Microbiology Lab Review: Chapter 15

Lab Review

Chapter 15: Direct Counting Using Hemocytometer

1. [CH-15-01] Explain term “cell concentration” –

Concentration of the number of cells per measure of volume. In this lab, the number of cells per milliliter (#cells/mL).

2. [CH-15-02] Explain the term “cell viability” –

Percentage of live cells out of the total number of cells.

Cell viability = {(# of live cells)/[(# of live cells)+(# of dead cells)]}*100

3. [CH-15-03] Why is it important to shake up the sample before loading it on hemocytometer? –

Yeast samples might have settled on the bottom of the container especially since the cells are non-motile. To get an even spread of cells throughout the liquid apply a thorough stirring.

4. [CH-15-04] Why is it necessary to close the diaphragm during direct microscopic counting of the cells? –

To decrease the amount of light and increase the contrast of the field of view. Decreasing the contrast will enable the viewer to find the grid lines of the hemocytometer much more easily and count the live (clear cells) from the blue (dead cells).

5. [CH-15-05] If there are 20 cells in a volume of fluid 0.02 mm x 0.5 mm x 0.25 mm, how many cells are in one milliliter?  –

20 cells in (0.02mm*0.5mm*0.25mm) = 20 cells in 0.0025mm³

Conversion factor (1000 mm³)/(0.0025 m³) = 400000x

(20 cells)(400000) = 8000000 cells/mL = 8.0*10⁶ cells/mL

6. [CH-15-06] What accounts for the inaccuracy of direct cell count? –

The volume of liquid that the cells are dispersed in does not provide the perfect even distribution to the cells. Thus each sample taken will NOT always have the same number of cells. Same goes for the hemocytometer distribution of cells on the slide.

7. [CH-15-07] What is a theoretical minimum concentration of cells detectable by the hemocytometer? –

About 1 cells/mL is the theoretical minimum concentration detectable by the hemocytometer. However standard readings, it should be about 10,000 cells/mL. For the best results 1,000,000 cells/mL.

8. [CH-15-08] What is the purpose of adding methylene blue to the sample in direct count? –

To differentiate between the live and dead cells present in the sample of solution. Thereby allowing us to get the percent viability of the sample.

9. [CH-15-09] Why do living cells appear colorless and dead cells stain blue during direct microscopic count in samples mixed with methylene blue?
   The live cells have enzymes to break down the methylene blue dye, while the dead cells no longer produce the enzymes. Therefore, the live cells will appear clear and without the dye, while the dead cells will be dyed blue.

10. [CH-15-10] Would living cells stay colorless if left with methylene blue for 20 min? Why?
NO,after being dyed and placed on the hemocytometer the yeast cells will usually die in about 10 mins. By 20 mins almost all the living cells will be dead and not produce the enzyme to break down methylene blue anymore. Thus, the living (now dead) cells will be dyed blue.