Sunday, January 17, 2010

Double agar troubles

Although most of the recent posts where about receiving new equipment and plans to buy additional ones, we also have to push the science forward. One of the things that worries the last few days is getting reliable agar plates for growing yeast strains.

The troubles come in two different areas. I wrote before that we have contamination in plates. Well, we still have contamination in the plates we pour. Moreover, even when we do not have contamination, the plates start drying when we incubate them. For example, here the agar dried and then cracked.

In another plate, it didn't crack, but we see it drying toward one corner (the agar is thinner).

We know that the square plates are prone to water loss (much larger perimeter than round plates). For this reason we seal the plates either in plastic wrap (aka Saran wrap) or inside plastic bags. The two plates above were in the incubator for two days, one with plastic wrap and the other inside a plastic bag and both show bad signs of dryness.

We believe that part of the problem is the aggressive air circulation in our incubator. We called the company and they claimed that there is no way to turn down the fan speed. We now need to consider how to address this. One possible solution is to humidify the incubator by putting a container with water. If this will help, we might consider a more intelligent way of ensuring that it is humid.

The problem of contamination is a serious one. Since we want to grow libraries that will serve as resource for further examinations, having contaminants grow on the same plates is an issue. Since we first noticed the problem we talked with other yeast people in the building and they report similar problems.  All of them recommended shutting down the A/C circulation before pouring plates. Apparently the air supply introduces yeast and bacteria from outside the building into the lab.

One of the debates in yeast sterile techniques is whether to use flame. On the one hand, working with a flame you sterilize the opening of the bottle before you close it, reducing chances of contamination. On the other hand, an open flame heats air, which in turn goes up, leading to a circulation of air toward the area you are working in.

To test the effectiveness of different techniques, Ayelet tried a test where she compared pouring inside a biological hood, without flame sterilization and with flame sterilization. The results are inconclusive (small numbers) , but it seems that flame sterilization is a bit worse. It also seems that with A/C off the total amount of contamination was down. Surprisingly, even working inside a hood we got contaminants. This experience might be due to the fact that while waiting for the hood the agar cooled a bit (although it was in a sealed autoclaved flask).

To make sure that the source of contamination is the air and not the plates (that are supposed to be sterile), we tried today another round. This time we exposed half of the plates to U/V (using the Singer RoToR as a UV "oven") and the other half without exposure. 

We need to wait another day to see how this batch fared. In the mean time, the number of plates we throw is alarming, and so we hope to solve these issues soon.

1 comment:

Sourav said...

hey Nir, we always incubate those plates in a plastic bag. They have more surface area and thus loose moisture quickly!