In grade 11 I did a Biology EEI (extended experimental investigation) on the effects of environmental stress on cell membranes, using beetroot as a test subject. Beetroot cells contain brightly coloured betalain and anthocyanin pigments in a vacuole, thus the damage to the membrane can be measured by the amount of pigment leaked out of the beetroot.
Effect of Environmental Stress on Beetroot Cell Membranes
High temperatures should denature (change the shape of) the proteins in the beetroot membranes, as proteins are denatured by high temperatures. This will mean that the proteins will not fit in the cell membrane properly, or may come out of the membrane completely, creating holes in the membrane, and the betalain pigment will leak out. The amount of betalain leaked should be proportional to the amount of damage to the membranes. On the other hand, cold temperatures (below 0°C) will cause ice cystals to form inside the beetroot, puncturing the cell membranes and physically creating much larger holes, so more damage should be evident.
It was predicted that the acids and alkalis would damage or denature the proteins in the cell membrane, as proteins are denatured by changes in pH. Ethanol is a polar solvent, so it was predicted to disrupt the polar phospholipid molecules in the membrane. The detergent was predicted to disrupt the membrane by attaching to the non polar phospholipid tails of the cell membrane as well as to surrounding water molecules, pulling the membrane apart.
The solutions should become darker if the beetroot is left for longer in the solutions, as it takes some time for the chemicals to diffuse to the interior of the beetroot slices. Thus the longer the beetroot is left in the chemical solution, the more cells would be affected, and the more betalain would leak out. In addition, it would also take a while for the betalain to diffuse out of the damaged cells into the solution.
Equipment and Chemicals
- Microwave oven
- 1 L Pyrex jug
- Thermometer (immersion or infrared)
- Pen and labels
- 10 test tubes or other identical containers (such as baby food jars), with lids or stoppers if possible
- Test tube rack (if test tubes are used)
- Refrigerator and Freezer
- Raw beetroot
- Distilled water (fairly cheap from the laundry section of a grocery store, or tap water can be boiled to remove the acidic carbon dioxide)
- pH test strips (4) or pH meter
- Measuring jug (up to 100 mL)
- Measuring cylinder (e.g. for measuring medicine)
- Medicine syringe
- Methylated spirits (or other form of ethanol) (35 mL)
- Dishwashing detergent (6 mL)
- White vinegar (or other vinegar) (40 mL)
- Sodium bicarbonate (from baking section of grocery store) (10 g)
- Scales (accurate to 1 g is sufficient)
- Knife, cutting board, potato peeler
- For colorimeter: Ardiuno board, light sensor, computer, sheet of hard plastic, white LED, cardboard box
A fresh beetroot was peeled and washed, then cut into slices approximately 50 mm in diameter, and 2 to 3 mm thick. The slices were trimmed to the same thickness using their flexibility as a guide to how thick they were. Each slice was then washed under running water for 15 seconds, and stored in a jug of water to prevent dehydration.
Approximately 8 small jars or test tubes are required for the experiment. As the beetroot did not fit in the chosen jars (which were around 30 mm diameter and 40 mm tall), the slices were cut into quarters and rewashed.
The jars were labelled -5°C, 5°C, 30°C, 40°C, 50°C, 60°C, 70°C and 80°C. Four quarters were placed in each jar and completely covered with water, except for the 5°C and -5°C jars, which received no water.
One jar was placed in the freezer, and one in the refrigerator (both for 30 minutes); the rest were heated according to the following procedure. A 1 L Pyrex jug containing around 250 mL of water was heated in the microwave to the required temperature (it may help to calibrate the microwave beforehand, e.g. what temperature does the water reach after 45 seconds?).
The jars were placed in the corresponding heated water, and held there with a pair of tongs for 90 seconds, then removed. The 50°C jar, for example, was placed in the water bath that had a temperature of around 50°C, then set aside. Care was taken to ensure that none of the water from the hot water bath entered the jar that contained the beetroot, as that would dilute the colour. After the jars had sat for 70 minutes (any time over 30 minutes would do), their lids were screwed on and they were individually shaken for 10 seconds. After this, the beetroot was removed with a pair of tweezers, and the intensity of the betalain pigment in the water was assessed. The jars from the fridge and freezer were removed after they had been there for 30 minutes, then enough water was added to cover the beetroot, and they were left to stand until the other jars were ready. They were then shaken, and the beetroot was removed, as with the other jars.
Each jar was given a colour intensity rating from 0 to 5 stars, with 0 being clear water, and 5 being the colour of the darkest jar present. The figure below shows this qualitative colour intensity rating for the different temperatures tested.
|Temperature||Colour intensity – from 1 (light) to 5 (dark)||Star Scale – from 1 (light) to 5(dark)|
Photographs of the jars, such as those below, were taken from above and the side to assess and compare the darkness of different jars. Drops were also placed on a white plate, to confirm the difference in colour.
The procedure was repeated for different concentrations of ethanol/methylated spirits (1%, 25% and 50%), detergent (1%, 5%) and pH (4.2, 5.2 and 9.2), as well the control jar of boiled water and beetroot.
For the ethanol solutions, 1 mL of methylated spirits was added to 100 mL of distilled water to make the 1% solution, which was then used the fill the 1% ethanol jar. The 25% solution was made of 10 mL methylated spirits and 30 mL distilled water; the 50% solution used 20 mL methylated spirits and 20 mL distilled water.
For the 1% detergent solution, 1 mL of detergent (measured with a syringe) was added to 100 mL distilled water; for the 5% solution, 5 mL of detergent was added to just under 100 mL of distilled water.
The pH of the boiled water was around 6.5. One jar was filled with white vinegar (pH 4.2), and 7.5 mL of vinegar was added to 100 mL of distilled water to make a pH 5.2 solution. 10 g of sodium bicarbonate added to 100 mL of distilled water made a solution with a pH of 9.2.
The beetroot slices were cut into eights, and 4 were placed in each jar.
The jars were left overnight (about 12 to 14 hours), then the light transmission through each jar was measured with a homemade colorimeter. The colorimeter consisted of an LED which shone through the top of the jar, which was sitting on a piece of clear plastic. Underneath the plastic was a light sensor attached to an Arduino board (further details and photos available in this document).
The sensor gave a reading between 1 (dark) and 1025 (light). The results are shown in the graph below.
|Environmental Stress||Intensity from 1 (light) to 7(dark)||Star Scale from 1(light) to 7(dark)||Light Sensor Readings (approximate % of light transmission through the solutions)|
The results were very similar to the qualitative results (below), but were much more useful as they were non subjective measurements. Side note: this shows that the subjective measure of colour intensity can be quite reliable.
For acids, ethanol and detergent, the results obtained were quite similar to those predicted. It had been predicted that acids would damage membranes, and the results showed that even relatively weak acids – with a pH of 5 – caused damage. The predictions that high levels of ethanol and detergent would damage membranes were also supported. It had not been predicted that weak detergent concentrations would cause damage to membranes; however the results showed that even a 1% detergent solution caused significant damage to membranes.
The only major difference between the predicted results and the results collected was the effect of strong alkalis. It had been predicted that strong alkalis would damage cell membranes; however beetroot cells subjected to high pH levels did not appear to leak any more betalain pigment than the control (pH 6.5). After researching, it was found that high pH levels do cause damage to proteins in cell membranes. It was also found that over time high pH levels denature betalain pigments, and cause them to look light yellow or brown instead of purple. As this experiment entailed leaving the solutions to sit for half an hour, it may have given a misleading result about the effects of strong alkalis. In addition, the solutions sat for up to 14 hours before being measured with the colorimeter.
This experiment shows that the cell membrane is a relatively fragile structure, and can be easily damaged. The proteins in the membrane appear to be the structures most susceptible to be damage by environmental stress.
High and low temperatures, acids, ethanol and detergent were all damage cell membranes. Although the results did not show it, alkalis also cause damage to membranes. Temperatures below freezing destroy the lipid bilayer of the membrane when water crystals freeze and expand, puncturing the membrane. On the other hand, high temperatures increase phospholipid fluidity, and can cause proteins in the membrane to denature, creating ‘holes’ in the membrane. Acids and alkalis also denature proteins in the membrane, because they interfere with the hydrogen bonding in the proteins. Ethanol, being a polar solvent, dissolves the phospholipids in the membrane, and increases the membrane’s fluidity. Detergent is amphipathic, meaning it has both polar and non polar ends. This means that it attracts both the non polar tails of phospholipids as well as water molecules, breaking up structure of the lipid bilayer.
Some applications of these findings are outlined below.
- When parts of our body are very cold, they become numb. This is due to a decrease in permeability of our nerve cell membranes to ions, which prevents a potential difference forming, and thus no nerve pulses are transmitted (Becker & Deamer, 1991).
- Detergent kills some bacteria because it breaks up their cell membranes, increasing its usefulness for cleaning.
- Strong acid rain causes damage to cell membranes and may kill the cells.
- Ethanol may damage cell membranes in the human body when it is consumed.
The findings also have relevance to policymakers when considering the effects of adding waste water to waterways.
Many studies have been made into the effects of adding hot water or chemical wastes to waterways; however, few of these studies looked at the effects on actual organisms or cells.
A recent experiment performed on beetroot cells showed that cell membranes were easily damaged by temperature levels and chemicals commonly found in industrial and domestic effluent. Damage to cell membranes can result in cell death, altered cell function or uncontrolled cell division, causing disastrous consequences for the affected organism. Therefore the effect of the effluent upon cell membranes needs to be considered when making decisions about the acceptable contents of wastes entering waterways.
Moreover, the results showed that even small amounts of these chemicals could severely damage cell membranes. For example, when the beetroot cells were exposed to a 1% solution of detergent, the light transmission through the solution was 23%, compared to 43% for beetroot in pure water. The fact that the 1% detergent solution was almost twice as dark as the pure water solution indicates a very high leakage of betalain pigment, and therefore significant damage to the cell membranes.
Although the results about the effects of temperature were inconclusive, Becker and Deamer (1991) observed that “most poikilotherms (cold blooded animals) are paralysed by temperatures much above 43°C… nerve cell membranes become so leaky to ions that overall nervous function is disabled.” As the body temperature of aquatic organisms depends entirely on the temperature of the water they are in, hot water from industrial cooling systems can paralyse and kill animals and plants in waterways.
Other parts of the investigation showed that cell membranes were also damaged by small amounts of acid and also moderately diluted ethanol.
Decision-makers need to consider the damaging effects of chemicals and temperature on cell membranes when regulating effluent that enters waterways.