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Submitted by Jennifer Newsted on Tue, 10/02/2012 - 6:35pm
This week's focus in cell culture is on aseptic transfers because we will be getting our cells soon (Eek! I'm so excited).
On Monday we were assigned 100 mL of Luria Broth (LB in a 250 mL glass bottle) and were tasked with aseptically transferring 10 mL to 2 conical tubes, 10 mL to a glass bottle, and 5 mL to a cell culture (treated) petri dish. Here's a quick breakdown of the procedure for those not familiar:
- Wearing gloves: Connect Bunsen Burner to gas and use striker to ignite. Then adjust flame for optimal height and inner cone.
- Loosen caps on conical tubes (so you don't spend a lot of time trying to get the cap off with one hand while transferring)
- Break open wrapper to 10 mL pipette (sterile) and insert into pipette aid without setting down, without allowing pipette to touch anything other than pipette aid, or touching the pipette directly yourself
- While holding the pipette aid & pipette, use free hand to flame (pass through the flame to help keep sterile) sterile bottle of Luria Broth, remove the cap (pref. without setting cap down), flame bottle opening without cap
- Insert pipette without making contact with anything other than the broth and extract 10 mL of broth while holding bottle at an angle away from the face to help prevent settling particulates in the air from compromising sterility of broth
- Remove pipette, flame bottle opening and replace cap on glass bottle
- Remove tube cap, hold tube at an angle again, and insert pipette. Release broth into the tube against the side of the tube to prevent splash and minimize bubbles.
- Re-secure the cap and extinguish flame (assuming you are finished with all of your transfers)
Ideally, I do not want to see any signs that I contaminated my broth transferred into the new containers. Within 72 hours, uncontaminated broth equals excellent aseptic technique. You shouldn't speak if possible while transferring or working with sterile equipment. Also, clearly it's not advisable to touch anything unnecessary during aseptic work especially not the skin, bench, coat, glasses, etc.
In my case, I was meeting this challenge while I may or may not have had a fever and was dealing with sinus drainage from being ill all weekend. In fact I'll live on in the BIT lab long after I'm gone as long as the glass bottle I transferred to is not broken. I failed to use tape on the bottle before I labeled it. Remember I may or may not have had a fever.
Good times practicing my technique when all I wanted to do was blow and or scratch my itchy nose! And for the record, had we been actually working with our cells I would have been unable to handle them because I would have been paralyzed with fear I would contaminate them.
Today was our 24 hour check of our transfers. So far, so good. No change in media color, no turbidity and no colonies showing on my LB agar plate which we streaked after cotton swabbing our working area at the benches.
Safe for today. (Insert cliffhanger Hollywood score music here.)
Does anyone have any other aseptic technique tips for the procedure described above? Please do share! I'd really love to read them!
Also today, we innoculated two new tubes with E. Coli using innoculation loops in order to practice our streaking on plates tomorrow. These projects/procedures will be added to my website listed above.