Objectives:
1. To explore the environmental factors (temperature, organic solvents) that may cause cellular membranes to rupture or fail.
2. To determine the extent of damage done to the vacuolar membranes of beet cells by measuring the amount of colored betacyanin exuded from the cellular tissue.

I. BACKGROUND MATERIAL

If you have ever cooked fresh beets, you know how much beet dye pours out of the cells into the cooking water. Why it is so potent? It’s because of betacyanin, a pigment found in the vacuoles of beet cells. In this laboratory, we will explore the structure of cellular membranes using beet dye as an indicator of the stress on the cellular tissue.

A. MEMBRANES
Mem·brane n: a lipid, protein bilayer covering all living cells and most internal organelles.

Biological membranes separate and organize the myriad reactions within cells and allow communication with the surrounding environment. Although quite thin (6-10 nm), membranes mediate the transport of most molecules into and out of cells, contain receptor molecules that detect other molecules or cells, and provide a covering to protect and facilitate cellular functions.

Membranes surround both cells themselves as well as the organelles within cells. For example, vacuoles are surrounded by a vacuolar membrane called the tonoplast. The entire cell is surrounded by the plasma membrane.
The structure of biological membranes is the basis of their many functions. The physical and chemical integrity of a membrane is crucial for the proper functioning of the cell or organelle that it surrounds.

Certain treatments can stress and damage the cell’s membranes. High temperatures cause violent molecular collisions that can physically destroy a membrane, whereas freezing causes water to crystallize as ice and expand because of hydrogen bond alignment, often rupturing membranes. Application of physical stress, such as cutting, or organic solvents to cells can also rupture membranes. Organic solvents dissolve a membrane’s lipids, in effect reducing the membrane to tatters.

Refer to your Text for information concerning organic molecules (solvents), lipids and membrane structure.

B. BEET CELLS AND BETACYANIN

Beet tissue will be your model to investigate membrane integrity. Roots of beet (Beta vulgaris) contain large amounts of a water-soluble reddish pigment called betacyanin, which is localized almost entirely in the large central vacuoles of cells. In intact, undamaged cells, betacyanin remains inside the vacuole, not being able to pass through the tonoplast.

Sources of stress can cause betacyanin to leak through both the tonoplast and plasma membrane. This leakage will produce a red color in the water surrounding the stressed beet cells. The amount of membrane damage is directly related to the intensity of the color, and the intensity of the color can be quantitatively assessed using a spectrophotometer.

D. SPECTROPHOTOMETRY

Spec·tro·pho·tom·e·ter n: An instrument used to measure the relative intensities of wavelengths in a spectrum.

Spectrophotometry is based on the principle that some substances absorb light of a particular wavelength better than of another wavelength. Each substance has an “absorption signature,” where it absorbs a certain amount of one wavelength of light, a different amount of a different wavelength of light, and so on. A spectrophotometer projects a beam of light of a particular wavelength through a solution contained in a glass cuvette, a test tube made of optically clear glass. The pigment will absorb some of the light, and the amount absorbed is proportional to the quantity of pigment present in the cuvette. This proportionality is called the Beer-Lambert Law:

A = Elc

The Beer-Lambert Law states the absorbance (A) of light is proportional to the light path (l) times the concentration (c) of the substance. The molar extinction coefficient (E, epsilon) is