YEAST PLATE COUNT PROTOCOL ©David B. Fankhauser, Ph.D., Professor of Biology and Chemistry              University of Cincinnati Clermont College,  Batavia OH 45103
Yeast suspension is diluted	This page has been accessed  times since 18 July 2001.	diluted yeast is spread on a nutrient agar + 4% glucose plate

The plate count is one of the most accurate means of enumeration of viable microbes because you get a visual indicator for every cell in the specimen.  The technique stems from Robert Koch's insight gained from viewing colonies growing on the surface of a spoiling slice of potato.  In practice, a small aliquot of a liquid suspension of microbes is spread on the surface of  solidified nutrient medium, which when incubated, leads to each cell 'developing' into a visible colony through repeated fission.  Many samples may have so many microbes that even a small aliquot would have more than the 30-300 colony forming units per aliquot.  In that case, the sample must be diluted to roughly 300-3,000 CFU per mL of which 0.1 mL can then be plated out.

This exercise employs all of these techniques to assay the number of yeast CFUs in a package of yeast.  The stages include

    1)  Suspension of the entire contents of a package of yeast in 100 mL dH2O
    2)  Dilution of this suspension by serial dilution to a dilution factor of 10^6 (a million fold dilution)
    3)  plating out aliquots of the 10^6 dilution on nutrient agar + 4% glucose
    4)   Incubation at 35 (or 37) C for 48 hours to develop the colonies
    5)  Counting all colonies formed, and calculating the number of CFUs in the original package.

REQUIREDSUPPLIES:
        fresh package of baker's yeast
        250 mL beaker with 100 mL dH₂O
FOR EACH STUDENT:
        two nutrient agar plus 4% glucose plates

THREE PROGRESSIVE WORK STATIONS ARE SET UP.

1. Repipet Station :
          sterile capped 16 x 150 mm test tubes, three per student
          test tube rack, one per student
          2 repipets with sterile dH₂O, set for 9.9 mL
          flame

2. Two Serial dilution stations (2x two adjacent, sharing equipment):
          displacement pipetters, 20-200uL, and sterile tips
          flame
          yeast suspension on Magnet-o-stir to keep suspended
          vortex
          plastic used pipet container

3. Two Plating out stations with sterilized field on which to work, (2x two adjacent, sharing equipment):
          2 turntables
          vortex
          2 spreaders in:
          2 250 mL beakers,1/2 filled with 95% EtOH
          displacement pipetters, 20-200uL, and sterile tips
          flame
          used pipet container with 1 inch deep diluted Lysol

PROTOCOL:  Here is a chalkboard diagram of the protocol
 

	Prepare yeast suspension:   First weigh full package of yeast, then suspend contents in 100 mL water. Mix thoroughly (magnet-o-stir works well) for 5-10 minutes . Weigh empty package, determine the dry weight of the yeast added. Create a data table in your notebook in which you list the plate number, the specimen, its dilution factor, the aliquot plated, a space for the number of CFUs on each plate, and room for calculation of total CFUs in the original sample.
	Prepare dilution tubes: Repipet 9.9 mL sterile dH2O into each of three sterile 16 x 150 mm capped tubes . Here is a close up of a repipet set for 9.9 mL .  Note the calibration stem with moveable clamp, the plunger (which is raised up all the way and then depressed all the way to deliver the volume), and the spout.    Label the tubes  2, 4, 6 (for 10 2, 104 and 106 dilutions).
	Perform a 10 6 serial dilution of the suspension as follows, using a fresh tip on a displacement pipetter for each stage:         a. Deliver 0.1 mL of original yeast suspension into first tube (#2)                       vortex to mix         b. Deliver 0.1 from #2 into #4 tube, vortex to mix         c. Deliver 0.1 from #4 into #6 tube, vortex to mix
	Determine the A660 of the 102 dilution , record in your book and on the computer.
	Label two 4% glucose nutrient agar plates on their bottoms with four bits of data: date your initials mL aliquot plated (0.1 or 0.2), and  dilution factor (106)
	Plate out samples:  Measure out 0.1 mL (and 0.2 mL) aliquot of the 106 dilution        Insert the tip just below to surface and release the thumb plunger to draw up the aliquot. apply to the surface of the appropriate plates (do not gouge the agar surface)           spread evenly .
	Incubate inverted at 37C for 48 hours.
colonies from the 0.2 mL aliquot:	Count the number of colonies .  Here are sample plates: a 0.1 mL plate (41 colonies), and a 0.2 mL plate (89 colonies). Calculate the original number of colony forming units (CFU) in the package:                 CFU/package = CFU/plate x dil factor x 1/aliquot x 100 mL/pkg For instance, for the 0.1 mL plate:                  41 CFU/plate x 10^6 x 1/0.1 mL x 100 mL/pkg = 4.1 x 10^10/package

 
9.     Smear and stain examples of a yeast colony (Note how the colonies appear "waxy," from where baker's yeast gets its specific name, cerevisiae) and a contaminate, staining with methylene blue, illustrate at 1000x .  Note in this closer view the staining of the organelles in the yeast cytoplasm .  Here is yeast stained with the Gram stain , a closer view , both of which show budding. This view of yeast plus E. coli contrasts their different sizes and staining properties.