Index
Practice
Sterile Technique
Objectives (Sessions 1 and 2)
· Learn and practice sterile procedures to keep cells alive and growing under cultured conditions.
· Recognize the many steps in passaging cells that can lead to contamination and be aware of procedures to minimize that contamination.
Materials (Sessions 1 and 2)
NOTE: Use pipets, flasks, etc from “Practice Box.”
15 mL, 50 mL centrifuge tubes
Aspirating, 5 mL, 10 mL pipets
Ethanol squirt bottle containing water
Micropipette, micropipette tips
24-well plate
T-75 flasks
Water
Tape and/or plastic shield
Safety
1. Wear disposable latex gloves at all times.
2. Wear safety glasses at all times.
Session 1
Procedure/Practice
** Read Tips for Sterile Technique and Cell Manipulation. **
** Read Feeding Cells. **
· Do this exercise on a table in the teaching lab.
· Work with a partner. Critique him or her. Communicate to your partner if you see her or him make a mistake or pull their hands to the “hand out of the hood” line. Good coaching at this stage will dramatically improve cell culture technique.
1. Place a line of tape 24 in from the edge of the table to simulate the depth of the hood. Also, place a line of tape 6 in from the edge of the table to serve as the “hand out of the hood” line. If available, place plastic shield at front ledge of table to simulate hood height in tissue culture hood. Remember all your work should be done at the middle to back of hood.
2. Transfer water from one centrifuge tube to another using a 10 mL pipet.
3. Aspirate water from flask. (There won’t really be any vacuum.)
4. Add 25 mL water into T-75 flask in 5 mL aliquots.
5. Feed flask of (imaginary) cells with 10 mL water following full instructions given on Feeding Cells protocol. Repeat as needed until you feel comfortable.
Session 2
Procedure/Practice
** Reread Tips for Sterile Technique and Cell Manipulation. **
** Reread Feeding Cells. **
** Read Cell Passage. **
· Do this exercise on a table in the teaching lab.
· Work with a partner. Critique him or her. Communicate to your partner if you see her or him make a mistake or pull their hands to the “hand out of the hood” line. Good coaching at this stage will dramatically improve cell culture technique.
1. Place a line of tape 24 in from the edge of the table to simulate the depth of the hood. Also, place a line of tape 6 in from the edge of the table to serve as the “hand out of the hood” line. If available, place plastic shield at front ledge of table to simulate hood height in tissue culture hood. Remember all your work should be done at the middle to back of hood.
2. Review procedure for feeding a flask of cells. Repeat until you can do without looking at your notes.
3. Mix water in flask using 10 mL pipet. (Flask should contain ~10 mL water.)
4. Passage flask of (imaginary) cells following full instructions given on Cell Passage protocol. Repeat until you can do the procedure comfortably without looking at your notes.
5. Transfer 1 mL of water using micropipette from 15 mL centrifuge tube to each of 6 wells in a 24-well plate. Aspirate water from wells using glass Pasteur pipet.
NOTE: When adding cells to a well, squirt cells into center of well.
NOTE: When adding rinse solution to a well, squirt the liquid on side wall of well (where there are no attached cells) rather than in center of well (where there are cells).
NOTE: When aspirating, place tip of Pasteur pipet along side of wall.
Questions
1. Why do the protocols recommend NOT spraying the T flasks with ethanol?
2. Identify 5 steps in the Cell Passage protocol where a mistake on your part could lead to contamination.
3. T-25 flasks (having 25 cm2 surface area for cell attachment) are also commonly used in tissue culture applications. Develop a table like Table 1 in the Cell Passage protocol that would be appropriate for 1:2, 1:6, and 1:12 dilutions. Assume a total volume in each flask of 3 mL.
Answers to questions should be recorded in your laboratory notebook. The answer to each question is worth 5 pts.