20 December, 2002

Preparing for the Samples

Before we left to collect our samples in the Taylor Valley, we prepared a lot of small growing plates on which to grow yeast and bacteria. If you want to try taking some soil samples and see if there are any yeast or bacterial living in the soil in your area, here is the protocol (that means something like a recipe) for the growth media that we used. We got the protocol by reading papers published by others who have worked with cold environment organisms. If you want to try this, it might be best to get a soil sample from where it is really cold right now. Maybe that is in your back yard or your schoolyard, or maybe you have to go to the mountains where it is likely to be colder. What difference do you think it might make if you try to grow yeast using this protocol but get the sample from a warm climate?

We made two different kinds of growth media.

1. Yeast media

Yeast Peptone Dextrose media (YPD)

Dextrose – 2%

Peptone – 1%

Yeast Extract - .5%

We want to make a liter of media – that is 1000 mls

How much in grams do we need of each material?

How much water will we need? We used deionized sterile water. To figure this out, you just need to use your knowledge of percents.

The media is autoclaved for 30 minutes at 115degrees celcius/110 PSI pressure

2. Bacteria media

Luria-Bertani Broth (LB Broth)

We used premixed capsules – each capsule is mixed with 40 mls of sterile distilled water. We want 1 liter (1000 mls). How many capsules do we need?

If you make LB broth from scratch, this is what you need:

10 grams tryptone

5 grams yeast extract

10 grams NaCl (salt)

Put all weighed ingredients in a 2000 ml flask

Add sterile distilled water up to 1000 mls

Add .5 mls of 4 millimolar NaOH This is to adjust the pH of the solution You need to ask your science teacher to help you figure out molarity or look in a chemistry book or on the web.

Autoclave the same as the Yeast media.

As you can see, it is much easier to use premixed LB capsules. Ask you science teacher about helping you make the broth if you try this experiment.

While the media is being autoclaved (sterilized) we make the antibiotic to add to the yeast media. This will help kill any bacteria that might grow on our plates.

We used amphicillin and Chlorophenicol solutions.

We want a final concentration of 100 microliters per ml.

We need to first dissolve the chloroamphemicol in alcohol because it is not soluble in water. We also need to add the antibiotic to the media after is cools from the sterilization process. If the antibiotic becomes too hot, it does not kill bacteria. This is a lot to think about, but we what to have a good chance of finding yeast cells from the soils.

When everything is ready, we label the plates that have a sterile absorbent pad. The plates are 47mm plates.

We add 2 mls of media to each plate and stack them up to be used when we filter the preparation form the soil sample.