YM agar plate (acidified)

• Mix the following in a bottle. Use pure water (or distilled water) to dissolve.
• Yeast extract   0.3%
• Malt extract   0.3%
• Bacto peptone   0.5%
• Glucose (dextrose)  1%
• Plain agar  2%
• Shake 2-3 times.
• Autoclave for 20 min at 121C.
• When the autoclaved medium cools to 55-65C, add 0.7% (v/v) of 1N HCl to the medium (final pH 3.0-3.5). [Use 0.7ml of concentrated HCl (10N)/liter]
• Make agar plates by putting 20-25 ml of the medium in each Petri dish.

YM broth

•  Make solution of yeast extract [0.3% in water].
•  Put 1 ml of the solution in 1.5 ml Eppendorf tubes.
•  Autoclave for 20 min at 121C.

2% Malt extract agar slant.

• Dissolve 2% of malt extract.
• Add 2% of agar.
• Dissolve agar by heating with microwave.
• Put 5 ml of the media in each test tube, and plug.
• Autoclave for 20 min at 121C.
• Slant test tubes at 30-35 degree until solidify completely.

0.7% Saline solution

•  Make 50 ml of 0.7% saline solution [0. 35g NaCl to the water].
•  Put 1 ml of the solution in 1.5 ml Eppendorf tubes, and 100 ul in 0.5 ml Eppendorf tubes.

•  50 mM Tris-HCl (pH 7.2)
•  50 mM EDTA
•  3% SDS

The isolates in the preliminary study were tested for assimilation of 19 major carbon compounds to provide information to supplement the sequence data for identification. This method has provided us with information to separate closely related yeast strains. Morphological observations will be made at two months after the initial culturing to allow enough time for possible spore formation. When appropriate cultures of non-sporulating yeasts are available, we will attempt to establish mating competence by mixing cultures. Because we were interested in yeast enzymatic activity that might be of use in the beetle habitat, we investigated the capacity of the cultures to degrade a variety of substrates (Untereiner and Malloch, 1999).  One test, the difference in lipid degradation also distinguished closely related strains in one case.DNA sequencing  Sequences will be obtained for all yeasts isolated in the study for approximately 600 bp from the 5’ end of 26S rDNA (D1/D2 region).  We have have used readily available primers for the PCR reactions and sequencing (Kurtzman and Robnett, 1998). After initial screening one or two isolates of each group will be used to determine the entire 18S rDNA sequence (Suh and Blackwell, 1999). The species of previously known yeast-like fungi in Coleoptera, Symbiotaphrina kochii CBS 588.33 and CBS 250.77, Symbiotaphrina buchneri CBS 420.63, Candida karawaiewii ATCC 22994, C. xestobii ATCC 24001, C. rhagii NRRL Y-2596, and C. tenuis NRRL Y-2597, were sequenced in these regions as well and will provide a conserved region for comparison to ensure that we do not have contaminating DNA, often a problem when endosymbiotic systems are studied.

The purified double stranded PCR products were used directly as templates for sequencing with an ABI PRISM™  Dye Terminator Cycle sequencing kit. The DNA sequences will be determined by an ABI PRISM 310 Genetic Analyzer. The 26S rDNA sequences will be aligned in the database of more than 600 D1/D2 sequences of almost all yeasts in Saccharomycetales (Kurtzman and Robnett, 1998, and unpublished results) using Clustal W (Thompson et al., 1994). Phylogenetic analysis is designed for identification, but the same process also allows for discovery of close relatives, and informs subsequent sampling. We use the parsimony options in PAUP* (Swofford, 1999) to compare the yeast sequences.