dip station (circled area in Figure 6.9). This is not easy to do if you are a beginner. This is not easy to do, but it is a necessary condition for success and can be learned in practice.9 Tilt the reagent bottle toward the pipette so that the operator's hand does not move over the open top of the bottle. The bottle should also be tilted if a pipette with a capacity of [sm] is used to draw up a culture solution of sm! and transfer it to a 25 cm2 bottle.
10. Repeat the above procedure and transfer the liquid to 4 more culture flasks. If the liquid is to be transferred to several bottles, the bottles can be placed upright. It is important to place them in the inner area of the purification table and not to move your hand over the top of the bottles (for Exercise 1, record the duration of the pipetting to 5 bottles).
11. Dispose of the used straws into the sterilized straw tube. If the straws are plastic. Dispose of them in a double-thick, autoclavable biohazard bag.
12. Replace the caps on the culture bottles.
13. Repeat the procedure, transferring 5 ml of solution to each of the five culture bottles using a 25 ml pipette.
14. Repeat with the 25 ml pipettes to transfer 5 ml of solution to each of the 5 culture bottles. 14. Replace the caps on the test or culture bottles.
15. When the experiment is finished, tighten the caps on all bottles and remove all unneeded solutions and laboratory materials from the work surface.
16. If cell culture operations are to be performed, weigh the culture bottles, check the accuracy of the media dispensed, and allow to stand at 37°C for 1 week to check for possible contamination.