As with most of the experiments in this course, you will be handling a variety of undomesticated organisms of unknown identity or pathogenicity. Handle all cultures with respect and using standard microbiological procedures.

The media used in this experiment contains ampicillin, an antibiotic to which some people are allergic. Students should not come into contact with the media in any case, but the concentration of antibiotic is relatively high, and so if you are allegic to ampicillin or any penicillin derivative, please handle this media with care.

Although the focus of this course is on Bacteria & Archaea, most eukaryotes are also microbial. One of the most important of the microbial eukaryotes to humans has been the unicellular fungus Saccharomyces cerevisiae, the brewers & bakers yeast. In this lab, we will attempt to isolate yeast from rotten fruit using two general properties: preference for acidic environments and resistance to broad-spectrum antibiotics.

We often also get filamentous fungi in this isolation. In addition, we get ampicillin-resistant Bacteria, usually members of the Cytophagales, that are inherently resistant to penicillin-type antibiotics.

• Rotten fruit or 'fresh' compost
• Loops, slides, coverslips, etc
• Metal spatula, tweezers, Pasteur pipets
• YPD-Amp media:
  

  Add to 1 liter of distilled water:

  • 20 grams dextrose
  • 10 grams peptone
  • 5 grams yeast extract

  Adjust to pH 5 w/ dilute HCl

  For tubes dispense 10ml into each of about 100 16x150mm tubes, cap (USE YELLOW CAPS), & autoclave. Allow to cool, and add 40ul 25mg/ml ampicillin to each tube

  For plates, add 15g agar per liter, mix, and autoclave. Cool to 50C, then add 0.1 gram ampicillin powder, mix and pour into plates

1. Use a loop to innoculate 10ml tube of YPD-Amp. A small piece of fruit is sufficient - don't stuff the whole orange in! Incubate 1-3 days at 30C.
2. If your tube has grown up, transfer 1 drop of the enrichment into a fresh tube of YPD-Amp.
3. Incubate again 1-2 days at 30C.
   
4. Streak a sample of the enrichment onto a YPD-Amp plate and incubate 1-2 days at 30C.
5. Check isolated colonies microscopically (low power) to identify yeast. Look especially for non-fuzzy opaque pasty-looking white or pink colonies. Pick a well-isolated colony and restreak onto a fresh plate.
   
6. Pick a well-isolated colony and make detailed drawings of different-shaped cells seen microscopically. Examine also at high-power/phase-contrast

Examine a sample of the enrichment microscopically at low power - do you see any yeast or filamentous fungii? Can you identify any internal parts of the cells? Do you see budding or sporulating cells?

The spores of a mold will look much like a yeast; the distinguishing trait is the presence of buds - yeast will bud, spores will germinate, and these are readily distinguished.

In the case of molds (filamentous fungi), make detailed notes about the filament structure, especially whether or not the filaments are septate (divided in to individual cells), branched (and the morphology ofthese branches), &c.

Some of you will get Bacteria. Many types of Bacteria are innately resistant to ampicillin. These are great choices for further investigation in the Term Project!