Equipment and Aseptic Technique

1. Equipment and Aseptic Technique Helping our cells grow

2. Automatic Pipet • Pipettor or Pipet-Aid • used for transferring large volumes of liquid (1-50mls) • Important not to draw liquid up into the pipettor

3. Sterile serological pipets • Individually wrapped • Multiple sizes • Numbered along the sides • Be sure numbers face you when putting pipet into pipettor

4. Tubes, bottles • Various sizes • Some plastic, some glass • Loosen caps before starting to work in the hood

5. Micropipettor • For dispensing small volumes of liquid • 1-1000ml • Different pipettors dispense different ranges of liquid • Always use a disposable tip with the micropipettor

6. What is a microliter? • In the metric system, used by all scientists • 1 gram is the weight of a cube of water that measures 1cm3—this volume is 1milliliter • 1 liter (l) =1000 milliliters (ml) • 1 milliliter (ml)=one-thousandth of a liter =0.001 l =1 x 10-3 l • 1milliliter =1000 microliters (ml) • 1 microliter (m l)=one-millionth of a liter (l) =0.000001 l =1 x 10-6 l • 1 microliter (ml)= one-thousandth of a milliliter (ml) = 0.001ml = 1 x 10-3 ml

7. Culture Dishes (plates) and Flasks • Flasks • Capped vessels • Prevent spills • Well-plates • Multiple areas for experiments with multiple variables • Different sizes available • Dishes • Easy to manipulate cells • Easier to spill or contaminate

8. “Invisible” life forms are everywhere! • Present on skin, fall off of hair, clothing, in saliva, tears, breath. • Present on surfaces and in water • Especially warm water

9. Micro-organisms • Bacteria • Most are harmless to us • Cell culture media will help them grow • Bacteria will compete for nutrients in media • Fungi • Mold, yeast • Float in air and are on surfaces bacteria yeast mold

10. More microoganisms • Viruses • Enter into cells and destroy them in their quest to replicate • Mycoplasma • A bacteria that is hard to discover and hard to get rid of. Viruses-transmission Electron microscopy Mycoplasm-scanning electron microscopy

11. Sterile vs. Aseptic • Sterile: No life at all (bacteria, fungi, viruses) • Hard to keep an area perfectly steril • Aseptic: Reduced numbers of life forms • Most equipment and reagents provided to you comes sterile • gamma irradiation • autoclaving • High temperature and high pressure steam • filtration

12. Controlling microorganism growth • The goal: Prevent microbial contamination • Simple, common sense guidelines • Handwashing, using 70% ethanol to clean all items being used near cells • Additives to media • Antibiotics • Antifungals

13. Biosafety Cabinet • Laminar Flow hood • HEPA filtered air • Air blown down the back of the hood and then across the surface • Aseptic area • Must be wiped clean before and after use

14. Aseptic Technique • work in culture hood • roll back long sleeves, no rings, watches, bracelets, etc • hair pulled back, caps off • wash hands with antibacterial soap, spray down with 70% ethanol • wash all surfaces with 70% EtOH • don’t cough, talk, sneeze, sing into hood, no eating or gum chewing • lids on bottles when not in use • sterile tips: change if they touch anything other than media. • Keep serological pipets IN hood while working with them…don’t swing them out when you turn around to ask your partner questions. • back surface of hood is cleanest

15. CO2 incubator • maintains CO2 level (5-10%), humidity and temperature (37o C) to simulate in vivo conditions.

16. Water bath • To warm media, TRED and PBS before placing on cells • Can harbor fungi and bacteria, • spray all items that were in the waterbath with 70% ethanol before placing in the hood.

17. Vacuum pump • For permanent aspiration of liquids (media, PBS and TRED). • Use unplugged glass pasteur pipets, throw into sharps box when done.

18. Inverted Phase Microscope • A phase contrast microscope with objectives below the specimen. • Creates contrast in a sample without staining it. • Staining cells will kill them.