Pins and Needles AND Aseptic Transfers

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!

 

jennifernewsted.wix.com/biosciencetech

 

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. 

 

Bio-Link Program: 
Portland Community College

This is going to sound

This is going to sound incredibly geeky, but my advice is to practice with your tooth paste cap.  

I posted some pictures below to show how this works:

 

 

 

That's fantastic advice and

That's fantastic advice and nothing is too geeky at this point! Everything helps!!

Yep, Sandy's suggestion is

Yep,
Sandy's suggestion is good. Practice with ANYTHING that has a cap. (That was my homework to a couple thousand HS freshmen!)

1. Completely loosen the cap being careful not to touch the bottom rim.
2. Using your index finger and thumb, take the cap off slightly so you are sure you can reset it and remove it easily.
3. ASEPTICALLY remove the cap by encircling it with your pinky. (Practice holding the cap in your pinky as you snap your fingers.

With this method, the opening of the cap stays down as you perform the rest of your task.
4. Right after you remove your sample, you can set the lid back over the vessel, but you can again pick up the cap with your pinky.

You can practice this on your kitchen bench or dining table.
Put out a drinking glass, a dessert plate and a small bowl (and fit the plate over the bowl), a knife or spoon or a straw and some ketchup or mayo or mustard, even if the bottle has a squirt cap. Loosen the squirt cap to expose the contents. (You can use the plate and bowl as if it were a Petri plate and lid.)

Keep in back anything you are NOT using at the moment.

Move forward anything you are transferring from or to.

In my lab, we stopped spraying caps with EtOH because there was one spark-induced fire in a TC hoods (that was used nearly constantly). You can keep a bottle of 70% right outside and use a dampened paper towel, but it's really not necessary unless your company's GMP requires this.

Loading serological pipettes into their pipettors:
Consider the double lines near the plug as the line you do not cross.
Hold the pipette between the double line and the plug so your hand is UNDER the pipette.
Plug into the pipettor so it feels relatively firm. The double line should still be easily visible.

All the aseptic handling should be performable with your elbows near your torso.

Before I start an aseptic procedure, I sit or stand where the work will be done and go thru in my mind how each step will go and look including where my hands will be, then reset whatever I notice could be placed better (and have been doing cell culture since 1967). Mindful aseptic handling.
Regards,
Toby Horn

Busted!! I seem to have a

Busted!! I seem to have a habit of touching the bottom rim of the caps. Thank you for bringing this up or I may not have noticed for a long time. Now I can watch for it. Fantastic tips!!! Nothing is too basic. I really appreciate the input. These are also great exercises for me to do at home. Thank you!

Also, you just solved the mystery of why my instructor stands there and touches everything before she demos a procedure. She has to be walking through it in her mind and placing for efficiency. Brilliant!

I'm still at the stage where I am trying to juggle and therefore have no idea where things should be placed for maximum efficiency but I'll keep working on it and get better!

Thanks again!

Jenn

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