Vivian Marlowe
The root tip is placed in acetic alcohol to preserve it for observation under a microscope. Acetic acid causes the swelling of the protoplast, while ethanol causes shrinkage, so the two are often combined to create an effective fixative for plant tissues. This mixture is also known as Farmer's Fluid. The root tips are cut at a specific time of day, usually around noon, to ensure the highest likelihood of finding actively dividing cells. This technique is often used to investigate mitosis in plants, where the chromosomes in root tip tissue are made visible through staining.
| Characteristics | Values |
|---|---|
| Purpose | To investigate mitosis in root tips |
| Fixative | Acetic acid and ethanol |
| Ratio | 3:1 (ethanol:acetic acid) |
| Alternatives | Ethanol IDA or 95% ethanol |
| Root type | Allium, garlic, onions, hyacinths, bean/pea seedlings |
| Preparation time | 1-10 days in advance |
| Cutting time | Around midday |
| Cutting length | 2mm from the growing root tip |
| Staining | Small drop, leave for 2 minutes |
| Microscope magnification | x400 |
| Area of interest | Meristematic zone |
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What You'll Learn
To observe cell division in root tips
The process of mitosis involves the division of the nucleus of the Eukaryotic cells into two, resulting in the splitting of the parent cell into two daughter cells. The stages of mitosis include the coiling and thickening of chromosomes, the shrinking and disappearance of the nucleolus and nuclear membrane, and the formation of spindle fibres from a cluster of fibres.
To observe mitosis in onion root tips, the onion root is placed in a vial containing freshly prepared aceto-alcohol. About 2-3 drops of acetocarmine stain are added, and it is heated lightly on a burner. The excess stain is removed, and the stained part of the root tip is retained. The meristematic tissue of the root tip is then squashed under a coverslip using a needle to straighten it out as a fine cell layer. The slide is then examined under a compound microscope, and the changes are noted and sketched.
Another method to observe cell division in root tips is to place the slide and coverslip on a double layer of paper towel and fold the paper over the coverslip. The slide should be on a flat surface, and strong vertical pressure is applied to the coverslip with the thumb. The root tips are then viewed under a microscope at 400x magnification to observe the chromosomes within the actively dividing cells. Sketches or photographs of the cells showing the stages of mitosis can be made.
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To prepare root tips for microscopy
After cutting the root tips, discard the rest and retain only the 2mm tips. Add a small drop of stain and leave it for about 2 minutes. Then, place the slide and coverslip on a double layer of paper towel and fold the paper over the coverslip. Ensure the slide is on a flat surface, then use your thumb to apply strong vertical pressure to squash down on the coverslip. Avoid twisting or rolling your thumb.
Alternatively, tap the coverslip about 20 times by dropping a wooden-mounted needle or a pencil, blunt end down, from a height of 5 cm onto the middle of the coverslip. Now, you're ready to view the root tips under a microscope. Use 400x magnification to observe the actively dividing cells and locate the meristematic zone, characterized by small, square-shaped cells with large nuclei.
The preparation of root tips for microscopy is a delicate process that requires precision and attention to safety. By following these steps, you'll be able to effectively observe and analyze the cellular structure of plant roots.
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To fix the root tips
The next step is to add a small drop of stain and leave it for 2 minutes. The slide and coverslip are then placed on a double layer of paper towel, and the paper is folded over the coverslip. It is important to ensure that the slide is placed on a flat surface. Then, using your thumb, apply strong vertical pressure and squash down on the coverslip. Do not twist or roll your thumb from side to side.
An alternative method involves tapping the coverslip about 20 times by dropping a wooden-mounted needle or a pencil, blunt end down, from a height of about 5 cm onto the middle of the coverslip. The root tips are then viewed under a microscope at 400x magnification to look for actively dividing cells and locate the meristematic zone.
The root tips are fixed in acetic ethanol or ethanoic alcohol, which is prepared by mixing 3 parts absolute ethanol and 1 part glacial ethanoic acid just before use. The ethanol can be substituted with ethanol IDA or 95% ethanol, but the chromosomes may not be as clearly defined. Acetic acid causes the swelling of the protoplast, while ethanol causes shrinkage, so the ratio of the two is adjusted in most fixatives to get good images.
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To preserve the root tips
The fixative for light microscopy of plant tissues is typically FAA—a mixture of Formalin, Acetic acid, and Alcohol. It is prepared as 90 ml of 50% ethanol, 5 ml of glacial ethanoic acid, and 5 ml of commercial formalin.
Ethanoic alcohol is made by mixing three parts absolute ethanol with one part glacial ethanoic acid. It is important to note that both components are flammable, and ethanol is also corrosive. The mixture should be prepared just before use, adding the acid to the alcohol. An alternative is to use ethanol IDA or 95% ethanol, but the chromosomes may not be as clearly defined.
Practitioners may choose to have a technician cut and fix the root tips in ethanoic alcohol in advance, rather than having students perform this step. This is because cutting the root tips at a specific time of day (around noon) can impact the number of dividing cells and the mitotic index.
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To compare mitotic indices
The mitotic index is the proportion of cells in a given population that are undergoing cell division. It is a measure of how actively cells are dividing. To compare mitotic indices, one can use root tips from alliums that have been sprouting for different amounts of time (e.g., 1, 2, 5, and 10 days). The root tips are then viewed under a microscope at 400x magnification to identify actively dividing cells and locate the meristematic zone, which has small, square-shaped cells with large nuclei.
The meristematic cells in the tip of the roots are particularly useful for studying the different stages of mitosis because they are undifferentiated and undergo repeated cell division by mitosis. The root tip meristem is usually denser and more rounded than the cut end. To prepare the root tips for observation, they are placed in acetic alcohol, which fixes them in place and preserves them. This mixture of glacial acetic acid and ethanol helps to keep the root tips intact so that they can be examined under a microscope to compare mitotic indices.
Additionally, stains such as acetocarmine or toluidine blue can be used to help visualize the chromosomes within the cells. These stains give a clear image of the chromosomes without staining the cytoplasm, allowing for a better comparison of mitotic indices. The prepared slides are then examined under a compound microscope, and the changes during mitosis are noted, sketched, or photographed. By comparing the number of actively dividing cells and the stages of mitosis in root tips with different sprouting times, one can compare the mitotic indices and understand the timing of cell division in plants.
Overall, the use of acetic alcohol and specific staining techniques in combination with microscopic observation allows for a detailed comparison of mitotic indices in root tips, providing valuable insights into the process of cell division in plants.
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