Observations for November 21-26

Actually all the flasks contained green fronds by now - including the 1m30s sample flask! All showed signs of budding, no contamination present whatsoever.

The Next Day...

The plants had no contaminant film surrounding them! 

*Cue celebratory music*

The 30s seemed to be the only one that was really healthy - the 1 min 30s seemed to be too long of a bleach time, as the frond had sunk to the bottom (although there was still a green bud zone.)

Sterilization Procedure

We were going to use 10% bleach solution to sterilize the duckweed fronds before placing them to grow in the sterile media. However, the problem with this method is that if you bleach the fronds for too long, the plants will die - if you bleach them for too short of a time, not all the bacteria will be killed. In order to combat this issue, we bleached each sample for a different amount of time: 15 seconds, 30 seconds, 1 minute, and 1 minute 30 seconds.

So the methods:
A few duckweed samples were transferred into a petri dish filled with tap water using an inoculating loop. An inoculating loop was then used to pick one clone and transfer it to a small watchglass filled with about 10 mL of 10%bleach solution; as soon as the plant was immersed in bleach, the timer was started. After 15/30/60/90 seconds, the plant was immediately removed with a new inoculating loop and placed into a new petri dish with sterile water for rinsing and observation (I was looking for a small green bud zone which would indicate that the sample was still alive) Then using a new sterile inoculating loop, the samples were transferred into their appropriate sterile flasks with minimal exposure time to air. A new sterile rinsing petri dish was used for each clone.

This Calls for a New Game Plan

To combat bacterial and fungal contamination, we were going to make the following changes:

1. Use giant cotton balls to stuff the flask openings instead of tinfoil (Dr. B said when she did research in her lab, this method worked quite well.)
2. Autoclave the media longer, with emphasis on listening for a very high whistle
3. Most important change: Sterilize the plants before transferring them to the media to grow

On November 18, I redid the autoclaving procedure:

8:18: started heating the pressure cooker
8:33: faint gas noise readily heard, first signs of bubbles foaming at the valve
8:37: started whistling
8:42: louder, but still no "chugging"
8:43: shut heat off
*Here I had to dash off to Multi class

10:01: turned heat on again
10:30-10:50: let it run

Ahhh, Contamination!!!

We let the duckweed sit for a few days. This was the 2 days after the media sterilization procedure; as you can see, signs of contamination are already apparent:

...This, is 9 days afterward:

Ick, I know!

... Clearly, sterilization is going to be a bigger challenge for us than I'd thought. 

(But hey! This is all part of the experience - might as well let it mold me! Ahahaha...)

"Autoclaves" and Trying Days

First thing to do when the SH media arrived was to reconstitute 1L of it, and get my samples growing. However, sterilization was going to be a big issue for us, as we had no autoclave. Fortunately, we were able to make do with a makeshift autoclave - Dr. B's pressure cooker.

Below is the methods write-up:

Wednesday, 10/30

Using a tin weigh boat and metal spatula, the entire contents of the SH Media bottle was massed on an electronic balance and determined to be 32.0 grams. If 32.0 g can be reconstituted to from 10L, then it follows that to make 1L would 3.2 g. Accordingly, all powdered media was placed back into the original container except for 3.2 g, and stored at 2-8˚C. To a 2 L Erlenmeyer flask was added 3.2 g of SH Media and 1 L of distilled water, and swirled until dissolved. 150 mL of the solution was apportioned into four 250 ml Erlenmeyer flasks each; the large Erlenmeyer flask containing the remaining solution was covered over the opening with tinfoil and left to stand at room temperature.

Then, the 250 ml flasks containing the media were each covered with tinfoil at their openings and placed into a small-sized safety pressure cooker. The pressure cooker was then sealed and placed onto a hot plate, with the heat setting turned on high. The pressure cooker was heated until whistling; the heat was then turned down to 8 and let sit for 20 minutes. After 20 minutes, the heat was turned off, and the flasks were left to cool.

Friday, 11/1

A jar of samples were taken out of the greenhouse. Forceps were used to pick up several colonies  by transferring them to a petri dish, and then isolating four separate fronds (each their own colony). One colony was transferred to their respective flask, and labeled. The flasks were then taken to the greenhouse, covered lightly with tinfoil, and exposed to normal intervals of sunlight and darkness.