 |
YEAST PLATE COUNT PROTOCOLProfessor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103 |
 |
| Yeast suspension is diluted |
|
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. 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. |
 |
 |
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)
b.
Deliver
0.1
from #2 into #4 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.
Spread
evenly . (Do not press too hard into the
agar. When the sample has been absorbed, the
spreader will drag slightly over the agar.) |
|
|
| 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).
(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 |
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.
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?
