Do the different dilutions of yeast cell suspension affect the number of yeast cells per cm3 that counted using haemocytometer under the microscope?

Dependent variable :     Number of yeast cells per cm3 that counted using haemocytometer under the microscope.

Controlled variable :  

1. Type of yeast cells

• The same type of yeast cells are used to make different dilutions of yeast cell suspension.

2. Volume of distilled water added in each serial dilution

• 9 ml of distilled water are used in making every each of dilution of yeast cell suspension.

3. Way of counting the number of yeast cells

• 4 squares are considered in counting the total number of yeast cell for every group.

Apparatus and Materials

Procedures

1. Setting up the slide

1. The haemocytometer is cleaned with alcohol and wiped with lens tissue to ensure it is grease-free.
2. A damp tissue is used to moisten the shoulder of the haemocytometer to allow the cover slip to stick on it.
3. The special cover slip is pushed on to the slide and the outside edges of the cover slip is pressed down at the same time until Newton’s rings is seen.  

2. Loading the haemocytometer

4. The yeast cell suspension is gently shaken and well stirred to ensure the suspension is properly mixed.
5. The end of capillary tube is inserted into the suspension and risen the liquid into the tube.
6. The end of capillary tube is run along the edge of the cover slip between the arms of the ‘H’. The area between the cover slip and the top half of the ‘H’ is filled with the suspension.
7. The slide is turned through 180O and repeated for the opposite edge of the cover slip.
8. The haemocytometer is placed on a damp tissue in a petri dish for at least two minutes to equilibrate.

3. Counting the cells

9. The haemocytometer is placed on the microscope stage.
10. The grid lines of the haemocytometer are focused using the 10X objective of the microscope.
11. The numbers of cells in the area of primary squares are counted using a hand tally counter.
12. The haemocytometer is moved to another primary squares and the counting is carried on until all 4 sets of primary squares are counted.
13. Steps 1-12 are repeated using each of the serial dilutions.
14. The number of yeast cells in the original yeast suspension into cells/cm3 is estimate.

Quantitative Data

Table 1 : shows the number of yeast cells per cm3 that counted using haemocytometer under microscope from different dilutions of yeast suspension.

* Test tube A which is one-to-ten dilution of yeast cell suspension is not included as one of independent variable.

* Test tube D is the data from our group. The rest of test tubes are pooled data.

Qualitative Data

1. The colour of 1 ml yeast suspension was milky-white and turned fairer when diluted with 9 ml distilled water. It becomes colourless when it reached to one-to-ten thousand dilution.
2. The yeast suspension produced a pungent smell.
3. The haemocytometer has small grids and squares that can be observed under microscope.
4. The yeast cells looked like small granules and irregular in shape when it was observed under the microscope.

Data Processing

1. Sample calculation to estimate the number of yeast cell concentration in one-to-hundred (1/100) dilutions.

Total number of cells in 4 squares = 394

                Number of cells in 1mm3                       = 394 x 2.5

                                                                        = 985

                Cell concentration in 1 cm3            = (394 x 2.5) x 1000

                                                                        = 22500 no. of cells per cm3

Presentation of Data Processing

Table 2 : shows total number of cells in 4 squares, number of cells in 1 mm3 and cell concentration in 1 cm3 in different dilutions of yeast suspension.

Discussion

Total number of cells in the 4 squares, the number of cells in 1 mm3 and cell concentration in 1 cm3 are constantly decrease when the yeast suspensions are more diluted.

Conclusion

The number of yeast cells per cm3 that counted using haemocytometer under the microscope decrease when the yeast cell suspension becomes more diluted. This means that there is a larger proportion of water compared to yeast suspension, which explains why there are less yeast cells per cm3 in a diluted suspension. Thus the hypothesis is accepted.

Evaluation Limitation and Weaknesses

1. The yeast cells did not spread equally in the central main grid of haemocytometer. Therefore, the result obtained is not precise and not reliable.
2. The process of counting the yeast cells under the microscope is not repeated.
3. The counting of yeast cells that touched the chosen two grid lines are not consistent in every observation. Some observation used the top and right grid lines while the other observations used the bottom and left grid lines.

Suggestion

1. The yeast suspension must be stirred thoroughly before being dropped into the haemocytometer. This to ensure that the cells would spread equally and consistently in the haemocytometer.
2. The counting should be repeated at least three times to get certain yeast cells counted by calculating the average of the counting from each observation.
3. Only two grid lines should be considered as the area for counting the yeast cell. It is either top and right lines or bottom and left lines.

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