Lab 4

Determination of cell counts in 1 g of

bakers yeast using:
1. Serial dilution and colony counting techniques
(viable cell counts only)

2. Direct microscopic counts using Thoma counting

chamber (total cell counts; viable + dead cells).

3. Cell volume measurements (total cell counts; viable

+ dead cells).
1. Serial dilution and colony counting techniques (viable cell counts
only): 0.5 ml
0.5 ml 0.5 ml 0.5 ml 0.5 ml

Stock yeast Dil. factor 10-1 10-2 10-3 10-4 10-5

suspension
10 g/L

1. Aseptically add 4.5 ml saline into each of the 5 tubes.

2. Aseptically transfer 0.5 ml stock yeast suspension into the first tube 10-1 (done by instructor).
3. Use 1ml pipette to mix by pipetting up and down then transfer 0.5 ml to the next tube (10-2
dilution) then discard the pipette (Why???).
4. Repeat step 3 till you prepare 10-5 dilution.
5. Aseptically transfer 0.5 ml from the dilutions 10-5 then 10-4 then 10-3 using the same pipette
(Why???) into an empty sterile Petri dish.
6. Add 20-30 ml soft agar (about 42C) and mix by carefully moving the agar plate in the form of an
eight. (Pour plate technique)
7. Leave to solidify and incubate at 30C.
1. Serial dilution and colony counting techniques (viable cell counts
only):
Next week

Count no. of colonies

Use plate of 20 -200 colonies (Why ???)

Calculate the cfu/ml in the original stock yeast suspension (10 g/L)
according to:

Calculate the no. of cells in 1 g yeast:

2. Direct microscopic counts using Thoma counting chamber (total cell
counts; viable + dead cells).
1. Use the 10-1 dilution (1 g/L).
2. Clean the Thoma counting chamber and the cover slip using 70% ethanol.
3. Breath onto the Thoma counting chamber and place the cover slip and press it onto the
counting chamber.
4. Use Pasteaur pipette to fill the counting chamber with the suspension.
5. Use 40X magnification and PH2 and count the cells in 1 b-field (16 c-fields).
Top view 25 b-fields Counting field
1 b-field (16 c-fields)

Side view

0.05 mm

Volume of 1 c-field = 0.05 X 0.05 X 0.1

= 0.00025 mm3

6. Get the average no. of cells in 1 c-field (no of cells in 1 b-field/16)

7. Get no. of cells/ml according to
Average no. of cells in 1 c-filed 0.00025 mm3
??? 1000 mm3 (1 cm3 or 1 ml)
8. Get the no cells in 1 Liter (1 g yeast). (Remember we used the 1 g/L suspension) according to:
No. of cells/ml X 1000
3. Cell count using cell volume measurements (total cell counts;
viable + dead cells).
1. Use the 10-1 dilution and place one drop of yeast suspension over a clean glass slide
and cover with a clean cover slip.
2. Examine under oil immersion lens (100x magnification) and PH3.
3. Determine cell dimensions (length; 2a and width;2b).

2b
2a

4. Determine the cell volume according to:

4 a (m) X b2 (m2)
Volume of 1 cell (m3) =
3

5. Determine no. of cells in 1 g yeast (1 X 1012 pg) =

1 X 1012 pg
Volume of 1 cell (m3) X Density (1.07 pg/ m3) Weight of
1 cell
 Next week
midterm exam
20%