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Biology 121 EXERCISE 1: ASEXUAL REPRODUCTION Many organisms are capable of reproducing without relying on the fusion of sperm and egg. Asexual reproduction may be accomplished in a number of ways but always results in offspring that are genetically identical to the parent. Under what conditions is asexual reproduction advantageous? A. Bacteria These organisms lack an organized nucleus and divide primarily by a simple splitting of one bacterium into two parts, a process known as binary fission (“division in half”). If you have ever had bacteria multiply in some food so rapidly that the food spoils before you have a chance to eat it, you know how effective binary fission can be in increasing the number of bacteria. 1. You will be shown a video on Aseptic Technique and Streaking Nutrient Plates. These will give you the basic information you will need on handling bacteria properly. Although the bacteria we will be using are not pathogens (disease causing organisms), most microbiologists believe that any bacterium in the wrong place is a potential cause of trouble. 2. A typical sample of bacteria taken in the wild or from a patient in a hospital, for instance, will usually contain a mixture of different bacteria. The first step in dealing with such a mixture is to separate the bacteria into pure cultures. This is often accomplished by the streak-plate technique (see figure at right). The streak-plate technique is a dilution method used for rapid and qualitative isolation of bacteria. It involves spreading a loopful of a culture of mixed bacteria over the surface of an agar plate in a series of streaks that are increasingly more dilute until the last streak results in individual colonies of a pure culture (a single species of bacterium). This is an effective method for isolating discrete bacterial types for study. Streak-plate technique 3. Each pair of lab partners should follow the instructionsgiven in the video and streak an agar plate with a broth culture that contains a mixture of bacteria. 4. The rapid rate of fission by bacteria can be controlled through the use of antibiotics. Each pair of lab partners should obtain a clean agar plate and divide it into four quarters by marking the bottom of the plate with a wax pencil. The four quarters should be labeled 1, 2, 3, and CONTROL. Dip a sterile swab into the broth culture of a single type of bacterium, and rub it over the surface of the agar, completely covering the surface with a thin layer of bacterial suspension. Using forceps sterilized by dipping them in alcohol, obtain three different types of antibiotic discs and place one disc in each numbered quarter. Place a plain paper disc on the quarter labeled CONTROL. Use an alcohol-sterilized forceps to tap the discs down. Record below the bacterium and the three types of antibiotics that you selected: What is the purpose of the control section containing a plain paper disc? _____________________________ ________________________________________________________________________________________ Ex. 1 Pg. 1 Write your initials, the date, the lab section, and the type of bacteria on the top of the plates and place the plates upside down in the place designated by your lab instructor. After 48 hours, the plates will be placed in a refrigerator to arrest growth. During the next lab period, examine the plates and record the results. If some antibiotics appear more effective against your bacterium, record the variation in effectiveness. ________________________________________________________________________________________ ________________________________________________________________________________________ ________________________________________________________________________________________ 4. On demonstration, under an oil immersion lens you will find representative types of bacteria. Bacteria are classified in one system by their shape: round (coccus), rod (bacillus), and corkscrew (spirillum). Draw each type below and be able to distinguish these types by their appearance. B. Single-celled eukaryotes (cells with a nucleus) Many one-celled plant and animal-like organisms reproduce by fission. The nucleus undergoes mitosis, followed by division of the cytoplasm. Although many protistans have sexual reproduction as well, some such as the amoeba reproduce only by fission. Examine the slide of fission in Paramecium. Sketch a Paramecium undergoing fission. Label the nucleus. How many organisms result from one fission? _____________________________________________________ How closely do they resemble each other? _________________________________________________________ C. Hydra Multicellular animals are unable to divide into two equal individuals, but some are able to produce a smaller individual by budding. A small bulge develops the characteristics of an adult, breaks away from the original, and gradually grows to full size. How does this compare to binary fission? Examine the slide of budding Hydra. The Hydra is a freshwater relative of the marine jellyfish. It looks like a testtube with tentacles around the mouth of the tube. When it reproduces asexually, it forms a small bud which eventually develops a mouth and tentacles of its own. Sketch the budding hydra, labeling the bud. Digestion in the hydra occurs inside the “tube” in the central (gastrovascular) cavity. Are the gastrovascular cavities of the adult hydra and its bud separate or continuous? ________________________________________________ Ex. 1 Pg. 2 How does the bud apparently obtain food for growth until it is large enough to catch its own? ________________ ____________________________________________________________________________________________ D. Dugesia As animals increase in complexity, their ability to reproduce asexually continues to diminish. A piece of the planarium flatworm, Dugesia, can regenerate missing parts to produce an entire individual (provided that the piece is not too small to start with)! If a planarian were cut in half, two individuals would result. This type of reproduction would be the exception rather than the rule in the life of the planarian. Normally it reproduces sexually. Dugesia Obtain a large, lively Dugesia. Place it in a glass petri dish half full of spring water or water which has been allowed to stand overnight. Why is tap water not used? _______________ ___________________________________________________________________________________________ Examine it under the dissecting microscope and observe its features. Note which end always goes first. Observe the diagram to the right and compare it to your specimen. With a clean, sharp razor blade, cut the animal into two. The cut may be made any way you wish. In the table below, record the appearance of the pieces. When you examine your Dugesia in 48 hours, examine the pieces and record any changes in their appearance. Examine again before the start of lab next week (7 days). Be especially observant of regenerating tissue which will lack the pigmentation of the original. Pieces at To Pieces after 48 hours Pieces after 7 days #1 #2 E. Fungi 1. An example of reproduction by budding in the Kingdom Fungi occurs in yeast. Yeast have been stimulated to grow by putting them in water with a good size pinch of sugar. Why sugar? __________________________ Place a drop of the yeast preparation on a slide, add a cover slip, and locate the yeast. You will eventually need to get up to high power to locate buds. These will be small cells attached to the larger ones. Because the yeast divide rapidly when warm and well supplied with food, you may find buds which produce other buds while still attached to their own parent cell. Look for strings of cells which would indicate that this has happened. You may get better contrast by reducing the light. Sketch a yeast cell with a bud. Ex. 1 Pg. 3 2. Black bread mold (Rhizopus nigricans) is a commonly occurring fungus which reproduces asexually by the production of asexual spores. The spores are extremely small and light, and are found nearly everywhere. The black color of the mold is caused by the spores. You should think about the efficiency of this type of reproduction the next time you find that a loaf of bread has been spoiled by this mold. Examine the Rhizopus tube-culture setup on demonstration under the dissecting microscope. Also study a prepared slide of Rhizopus. Microscopic examination will show you the threadlike hyphae which compose the diffuse “body” of the fungus. Locate a spore case (sporangium) which forms at the top of a vertically-growing hypha called a sporangiophore. The spores are loosely held in the sporangium and are readily dispersed through the air. When these spores land on a suitably warm and moist food substance, they each will produce hyphae which will grow into a large mass, such as you have seen on the bread. Why is it advantageous to have spores produced at the top of an upright hypha? _______________________ ________________________________________________________________________________________ What features of the spores make them well suited for dispersal through the air? _______________________ ________________________________________________________________________________________ What can be done to retard or prevent the growth of molds such as this on food substances? ______________ ________________________________________________________________________________________ F. Flowering Plants Unlike the higher animals (you, for example), the flowering plants have retained the ability to reproduce asexually, although some use it to a greater degree than others. 1. Stem cuttings can be made from just about any flowering plant, although some will root more readily than others. A stem cutting is a short length of young stem with 2-4 leaves. Kept moist, the lower end of the stem will form roots. From one choice plant, many identical plants can be formed. From the plants available, each pair of lab partners should cut a 2-4 leaf branch with a sharp razor blade. Dip the cut end into rooting hormone. While not absolutely necessary, this step will hasten the growth of the roots. Poke holes in the bottom of a small plastic cup and then fill with perlite. Pour water over the perlite in the cup until it flows out of the holes in the bottom of the cup. Place the treated end of the plant into the moist perlite. Although many plants will root in water, some which are more susceptible to rot will not root. Also, the roots which form in water tend to be more brittle. Cover the top of the plant with a plastic sandwich bag and place the whole setup into a tray designated by your lab instructor. Make sure your initials are on the cup, so that you can identify yours later. Why is the bag used? _________________________________ ____________________________________________________________________________________ At the next lab period, pull gently on the stem. If roots have developed, you will feel resistance. When the plant is well rooted, it should be transplanted into regular potting soil. Ex. 1 Pg. 4 2. Leaf cuttings These are similar to stem cuttings, except that a single leaf is used, roots form at the base of the leaf, and a young plant develops from the base of the leaf. Each pair of lab partners should cut a leaf complete with a leaf stalk from an African violet plant (Saintpaulia). Dip the cut end in rooting hormone and push the treated end gently into another cup of moist perlite until the wide part of the leaf (blade) touches the surface of the perlite. Cover with a plastic bag as before. Don’t forget your initials. Place in the tray provided. Opportunity will be given to you to examine your cutting at intervals to check its growth. 3. Demonstrations Some flowering plants reproduce asexually as a natural part of their lives, as opposed to the rather artificial methods employed above. Name some with which you are familiar. Examine demonstrations of Kalenchoe, Strawberrry, Begonia, Spider or other plants and describe the asexual reproduction occurring. Ex. 1 Pg. 5 TERMS FOR REVIEW asexual reproduction binary fission aseptic technique pathogen pure culture streak-plate technique colony antibiotics coccus, bacillus, and spirillum budding gastrovascular cavity regeneration asexual spores sporangium sporangiophore hyphae stem cuttings leaf cuttings TAXA bacteria Paramecium Amoeba Hydra Dugesia fungi yeast Rhizopus nigricans flowering plants Ex. 1 Pg. 6