Isotonic solution in Sheep Erythrocytes Isotonic

Every cell is selectively permeable to different molecules. This type of selectively is caused by a semi-permeable membrane, which allows the movement of certain molecules across it. Water exchange can be measured in two ways: RBC osmotic permeability is measured, and diffusional water permeability is measured (Benga and Borza 1995). Diffusion is the movement of high concentration to low concentration. The diffusion of water across a permeable membrane is called osmosis. Water concentration in red blood cells can cause three different situations: hypertonic solution, isotonic solution, and a hypotonic solution. A hypertonic solution is when there is a higher concentration of salts dissolved outside the cell and pure water inside the cell, the water rushes out of the cell to try to dilute the salt solution causing the cell to shrivel up. An isotonic solution is where the water has achieved equilibrium with concentrations inside and outside of the cell, so there is no alteration of the cell.

A hypotonic solution is where there is a higher concentration of salt dissolved inside the cell and a higher concentration of water outside the cell, so the water rushes into the cell trying to dilute it causing swelling of the cell. If this takes place too rapidly, it will lead to the cell bursting. Knowing the isotonic solution of red blood cells is important in many ways such as filtration because the membranes flux causes the cell to become larger or smaller so if it is too large it can not be filtered through certain size capillaries (Tuvia 1992). Also, red blood cells can only operate in an isotonic state, in a hypotonic solution the cell bursts leaving no cell at all, but in a hypertonic solution the cell shrinks, which decreases the surface area and its viability to uptake oxygen leaving it ineffective for its purpose. To measure the size and amount of red blood cells in each concentration of solutions, a hemocytometer was used.

Materials and Methods

Red blood cells of sheep were obtained through a company, which assured it was free of any contaminants, also standard salt concentrations were obtained (75ml of 1.0% NaCl and 25ml of 1.5% NaCl). We took 6 pairs of test tubes and labeled them according to the following concentrations: 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, and 1.5% NaCl. Then dilute each tube accordingly with the 1.0% NaCl and then made a 1.5% solution with just 1.5% NaCl.

NaCl (ml)Concentration

NaCl (%)Water (ml)Final NaCl (%)

2.0 1.08.00.2

4.01.06.00.4

6.01.04.00.6

8.01.02.00.8

10.01.001.0

10.01.501.5

Table 1: NaCl dilutions in water made according to concentration level to give Final

NaCl concentration.

Hemocytometer

The hemocytometer is calibrated with specific measurements in squares (Fig. 1).

Figure 1: Hemocytometer grid

Each side of the square is 0.1cm (1.0mm) and the space between the cover slip, which is placed on the hemocytometer and the surface of the grid, has a space of 0.1cm as well. So the volume of one grid square is 0.1cm x 0.1cm x 0.1cm= 0.0001cm3 = 10-4 cm3, given 1cm3 = 1.0 ml volume, so the volume of the square is 10-4 ml. Then place the cover slip on the hemocytometer, and added the drop of diluted blood under the edge of the cover slip using the Pasteur pipet. Once the grid was flooded we placed the hemocytometer on the stage for microscopy readings. First it was put at 4X and focused then we switched to the 10X objective and focused on 1 square. We then counted the number of cells in the square including the cells that overlapped with the top and left boundaries, and then we counted the number of cells in another square. Then repeat these steps with all 6 of the different concentration solutions. Afterwards rinse both the cover slip and the hemocytometer with 95% ethanol to sterilize any contaminants and dried them with a tissue.

Results

After counting the amounts of red blood cells in each square according to concentration, we obtained the averages shown in Table 2.

%NaClSquare 1 (# of cells)Square 2 (# of cells)Average (# of cells)

0.215510

0.4543745.5

0.612798112.5

0.8174203188.5

1.0857278.5

1.57119

Table 2: Number of sheep red blood cells in each square and averages.

With these results in place we can see that the highest count of red blood cells was at the 0.8% saline solution. We can graph the percent of saline
solution to the averages as shown in Fig. 2.

Figure 2: Average number of cells plotted versus percent concentration of NaCl.

Shown in the graph the highest number of cells was at 0.8% with an inverted parabolic graph.

Discussion

According to the results; we can determine the concentration of the isotonic solution at 0.8% because it contains the highest number of viable cells. This is what must be the saline concentration of blood because red blood cells must be able to sustain a stable concentration of water inside and outside the cell in order for them not to burst or shrink preventing the carrying capacity of O2. Using the count of cells and dilution factor we can determine the original concentration of the RBC/ml prior to dilution.

188.5 RBC x 1000= 188.5 x 107 RBC/ml = 1.885 x 109 RBC/ml

10-4 ml

So the original sample contained 1.885 x 109 RBC/ml at the isotonic 0.8% solution. We can safely then say the concentration saline in human blood is approximately 0.8% at isotonic state. The number of cells is a rough estimate relative to human RBC/ml because membrane permeability differs significantly from species to species (Benga and Borza 1995).

References

Benga G, Borza T. 1995. Diffusional water permeability of mammalian red blood cells. Comp. Biochem. Physiol. 112B(4):653-59.

Tuvia S, Levin S, Korenstein R. 1992. Correlation between local cell membrane displacements and filterability of human red blood cells. Eur. Biophys.
304(1):32-6.