Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Determining Protoplast Concentration with a Hemocytometer

Background Information
:
In order to calculate the anthocyanin concentration, we will need to know the number of protoplasts per mL of our protoplast preparation.  The number of cells (or other particles) in a sample is routinely determined with a hemocytometer.  This device, which was originally developed to count blood cells, is essentially a modified microscope slide that has two chambers. Each chamber is ruled into nine, 1 mm squares and these, in turn, are further divided. A typical ruling is shown below:

A cover glass placed over the chambers is supported 0.1 mm above the ruled area. The volume over any sized square can easily be determined once the size of the square to be counted is determined. Thus, when the chamber is filled, the volume of the sample over each of the 9 larger squares is:

0.1 mm3 (1 mm x 1 mm x 0.1 mm) or 1 x 10–4 ml

(remember that 1 mL = 1 cm3 = 1000 mm3)

The number of particles per unit volume in a sample can therefore be determined simply by counting the number of particles in several, appropriate-sized squares.

Question(s):  How many protoplasts mL-1 are in the protoplast preparation?

Hypothesis:  Depending upon yield, we can expect large numbers of protoplasts, at least 106 per mL.

Protocol:

1. Obtain a protoplast preparation. Protoplasts are prepared as described in the previous exercise. The protoplasts can be further purified by density centrifugation if necessary.
2. Place a cover glass over the hemocytometer counting chambers.
3. Gently mix the protoplast sample thoroughly to insure a uniform distribution of the cells and withdraw a sample with a Pasteur pipet. Dilute the sample if the cell count is greater than 107 cells � mL–1.
4. Place the tip of the pipette at the edge of the cover glass and fill both chambers by capillary action. Allow the cells to settle for five minutes.
5. Examine at the appropriate magnification (100 x should work for the protoplasts) and count the number of protoplasts in four, appropriately-sized grids in each chamber. Include all cells touching the middle line along the left or top margin of a square, but not the right or bottom. At 100 x, a 1 mm3 square will fill the field. Count a total of 16 grids from 4 chambers. Record your data in Table 1 & 2.
6. Clean and dry the hemocytometer gently after each use – avoid scratching the chambers by using lens tissue. Do NOT use Kimwipes or paper towels.

Data:

1. Complete Tables 1 & 2.
2. Calculate the number of cells per mL using the using the following equation (to be significant at the 5% level of confidence, the 8 counts cannot deviate more than 7% from the mean [Klein and Klein, 1970]):

cells/ml  =  cells/square x 1 square/volume (mm3) x 1000 mm3/ ml x dilution factor

 Table 1:  Hemocytometer Data grid size counted 0.1 mm x _____ mm x _____mm volume of grid counted (mm3) dilution (if any)

 Table 2: Protoplast density in a partially purified suspension of Red cabbage protoplasts measured with a hemocytometer Grid protoplasts grid-1 protoplasts ml-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 mean +/- std

Analysis & Conclusions:

1. Are your protoplast counts statistically significant? Show your work and explain.
2. What do you conclude about our hypothesis?  Did the protoplast suspension have a large yield?

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Last updated:  01/07/2009     � Copyright  by SG Saupe