Experiment #6 - Osmolarity and a Cytological Bioassay

Introduction

Background Information

Diffusion is the process by which molecules randomly move from an area of high concentration to and area of low concentration. When this process involves water it is called osmosis and can be demonstrated though the use of a selectively permeable membrane which allows water molecules to pass but prevents a larger molecule (such as sucrose molecules) from passing. The build up of pressure on one side of the membrane is termed the osmotic pressure and can be measure using special instruments. The osmotic concentration responsible for the build up of pressure is termed the Osmolarity (Osm) and is defined as the number of solute particles dissolved per unit volume. Osmosis may also be defined as the diffusion of water from an area of high water activity to a region of low water activity.

In living cells such as the mammalian erythrocyte the effects of osmosis can be seen if cells are placed in either a hypotonic solution (solute is greater inside the cell than out) or a hypertonic solution (solute is greater outside the cell than inside). The former will cause the cells to swell and perhaps burst (hemolysis) and the latter will cause the cell to shrink. In an isotonic solution there is no net movement of water into or out of the cell.

In plants a hypotonic solution will not cause the cell to burst because of the rigid cell wall. Instead osmotic pressure builds up on the inside (turgor pressure) providing a useful mechanical support for leaves and stems. In the case of a hypertonic solution water will diffuse out of the cell causing the cell membrane to shrink and pull away from the cell wall. This shrinkage is termed plasmolysis and is capable of being studied in a laboratory exercise called a bioassay.

Purpose

The purpose of this lab is to determine the concentration of sucrose in a sample by a bioassay procedure.

Hypothesis

As the concentration of the solution increases there will be increased evidence of plasmolysis.

Procedure

A reaction plate was obtained, wells labeled and filled with 1 ml of solution according to the chart below:

The outer epidermal layer of an onion bulb was obtained, cut into small segments and placed into each of the wells to incubate for approximately 25 minutes. During this time nine microscope slides were obtained and labeled. After the incubation period the onion bulb segments were transferred to their corresponding slides, drained, and stained with iodine. The segments were examined under the microscope and the relative number of plasmolyzed and unplasmolyzed cells were recorded.

Results

Discussion

In analyzing the results of the bioassay the initial hypothesis was supported in that there was a progressive increase in the relative number of plasmolyzed cells as the sucrose concentration increased in solution. Individual plant cells showed evidence of increased plasmolysis beginning with all normal cells in slide one to all plasmolyzed cells in slide six.

For the unknown samples there was a progressive decrease in the number of plasmolyzed cells in accordance with the decreasing strength of the unknown solution. Slide eight exhibited a granular appearance. Since the cells were still intact and showed evidence of plasmolysis it is unlikely that the granules were the result of lysis of the cellular membrane and release of the cells contents. The granules were more likely to have resulted from contamination of the well samples with particulate debris.

In determining the relative concentration of the unknown from the standard curve, the derived concentration did not match the pattern of expected values between the full strength and 1/2 strength unknowns. Reasons for this may be either contamination of the wells, experimental error in preparing the well samples, or perhaps a manufacturers' defect in the stock solution. The pattern of expected values between the 1/2 strength and the 1/4 strength unknown solutions matched expected value patterns.

In conclusion, this experiment showed that the there is a progressive increase in the relative number of plasmolyzed cells as the concentration of a solution increases. Using a set of standard concentrated solutions a standard curve can be generated in order to determine the concentration of an unknown solution. A comparison of class data is recommended for more conclusive results as well as a more precise determination of the concentration of the unknown solutions.

Study Questions

1. In order to prepare the unknown sucrose solution you would dissolve 1000 of the sucrose molecules into 1 liter of water in order to yield a 1 Osm concentration..

2. Excessive application of soluble fertilizers like ammonium sulfate will cause wilting of young plants because water will diffuse out of the plant cells (plasmolysis) and into the fertilizers due to the difference in osmotic concentrations.

3. The effects of placing young protoplasts in distilled water will be swelling of the cells and end with lysis. In contrast, the placement of normal onion epithelial cells into distilled water will cause swelling of the cells, but, because of the rigidity of the cell wall there will be a build up of pressure (turgor pressure) instead of lysis of the cell.