Basic cell culture technique II Sterile technique and

Basic cell culture technique

II. Sterile technique and contamination control A. Working within the laminar flow 1. Function and limitations of laminar flow Laminar flow creates a clean working environment, not a sterile one Filtered air may be prevent cultures from contamination Horizontal-flow hoods must never be used in work with pathogens or with human or primate cell lines

Perform all activities in the hood Hand arm can be serious source of contamination Organizing the hood for routine work

B. Pipetting and prevention of aerosols 1. mouth pipetting is never permissible 2. use automatic pipetting 3. only plugged pipette should be use 4. withdraw the pipette cleanly from its canister or wrapper 5. never use a pipette more than once 6. never draw so much fluid into the pipette as to wet the plug 7. clean up any spillage immediately with 70% Et. OH

C. Additional considerations for good sterile techniques 1. avoid wetting the mouth and lip of bottle, tubes, and flasks 2. never pour from a culture dish or flask 3. do not over fill culture flask or dish 4. never invert caps or lids 5. never work directly over an open culture vessel or bottle 6. do not allow more than one person to work in the hood

D. prevention of cellular crosscontamination 1. Eliminate aerosol or cell suspension aerosol 2. Never work in the hood with more than one cell lines 3. Separate bottles for different cells

Preparation : 1. Clean the laminar flow 2. Reagent preparation 3. PBS, culture medium 3. Sterilization of equipments 4. volumetric pipette 5. glass bottle for medium and reagent 6. disposable centrifuges 7. 4. 75% ETOH

D-PBSA g/L final concentration KCl 0. 2 2. 68 KH 2 PO 4 0. 2 1. 47 Na. Cl 8 136. 9 Na 2 HPO 4. 7 H 2 O 2. 2 8. 06 Make 500 ml/bottle, and autoclave before use

1 X DMEM growth medium 1 pack DMEM from GIBCO, add 1000 ml DDW filter through 0. 45 um filter Add 450 ml/bottle Add 50 ml FBS, 5 ml P/S