Whooping cough bacteria multiply through mitosis

According to K. BACHMANN: Instructions for the practical course "Biology for Medicine", University of Heidelberg, February 1974

Felt-tip pen (the ink must not be water-soluble). You will use the felt-tip pen several times during the internship.
razor blade
Drawing paper

Test tubes
Microscope slide
Cover glasses
Pasteur pipettes
Filter paper
Paper towels
Fixing liquid (3 parts methanol, 1 part glacial acetic acid)
Schiff's reagent (keep closed) = Fuchsin-sulphurous acid
1 N HCl
Hydrochloric acid water (18 parts water, 1 part 1N HCl; should also contain 1 part 15% sodium metabisulphate, omitted due to stench)
45% acetic acid
Sprouted onion seeds (laid out on moist filter paper 3 days before the internship date)
100 ml Erlenmeyer
Glucose (2%) dissolved in 0.1 M phosphate buffer pH. 6.7.
Preparation: 40 ml 0.1 M Na2HPO4 + 60 ml 0.1 M NaH2PO4

Water baths set to 60 ° C
Test tube rack
Incubators set to 37 ° C

This experiment deals with dangerous chemicals:

is highly flammable. It is toxic by inhalation and if swallowed. It must not get into the hands of children. Keep container tightly closed. Keep away from sources of ignition - No smoking. Avoid skin contact.
is flammable. It causes severe burns. It must not get into the hands of children. Do not breathe vapor. In the event of contact with the eyes, rinse thoroughly with water and consult a doctor.
Irritating to eyes and skin. It must not get into the hands of children. In case of contact with skin, wash off immediately with plenty of water.

Today's experiment is one of the easiest of the whole internship. If carried out properly, however, it can contribute several fundamental methodological and theoretical insights to the understanding of the division of cells and nucleus. Work carefully and make sure not only to carry out the experiment mechanically, but also to understand the individual processes.

There are basically two methodological methods of attack to obtain statements about the chemistry of biological systems, the biochemical and the histochemical. Biochemistry is "test tube chemistry": at some point in the course of the experiment, the chemical fraction to be examined is extracted from the tissues and examined using organic chemical methods. The difficulty with this is that it is often not possible to determine the exact location of the extracted fraction within the tissue or cell, and often enough the location of a molecule in the cell plays a fundamentally important role. Biochemists therefore spend a lot of work preparing cell fractions before extracting the various molecules, i.e. breaking open the cell and removing nuclei, mitochondria, basic cytoplasm, membrane fractions, etc. cleanly separated from each other and then examined individually biochemically. Obtaining really clean fractions of the individual cell components with the mildest possible methods is one of the most difficult tasks in biochemistry. You will learn more about this elsewhere. On the other hand, the tissues can also be left intact and chemical reactions can be carried out on tissue sections that can be viewed under the microscope (or the electron microscope).

You do that in histochemistry (when you examine tissue) or cytochemistry (when you look at individual cells). The location of the various chemical fractions is then much less difficult; but the chemical reactions with which one can visualize the small amounts of molecules that are present in a single cell are very limited. Usually a dye with a very high extinction coefficient is coupled to the examined fraction, of which even small amounts are visible under the microscope.

Biochemical and histochemical methods complement each other, and for many studies it is worthwhile to use both. In today's experiment we will specifically depict the DNA of the cell nucleus using the Feulgen reaction (FEULGEN and ROSENBERG, 1924).

The Feulgen reaction is based on the reaction of colorless Schiff's reagent with aldehyde groups, which leads to red-colored compounds. Schiff's reagent reacts with all aldehyde groups. If you do not work very cleanly, you will be surprised at how many aldehyde groups there are in your environment: hands, clothes, writing paper, table tops, tap water and much more give astonishingly positive reactions that can only be reversed after rough treatment with strong hydrochloric acid .

In the cell, too, there are aldehyde groups in a wide variety of molecules, e.g. in sugar. There are no free aldehyde groups in DNA. The sugar molecules (deoxyribose) that are built into the DNA structure are in a closed ring form, which are connected to one of the four bases (adenine, thymine, guanine or cytosine) on the first carbon atom, the sequence of which the DNA has its biological sequence Specificity. In the complementary strands, the bases A and T, as well as C and G are opposite each other, they are complementary to each other, the base pairs are held together by two or three hydrogen bonds. Before the cell is stained with Schiff's reagent, all smaller molecules with aldehyde groups have to be removed from the cell and the aldehyde groups of the sugar molecules in the DNA have to be released.

  1. Fixation: There are various substances that can be used to fix the cell before the histochemical treatment: formalin, alcohol, various mixtures. In all cases, the proteins of the cell are denatured, lose their function, are stuck together within the cell and remain in place as a protein skeleton of the cell. Lipids are mostly extracted completely. Since the cell membrane owes its structure to lipid molecules, the membrane structure breaks open and chemical reagents have free access to the macromolecules inside the cell. All smaller molecules are washed out of the cell. After the fixation, there is a network of denatured proteins in which the other macromolecules (carbohydrates and nucleic acids) are also fixed.

  2. Hydrolysis of DNA: When the DNA is subjected to mild hydrolysis with dilute acids, the hydrogen bonds that hold the two chains of the double helix together break. The "inside" of the molecule becomes accessible to chemical reactions. The purine bases (adenine and guanine) are then specifically removed by the hydrolysis. After the purine base has been hydrolyzed, the aldehyde group on carbon atom 1 of the sugar can be colored with Schiff's reagent. Each base pair of DNA provides an aldehyde group for the following reaction. Under carefully controlled circumstances, the reaction is so specific that it can be used to quantitatively measure the DNA content of individual cells. This is particularly informative when the DNA quantities in different nuclei vary within a tissue (tumors, pathological conditions of different tissues, tissues with a high rate of cell division and DNA synthesis, normal liver of different animal species and humans, especially during regeneration and under the Influence of various hormones). We can work faster for today's experiment if we forego quantitative accuracy. Hydrolysis in normal hydrochloric acid (1N HCl) for 10 minutes extracts many of the cellular proteins, all RNA and about 60% of the DNA. But since there are several million base pairs in each nucleus, the remaining DNA gives a clear Feulgen reaction.

  3. Staining: Complete staining of the aldehyde group of the hydrolyzed DNA takes about an hour. Most of the aldehydes are bound to Schiff's reagent after just half an hour. After staining, all non-stoichiometrically bound reagent should be washed out in an acidic sulfur dioxide solution. Since sulfur dioxide has an unpleasant odor, which could easily lead to the evacuation of the laboratory, we will again forego quantitative accuracy and wash it in slightly hydrochloric acid water. The cells can then be softened in 45% acetic acid and squeezed out on a microscope slide.

  1. Cut the first two to three millimeters off a few root tips of a budding onion. Place the cut pieces in the fixing liquid [3 parts of methanol, 1 part of glacial acetic acid (glacial acetic acid is concentrated acetic acid)]. Leave the root tips in the fixative for 30 minutes. Keep the fuser jar closed.

  2. Mark a test tube with the number of your workplace, fill the glass with a few milliliters of 1N HCl and place the glass in a water bath so that it is preheated to the temperature of 60 ° C.

  3. After half an hour, the root tips are removed from the fixation fluid. If you touch the tips of the roots, then only the cut ends. At the top of the root is the meristem, in which new cells arise through division. There you will find cells in mitosis that you will want to look at later. Place the root tips briefly on a piece of paper towel to dry them. Then they are placed in the warm hydrochloric acid for 10 minutes and left in the water bath.

  4. After 10 minutes of hydrolysis, remove the test tube and fill it with cold tap water. Carefully pour the tap water down the sink without losing the root tips. Repeat the wash several times until you are sure that all of the acid is washed out. Then the tips of the roots are placed on a piece of filter paper.

  5. Only now do you briefly open the jar with Schiff's reagent in order to insert the root tips. The jar is immediately closed tightly again. After half an hour, the tips of the roots should turn deep red. Now take the roots out of the solution. Schiff's reagent stains: hands, clothing, furniture. . . . . Wash the colored roots several times in acidic water.

  6. Now comes the process that makes the difference between a good specimen and a broken slide with a piece of dirt on it: Place the stained root tip on a slide. Add a few drops of 45% acetic acid to soften the tissue, place a coverslip over it and crush the tissue between the slide and the coverslip. Add a single root tip. In order to get a clean layer of individual cells, it is best to proceed as follows: First, very carefully tap the cover slip with the blunt end of a pencil or the end of a pair of tweezers (cover slips are extremely fragile). The root should already fall apart and spread under the cover slip. Then the slide is placed on a soft surface (flat folded paper towel) and the cover slip is covered with a piece of paper towel. Now press, carefully at first, then as hard as you can with your thumb on the cover slip. The pressure must be exactly from above. When the coverslip slips around on the slide, the root tip curls up into a useless little sausage before the coverslip breaks. You want to crush the tip of the root under the unbroken cover glass into a layer of individual cells. Please work with feeling.

  7. Wipe the underside of the slide dry before placing the slide on the microscope table. Do not let the preparation dry out. Occasionally you can bring a drop of water under the coverslip.

  1. Specificity of the Feulgen stain. Are only the kernels stained and nothing else? In plant material, other structures sometimes stain slightly. Which, why?

  2. At the tip of the root there is an embryonic tissue, the meristem of the root. Here all cells of the root arise through division. Some of them are released towards the tip and form a root cap that protects the growing root against friction in the earth. The cells of the root cap soon die, become slimy and are abraded by the growth movement of the roots in the soil. Other cells from the meristem are shed backwards and continuously form the front end of the growing root. Through further cell division in the meristem, these cells get further and further away from the front end of the root. They soon begin to differentiate into the various types of cells in the root tissue. You can find the different regions of the root tip on an uncrushed specimen under the slightest magnification of the microscope. Now look for cells from the different areas in your squeeze.

  3. The cells of the meristem are more or less square. Since cell divisions occur in the meristem, with a bit of luck you should find all stages of mitosis. If you've squeezed really well, you can find metaphases that are written so flat that you can count chromosomes. How many chromosomes does the onion have? Identify the different stages of mitosis.

  4. Interphase nuclei and nucleoli. Most of the nuclei in your preparation are interphase nuclei in which the chromosomes are not individually recognizable, but rather form a chromate structure. In this chromate structure you can clearly see a structure which, after Feulgen's staining, appears as a large empty hole in the core. That's the nucleolus. The nucleolus plays an important role in the synthesis of ribosomal RNA. If we had stained the nucleus with a basic dye (hematoxylin is often used for this) that reacts with the phosphate groups of DNA and RNA, the nucleolus would be the most strongly colored part of the nucleus. But since we removed the RNA during the hydrolysis period, little of the contents of the nucleolus is left. Note, however, that a particularly large amount of DNA is deposited around the surface of the nucleolus. A special DNA fraction of several (or all) chromosomes contributes to this "perinudeolar chromatin".

  5. Differentiation. After division, the cells of the root are displaced backwards from the advancing meristem and begin to differentiate into the different cell types of the root tissue. Most cells stretch in the process. When the cell is stretched, the nucleus is also deformed. The nuclei of differentiated cells are elongated and contain nucleoli (the ribosomal RNA of the nucleoli is of course required for protein synthesis to take place. A cell with a large nucleolus and a large amount of RNA in the cytoplasm is a synthetically active cell).

  6. Polyploidy. A typical property of differentiated plant cells is the appearance of polyploid nuclei. The DNA of the nuclei doubles without any cell division. It's a way of doubling the number of genes per cell. This is possible in plant cells because these cells no longer have to divide. All divisions take place in the meristem. In animals, the growth process is somewhat more complicated and polyploid cells therefore only arise in very special cases. Polyploid interphase nuclei appear to be reduced to diploid by mitosis only in rare cases, and mitosis of the polyploid nucleus into two polyploids also causes some difficulties. You can recognize polyploid nuclei in your preparations by their particular size and the fact that they have two instead of one nucleolus (tetraploidy). Normally, a diploid nucleus in the onion forms only one nucleolus, although it naturally contains two sets of chromosomes.

Yeast of the kind Saccharomyces cerevisiae multiplies by sprouting. This process can be easily induced and observed without much effort. Take a small piece of baker's yeast and suspend it in about 30-40 ml of glucose solution. We have chosen a weakly acidic buffer as the solvent for the glucose because fungi grow better in this area than in the alkaline one. The glucose serves us as a source of energy.

Observe the yeast cells under the microscope immediately after preparing the suspension. Above all, watch out for any sprouting cells. Add a drop of iodine - iodine potassium solution to your yeast suspension. How does it change color? Incubate the yeast suspension for approx. 3 hours at 37 ° C in the incubator. (Label your sample to avoid confusion.)

What do the cells look like after an incubation period of 3 hours? (Make a sketch)
What do they look like after adding iodine and potassium iodide?

Estimate the percentage of cells where you see a sprout.