Yeast Plate Count Protocol
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. Here are the steps and the experimental plan:
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
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.
- 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
- Two Serial dilution stations (2x two adjacent, sharing equipment):
displacement pipetters, 20-200uL, and sterile tips
yeast suspension on Magnet-o-stir to keep suspended
plastic used pipet container
- Two Plating out stations with sterilized field on which to work, (2x two adjacent, sharing equipment):
2 spreaders in:
2 250 mL beakers,1/2 filled with 95% EtOH
displacement pipetters, 20-200uL, and sterile tips
used pipet container with 1 inch deep diluted Lysol
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 Yeast Plate Count 10 2, 104 and 106 dilutions).
Perform a 106 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 10 2 dilution , record in your book and on the computer.
Label two 4% glucose nutrient agar plates on their bottoms with four bits of data:
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 . (Do not press too hard into the agar. When the sample has been absorbed, the spreader will drag slightly over the agar.)
Incubate inverted at 37C for 48 hours. (Here are plates which have been incubated, two sets of which were incubated upside down. Can you see why the agar side should be up?)
Count the number of colonies . Here are sample plates: a 0.1 mL plate (41 colonies), and a 0.2 mL plate (89 colonies).
(Here are two plates, one with 0.1 mL the other with 0.2 mL aliquots spread. 0.1 = 37 CFU, 0.2 = 97 CFU)
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
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.
In Summer of 2011, two students got tiny colonies on one of their two yeast plates. A gram stain showed them to be more spherical than normal, and the presence of numerous ‘ghosts‘. Here is another image of these affected yeast. Could they be infected with a Killer” virus which infects Saccharomyces?