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Transcript
-1 BACTERIA All bacteria are members of the kingdom Monera, the simplest organisms. Monerans are referred to as prokaryotes, "pro" meaning before and "kary" meaning nut. This is a reference to the lack of a nucleus in bacteria. The four other kingdoms of organisms are made up exclusively of organisms that have a membrane bound nucleus. These nucleated cells are referred to as eukaryotic, "eu" meaning true, a reference to their "true" nucleus. Other characteristics that can be used to distinguish bacteria from other organisms include: 1 2 3 4 Typically smaller size than other cells Division by binary fission instead of mitosis Peptidoglycan cell wall All bacteria are single celled rather than multicelled. Four general characteristics are commonly used to identify specific species of bacteria: 1 Colony morphology - Colonies may vary in color, texture and shape. Figure 1 shows some of the different shapes and textures colonies may have. 2 Cell morphology - Cells may vary in shape and also in the number of other cells they are joined to (Figure 2). The term cocci, meaning berry, describes spherical bacteria. Rod shaped bacteria are called, somewhat logically, rods. Spirillum describes spiral shaped bacteria. When two cells are joined together the prefix "diplo" meaning two is added. For example a "diplococcus" is a bacteria commonly found as two cocci joined together. A bacteria commonly found as a chain of cocci is called a "streptococcus," "strep" meaning twisted chain. -2 3 The outer covering of the cell Bacteria may secrete capsules that protect them from their environment. This was true of the Streptococcus bacteria used by Griffith in his study of the genetic material. All bacteria can be classified on the basis of whether they are Gramm positive or negative. The Gramm staining procedure takes advantage of the fact that Crystal Violet will stain the peptidoglycan cell wall of bacteria, but not their cell membrane. Bacteria that are Gramm positive have no membrane on the outside of their cell wall. Gramm negative bacteria have a membrane that covers their cell wall, so it is not stained by crystal violet and a negative test results. Figure 3 shows the different outer coverings of bacterial cells. 4 Biochemical pathways - Different bacteria can make different biochemicals and may used different carbon and energy sources. For example, some bacteria are anaerobic and lack the ability to break down hydrogen peroxide to oxygen and water while other bacteria can do this. 2 H 2 O2 _2 H 2 O + O2 Other bacteria can use manitol or lactose as their carbon and energy source, while others can only use glucose. When working with bacteria it is essential to maintain aseptic conditions. This means that everything that could possibly come into contact with your bacterial cultures must be sterile. Before doing anything else, you should wash your hands and the bench -3 area you will be working on with disinfectant. A Bunsen burner flame is used to sterilize the wire loops that are used to transfer bacteria between cultures. All glass culture tubes are "flamed" at the lip before and after a sample is removed. Care must be taken to not get hair or skin in contact with any of the culture media both before and following inoculation with bacteria. Always wash your hands with bacteriocidal soap after working with bacteria. WARNING: The bacteria you are working with are not typically pathogenic, but every precaution should still be taken to avoid coming into contact with them. This is particularly true in the case of individuals with weak immune systems. If you get cultures on yourself or the bench tell your instructor immediately, and wash the contaminated area thoroughly with bacteriocidal soap. WARNING: When working with open flames use extreme caution. Keep hair and loose clothing away from the flame. If your hair or clothing catches on fire smother the fire, DO NOT RUN, this will only fan the flames. If you are burnt, run the burn under cold tap water, do not put ice on the burn. Inform your instructor immediately if you are burned. Exercise 1: Colony morphology 1 A number of different bacteria are available in broth cultures at the front of the lab, select one culture per student (3 per group) 2 Obtain a nutrient agar petri plate and clearly mark it with the type of bacteria you have chosen and your name. 3 Sterilize a transfer loop by heating it to red hot then letting it cool. Be careful to avoid touching anything with the loop before it is cool enough to use in the transfer. 4 Open the broth tube and flame the top for a couple of seconds. 5 Dip the loop into the broth culture then lift the lid of the petri dish and streak along one side of the nutrient auger. 6 Flame the opening to the broth culture and replace the cap. -4 7 Flame your transfer loop then open your petri plate again and streak the loop across the plate at 90o to the original streak. The object of this is to get a few bacteria from the original streak and spread them out so that colonies resulting from one individual bacteria can be observed later. To spread out the bacterial even more, repeat this step twice more, flaming your loop between each streak. Each time rotate 90o from the previous streak. This is depicted in Figure 4. 8 Place your plate auger side up in a 37 oC incubator and leave it for 24 hours. 9 Check the morphology of your colonies the next day using a dissecting microscope and record the results. Experiment 2: Biochemistry Once you have checked the morphology of your colonies, place a drop of hydrogen peroxide solution on one of the colonies. If the enzyme catalase is present, bubbles of oxygen will appear. If the enzyme is absent, no bubbles will appear. Experiment 3: The Gramm Stain You will want to do a Gramm stain on all the live bacterial cultures available in this lab. 1 Place one loop-full of broth culture on a clean slide and smear it around in a small area. If you are using a colony from an agar plate, put a drop of water on the slide then get some bacteria on a loop and smear them around in the drop. 2 Allow the smear to air dry. 3 Pass the slide two or three times through a flame to heat fix the bacteria. This will cause them to stick to the slide and not be washed off. Do not over heat the slide or your bacteria will go up in smoke. -5 4 Be careful when using the following stains as they will stain you and your clothing just as well as they will stain bacteria. Take the slide to the sink and place several drops of crystal violet stain on your smear. Leave the stain on the smear for exactly 1 minute then wash it off gently with water. The crystal violet stains the peptidoglycan cell walls of Gramm positive bacteria, but cannot do the same to Gramm negative bacteria as the outer membrane prevents the stan from getting to the cell wall in these bacteria. 5 Cover the smear with Gramm's iodine and allow it to stand for 1 minute before rinsing off the iodine with water. The iodine helps to fix the stain to the cell wall. 6 This step is crucial. Flood the smear with decolorizing solution (95 % ethanol) for 10 - 20 seconds. The time should be just long enough to get the decolorizing solution to run clear, but not so long that everything is washed off the slide. 7 Stain the smear for 20 - 30 seconds with safranin. Safranin stains membranes so it will stain the outer membrane of Gramm negative bacteria, so that they will be visible as red bacteria on your slide. 8 Examine your smear under the oil immersion lens of your microscope. Gramm positive bacteria should appear dark purple while Gramm negative bacteria should appear red. Experiment 4: Bacteria in the environment Each individual in your group will be provided with a nutrient agar plate which you can expose to the surface, liquid, or environment of your choice. For example, you may want to compare water from the faucet with water from a puddle. Yogurt contains excellent cultures of lactobacillus. You may also want to check out the effect of washing your hands with soap versus not washing them. Use your own imagination, inoculate your plate and place it in the incubator to be checked in the morning. Materials Equipment Bunsen burners Flints or matches to light burners Inoculating loops Microscopes: Compound and Dissecting Prepared slides of bacteria -6 37 oC Incubator Supplies Gramm stain chemicals Lysol to sterilize benches Nutrient agar plates (2 per student) Three different bacterial broth cultures