LAB NOTES FOR EXAM 2 SECTION

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1 LAB NOTES FOR EXAM 2 SECTION EX. 3-5: PREPARATION OF BACTERIAL SMEARS We will work with the live cultures first and prepare our bacterial smears for staining before using the microscopes. All live cultures should be disposed of before getting your microscope out. Purpose: Learn to prepare bacterial smears from both liquid and solid media Organisms: agar slant culture of Staphylococcus aureus, agar slant culture of Escherichia coli, broth culture of Bacillus subtilis, agar slant culture of Rhodospirillum rubrum Equipment: Glass slides cleaned with water, inoculating loop, bunsen burner, lens paper and bibulous paper, lab marker, heating block YOU AND YOUR LAB PARTNER WILL BE PREPARING A TOTAL OF 10 SLIDES: 2 slides each of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Rhodospirillum rubrum, and 2 slides of Staphylococcus aureus and Escherichia coli mixed together. Notes: Always make two smears of each specimen, one for staining and one for back up. Stain one at a time. If you make a mistake, you can repeat the procedure with the back up smear. DO NOT STAIN BOTH SMEARS SIMULTANEOUSLY! Set up all the slides at the beginning of lab so they have time to dry. Make sure you label the slides with the intended stain and organism. There are many things to think about in this lab, however, the most important one is following aseptic procedure and handling cultures appropriately. Always flame your loop before setting it down. Preparation of a bacterial smear from an agar (solid) culture: 1) Turn on your heating block to the lowest heat setting. 2) Wash a slide with tap water and dry it well. With the lab marker, make a small circle on the slide. The circle will help you locate your specimen. Turn the slide over. You will place your culture on the unmarked side of the slide so that the markings do not get washed away during the staining process. Use the lab marker and label the slide with your initials and the initials of the organism. 3) Place a single, small drop of water from the dropper bottle onto the end of your inoculating loop. Smear this drop onto the slide within the circle drawn on the opposite side. 4) Sterilize the loop, pick up a small amount of inoculum from an agar slant or agar plate and spread it around in the water. Flame your loop when you have finished. Note: Water is used when the inoculum is from a culture grown on solid medium (agar) to separate the cells. Beginning students usually pick up much more inoculum than is needed. You only need a barely visible amount on the loop to get a good specimen. Using a large inoculum will make it difficult to observe the morphology of individual cells. 5) Heat-fix your preparation by placing the slide on a warm heating block until specimen is completely dry. When it is dry, the specimen may be slightly visible or it may not be visible at all. If visible, it will be dull in appearance, not shiny. Preparation of a bacterial smear from a broth (liquid) culture: 1

2 1) Clean and label a microscope slide as described above. Following the aseptic procedure for picking up an inoculum described previously, place a loopful of inoculum from a broth culture onto the slide on the opposite side from the circle. Gently spread the inoculum around within the circled area. Note that NO water is placed on the slide when the inoculum is from a broth culture. 2) Repeat the procedure and apply two more loopfuls to the slide. Make sure that you flame your loop and cool it between each application. 3) Heat-fix your preparation by placing the slide on a warm heating block until specimen is completely dry. When it is dry, the specimen may be slightly visible or it may not be visible at all. If visible, it will be dull in appearance, not shiny. NOTE: Once a specimen has been heat-fixed, it can be stored indefinitely until you are ready to perform your stain. Even though the majority of cells have been heat-killed, you should still handle your slides with care. Store them in an empty microscope slide box and keep them in your drawer. Preparation of mixed culture smear: Place a small drop of water on your slide if you are using agar slats cultures. Aseptically transfer a loopful of Staphylococcus aureus to a clean microscope slide, suspending the culture evenly in the drop of water. Flame your loop. Aseptically transfer a loopful of Escherichia coli to the same slide. Mix the two cultures together with your loop before sterilizing your loop. Heat-fix slide as directed above. Ex. 3-1: USING THE LIGHT MICROSCOPE Objectives: Identify the parts of the compound microscope and describe their function Demonstrate the correct use of the microscope for observing stained slide preparations with the dry and oil immersion lenses Identify the basic morphologies and structures of bacterial cells Observe your smears stained with simple stains Look at the prepared slides that are available while you are waiting for your smears to dry. Look at the specimens with all three of the dry objectives, beginning with the lowest power objective, and then go up to the oil immersion objective. Your best view of the cells will be with the oil immersion; however, you must find and focus your specimen with the three dry objectives before this is possible. If you have not had experience using the oil immersion objective, make certain that you have the instructor explain how to do this before you begin. Remember that once you have looked at a specimen with the oil immersion objective, you cannot go back to the dry objectives! PREPARED SPECIMENS All bacterial cells are prokaryotes and, in general, they are considerably smaller than eukaryotic cells. For this reason, they are usually harder to find and observe. Bacillus megaterium, however, is an exceptionally large specimen, chosen to make your viewing easier. Always begin your observations with the low power dry objective and then gradually work up to the oil immersion. All of the following bacterial preparations must be observed with the oil immersion objective because it is only with this objective that you can accurately observe the shape, arrangement, and stained color of the cells. Bacillus megaterium. This stained preparation contains large rod-shaped bacterial cells that remain attached to each other in long chains after cell division. The term for this arrangement of cells is "streptobacilli" in which the term "strepto" refers to cells in a chain and "bacilli" refers to the rod shape of the bacterial cells. One rod-shaped cell is a bacillus and two or more are bacilli. 2

3 The word bacillus is used in two ways: it describes the shape of some bacterial cells (eg. bacillus) and it also refers to a genus (a category used in classification) of rod-shaped bacteria (e.g. Bacillus). There are many, many rod-shaped bacterial cells (bacilli) that are not classified in the genus Bacillus. Coccus or Staphylococcus aureus. Observe the stained bacterial cells that are spherical in shape and arranged in clusters. The term for one spherical bacterial cell is "coccus" and many spherical cells are referred to as "cocci" (pl.). When spherical cells are arranged in clusters (often called "grapelike"), the arrangement is known as "staphylococci". In this example, as in the previous, the arrangement is also the genus designation of one group of bacteria, the genus, Staphylococcus. What term would describe the arrangement of spherical bacterial cells arranged in chains? When you are done, your microscope MUST be put away properly: Rotate the nose piece so that the lowest power objective lens is directly over the stage Lower the microscope stage to the lowest position Remove the specimen slide from the microscope stage Wipe immersion oil from the lens using lens paper Wrap the power cord around the base of the microscope Cover the microscope with its protective cover Return the microscope to microscope cabinet, matching the microscope number with the shelf number EX. 3-5: SIMPLE STAINING Purpose: Stain bacterial specimens using simple staining technique. Observe the shape and arrangement of stained bacterial cells. Staining solutions: Methylene Blue, Crystal Violet, and Carbol Fuchsin stains 1. YOU AND YOUR LAB PARTNER SHOULD HAVE 10 SLIDES THAT YOU PREPARED LAST PERIOD: 2 slides each of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Rhodospirillum rubrum, and 2 slides of Staphylococcus aureus and Escherichia coli mixed together. Use one set for Simple Staining and save the second set for Gram Staining. 2. Do a simple stain of each organism according to the procedure in your lab manual using a different stain for each slide. Note that the staining times could be different for each stain. 3. Observe your specimens under the microscope. Always begin your microscope observations with the low dry objective and work up progressively through the dry objectives to the oil immersion objective. The oil is applied directly on the specimen. Do NOT use coverslip with the stained smear. 4. You MUST use immersion oil any time you look at a specimen using the 100X objective lens. Do not get oil on any of the objective lenses other than the oil immersion objective. Once you have looked at a slide with oil on it, you cannot go back to any of the lower power objectives. Do not attempt to do so. Once you have used oil on a slide you have prepared, the slide is disposed of in the special SHARPS BIOHAZARD container. 3

4 EX. 3-7: GRAM STAIN Purpose: Understand the chemical and biological basis of the Gram stain; perform and interpret the Gram stain The Gram stain is a differential stain that separates bacteria based on structural differences in their cell walls. Gram positive cells retain the primary stain, crystal violet, and appear purple blue in color after completion of the procedure. Gram negative cells lose the crystal violet-stain after decolorizing and absorb the counterstain, safranin, and will, therefore, appear pink. Materials: YOU AND YOUR LAB PARTNER SHOULD HAVE 10 SLIDES THAT YOU PREPARED LAST PERIOD: 2 slides each of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Rhodospirillum rubrum, and 2 slides of Staphylococcus aureus and Escherichia coli mixed together. Gram stain reagents: Crystal Violet, Gram's Iodine, 95% Ethanol, Safranin NOTE: 1. Place a few drops of crystal violet on your specimen. Let the stain remain on the slide for one (1) minute. Do not allow the stain to dry, as this will cause the formation of stain crystals. 2. Gently rinse with a small stream of deionized water from a squeeze bottle by holding the slide at a slight angle with the stream of water hitting the slide ABOVE the area with the bacterial smear. Do not allow the water stream to hit the specimen directly. You can also use a stream of tap water if it is VERY gentle. 3. Cover the smear with Gram s iodine and let it stand for one (1) minute. 4. Gently rinse off the Gram s iodine with water. 5. Decolorize with 95% ethanol using the same technique you used to rinse with water: hold the slide at an angle and allow the decolorizer to flow over the stained area of the slide, without it directly hitting the smear. The decolorizer should run clear within a few seconds. This is the critical step! Do not over-decolorize. 6. Gently rinse with water. 7. Counterstain with safranin for one (1) minute. 8. Gently rinse off the safranin with water, blot with bibulous paper and observe your specimen under the microscope. Remember to put your microscope away correctly. The lowest power objective should be in position over the stage opening, the slide should be removed, no oil should be present on the stage or on any of the dry lenses, and the microscope should be placed into the correct box. NEVER use Kimwipes, paper towels or Kleenex to clean microscope lenses - only the lens paper in your lab kit. Wrap the electrical cord around the base of the microscope. EX. 3-8: ACID-FAST STAIN The acid fast stain distinguishes bacteria in the genus Mycobacterium, which are associated with the diseases tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The presence of unique waxes, mycolic acid, in the cell wall make it possible to differentially stain these organisms for diagnostic purposes. Simple stains do not readily penetrate these waxy cell walls, and heat must be used to melt the waxy substance. Once stain penetrates the melted waxy cell wall, these organisms are difficult 4

5 to decolorize, even when acid is added to the decolorizer. Acid readily disrupts the ionic bond formed between simple stains and cell walls composed of peptidoglycan when simple staining or Gram staining. Cells that retain the primary stain in the presence of acid are referred to as acid fast. Non-acid fast cell walls decolorize with the acid alcohol decolorizer and must be stained with the counterstain, methylene blue, to be visible. Cells that are acid-fast appear cherry red or magenta in color, while non-acid fast bacteria will appear blue. Organisms: Slant culture of Mycobacterium smegmatis Slant culture of Staphylococcus aureus Materials: Carbol Fuchsin, Acid Alcohol Decolorizer (NOT Gram stain decolorizer), Methylene Blue Hot plate and beaker setup in fume hood 1. Prepare two slides of a mixed specimen containing both Mycobacterium and Staphylococcus aureus, using the same procedure you used to prepare the mixed specimen during smear preparation. Remember to add a small drop of water to the slide to suspend the cells. Use the loop to break up any clumped cells and to spread out the cells evenly. If you fail to spread out the cells you will have large masses of cells and will not see individual cells. 2. Heat-fix your preparation by placing the slides on a warm heating block until specimen is completely dry. 3. Place one slide over a beaker of hot water (just below the boiling point) on a hot plate. Cover the slide with a small piece of bibulous paper. Remember, the second slide is a backup. Do not use it unless your first attempt is unsuccessful. 4. Flood the slide with Carbol Fuchsin and allow the stain to steam for 5 mins. Add more stain as it begins to dry along the edges. Make sure that you do not let the stain dry out and handle the hot slide and hot plate carefully. 5. After 5 minutes carefully remove the slide with a clothes pin (it will be hot!), cool it for a minute or two, then gently wash it with water. 6. Decolorize with acid alcohol for a few seconds. This is the critical step in this procedure. Add the acid-fast decolorizer drop by drop. Do NOT use the 95% ethanol Gram stain decolorizer. 7. Wash with water again. One or two more decolorizing and washing steps may be necessary. There may still be some residual stain left even after repeated washing and decolorizing. 8. Counterstain with Methylene Blue for 1-2 minutes. 9. Wash with water, blot dry, and examine under the microscope. Examine the slide(s) you have prepared. Which organism is the acid-fast organism? What color is it? Which organism is not acid fast? What color is it? EX. 3-10: ENDOSPORE STAIN Purpose: Perform the spore stain and identify bacterial endospores and vegetative cells The spore stain is a differential stain used to distinguish between metabolically active vegetative cells and dormant, resistant spores. Spores are formed by some bacteria when their nutrient supply is 5

6 exhausted. Like other differential stains such as the Gram Stain and Acid-Fast Stain, the spore stain requires the use of two contrasting stains, a primary stain followed by a counterstain of a different color. Organism: An agar slant culture of Bacillus cereus Materials: Malachite green stain and safranin stain Hot plate and beaker setup in fume hood NOTE: 1. Prepare two slides according to the procedure for a smear from a solid medium. The second slide is a back up. 2. Heat-fix your preparation by placing the slides on a warm heating block until specimen is completely dry. 3. Place one slide over a beaker of hot water (just below the boiling point) on a hot plate. Cover the slide with a small piece of bibulous paper. Remember, the second slide is a backup. Do not use it unless your first attempt is unsuccessful. 4. Flood the slide with malachite green and allow the stain to steam for 5 mins. Add more stain as it begins to dry along the edges. Make sure that you do not let the stain dry out and handle the hot slide and hot plate carefully. 5. After 5 minutes carefully remove the slide with a clothes pin (it will be hot!), cool it for a minute or two, then gently wash it with water. 6. Apply the safranin counterstain for 1-2 minute. 7. Wash the safranin off gently with water. 8. Blot with bibulous paper and examine. The spores will appear green and the vegetative cells will appear orange-red. Make certain that your microscope is put away correctly. The lowest power objective should be in position over the stage opening, the slide should be removed, no oil should be present on the stage or on any of the dry lenses, and the microscope should be placed into the correct box. NEVER USE KIMWIPES, PAPER TOWELS OR KLEENEX TO CLEAN THE LENSES - only the lens paper in your lab kit. 6

7 NUTRITIONAL REQUIREMENTS: DIFFERENTIAL AND SELECTIVE MEDIA Purpose: To become familiar with various types of commonly used selective and differential media and their uses. EX. 4-4: MANNITOL SALT AGAR Organisms: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus; E. coli Media: One MSA (mannitol salt agar) plate per pair 1. Divide plates into four sections. Label each section with the name of a different organism. 2. Using aseptic technique, do a straight line inoculation, with a loop, of each organism into the appropriate section of the plate. Incubate the plate, upside down, at 37 C. 3. Next lab period: Record your observations on the appropriate pages from the lab manual. Look for the growth on the surface of the agar and make note of the amount of growth using a 0-3 scale. Growth on the surface indicates salt tolerance. Note any changes in the color of the agar itself. A yellow color indicates the ability to ferment the sugar mannitol. EX. 4-5: MACCONKEY AGAR Organisms: Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus, and Proteus vulgaris Media: One MacConkey agar plate per pair 1. Divide plate into four sections. Follow same procedure as for Ex Incubate the plate at 37 C. 2. Next lab period: Record your observations on the appropriate pages from the lab manual. Look for the growth on the surface of the agar and make note of the amount of growth using a 0-3 scale. Growth indicates that the organism is Gram negative. Note the color of the growth and the surrounding agar. A bright pink/magenta color indicates lactose fermentation. A bright pink "cloud" in the agar indicates Escherichia coli, produced by the large degree of lactose fermentation. EXOENZYMES The following tests detect the presence of exoenzymes. Exoenzymes are enzymes that are secreted into the surrounding medium and work on substrates found outside the cell. In general, these exoenzymes are hydrolytic and break down large biomolecules that are too large to be easily transported into the cell. These biomolecules must be broken down into their smaller building blocks before they can be made available as a nutrient source for the cell. Starch must broken down into glucose, protein into amino acids, and triglycerides into fatty acids and glycerol. EX. 5-15: LIPASE TEST for lipase exoenzymes that breakdown lipids to glycerol and fatty acids Organisms: E. coli, S. marcescens, and Pseudomonas aeruginosa Media: Tributyrin (lipid) agar plate 1. Divide plate into thirds. Do a straight line inoculation of each organism into the appropriate section of the plate. Incubate, inverted, for at least 48 hours at 37 C. 2. A clearing of the agar around the inoculum indicates the breakdown of lipids in the medium. Lipid hydrolysis may take longer for some lipid + species. If you observe incomplete clearing around an inoculum, return your plate to the 37 C incubator and check it again next period. 7

8 EX. 5-16: CASEASE TEST This is a test for the casease exoenzyme that breakdown the milk protein, casein, into amino acids Organisms: E. coli, S. marcescens, and Pseudomonas aeruginosa Media: One Casein agar plate 1. Divide the plate into thirds and label each section of the plate with one of the three organisms. Do a straight line inoculation into the correct section of the plate. Incubate, inverted, for hours at 37 C. 2. Observe for a clear zone surrounding the inoculum which is indicative of casein hydrolysis. CATALASE AND OXIDASE TESTS After the Gram reaction and cell shape has been determined, the catalase test and the oxidase test are often the first tests performed to identify an unknown organism. If an organism is Gram negative rod, then a negative oxidase test indicates the organism is a Enteric bacteria; a positive oxidase test indicates a non-enteric organism. If an organism is a Gram positive coccus, a negative catalase test indicates it is a Streptococcus; a positive catalase test indicates is a Staphylococcus. EX. 5-6: CATALASE TEST This test is used to indicate whether a microorganism produces the enzyme catalase that breaks down hydrogen peroxide to water and oxygen. This enzyme is important to aerobic organisms because it detoxifies hydrogen peroxide. Hydrogen peroxide forms during aerobic metabolism when components of the respiratory chain donate electrons to molecular oxygen. The other enzyme produced by many microorganisms to detoxify hydrogen peroxide is peroxidase, however, no oxygen is evolved from the breakdown of hydrogen peroxide by peroxidase. This test is used to differentiate between Streptococcus and Staphylococcus species. Organisms: S. aureus, S. epidermidis, Streptococcus salivarius and any other organisms used in today s exercises. Use only cultures grown on agar for the catalase test. Materials Needed: Microscope slides, hydrogen peroxide in small bottles Place a drop of hydrogen peroxide on a microscope slide, add a loopful of the organism and observe for immediate bubbling. Catalase positive organisms will exhibit bubbling, catalase negative will not. EX. 5-7: OXIDASE TEST This test detects the presence of the enzyme, cytochrome oxidase, which transfers electrons from cytochrome c to molecular oxygen in the electron transport chain. Many aerobic bacteria, such as Neisseria sp. and Pseudomonas sp., have cytochrome oxidase. On the other hand, many facultative anaerobes such as those in the family Enterobacteriaceae (Gram negative facultative anaerobes), are oxidase negative because they lack cytochrome c in their electron transport chain, and therefore, do not have cytochrome oxidase. This test is often used to differentiate between enteric (facultative anaerobic) and non-enteric (aerobic) gram negative bacteria. Organisms: E. coli, Pseudomonas aeruginosa and Alcaligenes faecalis grown on TSA plates Materials Needed: Oxistrips (Tetramethyl-p-phenylenediamine dihydrochloride) Plastic sterile disposable transfer loops 8

9 Place a Oxistrip on a paper towel. Using a PLASTIC loop, smear the test strip (the part with a matte appearance) with a loopful of inoculum. A positive result will produce a dark purple color within 20 to 30 seconds. No color means the organism is negative for cytochrome oxidase. DIFFERENTIATION OF GRAM NEGATIVE RODS The following tests can be used to differentiate Gram negative rods. In particular, the Indole test, Methyl Red test, Voges-Proskauer test, and Citrate utilization are sometimes known collectively as the IMViC test and are used whenever preliminary tests indicate an unknown organism belongs to the family Enterobacteriaceae. The Enterobacteriaceae include pathogens such as Salmonella and Shigella, occasional pathogens such as Proteus and Klebsiella, and normal intestinal flora such as Escherichia and Enterobacter. These tests are not entirely inclusive and they may be used for other purposes as well. The table below summarizes the characteristics of a few common species. Laboratory Tests for Differentiation of Gram Negative Rods Organism Enteric/Nonenteric (E/N) Carbohydrate Fermentation H2S Production Indole Production MR Reaction VP Reaction Citrate Utilization NO3 Reduction Urease Activity Oxidase Activity Gelatin Liquification Starch Hydrolysis Lipid Hydrolysis Glucose Lactose Sucrose Enterobacter aerogenes E AG AG AG+/ Escherichia coli E AG AG A+/ Proteus vulgaris E AG AG+/ / Salmonella arizoniae E AG+/ A+/ Alcaligenes faecalis N +/ + Pseudomonas aeruginosa N A = acid G = gas +/ = result can vary between strains of the same species The tests presented in Chapter 5 of the Lab Manual are all differential. Differential tests are based on the reaction of an indicator reagent with a biochemical product that is produced in the presence of a specific 9

10 enzyme made by an organism. The differential media must contain the appropriate substrate for the reaction as well as the indicator. It is up to the organism to make the product which then reacts with the indicator. Selective media are used to enrich for a particular type of organism if you are working with a mixed culture specimen that may have, for instance, been collected from a patient. Remember that some media are both selective and differential, while others are selective only or differential only. Ex. 5-8: NITRATE REDUCTION TEST - Some microorganisms are capable of using inorganic molecules other than oxygen as the terminal electron acceptor during an energy yielding metabolic pathway such as respiration. When this occurs, the process is called anaerobic respiration. Carbonate, sulfate, and nitrate are examples of inorganic terminal electron acceptors other than oxygen. The process whereby nitrate is reduced to nitrite, ammonia, or molecular nitrogen, is known as nitrate reduction. Organisms: E. coli, P. aeruginosa, and Alcaligenes faecalis Media: Three tubes of nitrate broth per pair 1. Inoculate and incubate tubes at 37 C. 2. After the specified incubation time, add reagents A and B (IN THE HOOD) as indicated on the reagent bottles and make observations. Interpretation of the results can be confusing. Read the manual carefully! a. If nitrate is reduced to nitrite, the nitrite will react with reagents A + B and the medium will turn red. This color change will occur very quickly and can be recorded as a positive result. b. If the medium remains colorless there are two possibilities: 1) the nitrate was further reduced to nitrogen gas or ammonia or 2) the nitrate was not reduced at all.to differentiate between these two possibilities, zinc is added. Zinc will react with nitrate, the original substrate, and reduce it nitrite. This newly produced nitrite will then react with the reagents A + B that were added previously and turn red. If your medium turns red after adding Zinc, then the reaction is negative because the original substrate, nitrate, was not reduced. This reaction may take 5 to 10 minutes to develop.if there is no color change after adding Zinc, then the original substrate nitrate was reduced to a product that cannot be detected by reagents A + B. These products can be either N 2 gas or NH 3, ammonia. This is a positive result. Therefore: If A + B red, then organism is positive for nitrate reduction If A + B no change, then add zinc If A + B + Zinc red, then organism is negative for nitrate reduction If A + B + Zinc no change, then organism is positive for nitrate reduction EX. 5-9: CITRATE UTILIZATION TEST - This is a test to determine whether a bacterium can use citric acid as its sole source of organic carbon. Organisms that can use citrate as a carbon source produce the enzymes citrate permease and citrase. In a multi-step process, the citrate is broken down into pyruvate and CO 2. The CO 2 is converted into sodium carbonate (NaCO 3 ), which is alkaline. The ph indicator, bromthymol blue, changes from green to blue. Organisms: E. coli and E. aerogenes - broth cultures Media: Two tubes of Simmon s citrate agar Inoculate the slant of the citrate tubes with the appropriate organism using the stab-streak inoculation method. In this procedure the inoculated needle is first stabbed into the bottom (butt) of the 10

11 tube and then it is dragged across the slant surface of the tube. Incubate at 35 C for 48 hours or more. Make sure that the top is quite loose when these tubes are incubated. Look for a color change in the medium. EX. 5-18: UREASE TEST - This test is done to determine whether an organism has the enzyme, urease, the enzyme that breaks down urea to carbon dioxide, ammonia and water. The presence of the enzyme is indicated by a color change in the medium. The product ammonia is alkaline and causes the ph indicator, phenol red, to change color from pale orange to iridescent pink, indicating the presence of urease. Organisms: P. vulgaris and E. coli Media: Two tubes of urea agar Inoculate the agar tubes using the stab-streak method and incubate at 37 C. Observe any color change that occurs in the medium. The production of a bright pink color indicates that the organism is positive for urease production. EX. 5-20: SIM TEST - This test is used to determine whether an organism produces hydrogen sulfide (S), whether it can metabolize the amino acid tryptophane to indole (I), pyruvic acid and ammonia, and whether or not the organism is motile (M). These three tests are all done in a single tube, a deep known as a SIM tube. Organisms: Escherichia coli, Enterobacter aerogenes, Proteus vulgaris & Staphylococcus aureus Media: Four SIM tubes 1. Using a needle, inoculate the tubes using a stab procedure. Incubate at 37 C. When you read the results, make sure you do the observations for hydrogen sulfide production (blackening of the medium) and motility (cloudiness away from the stab line) before you test for indole production. 2. Testing for indole must be done under the hood. Never use the Kovac s reagent out in the laboratory. To your SIM tube, add 10 to 20 drops of Kovac s reagent. You do not need to agitate or shake the tube. The Kovac s reagents will react immediately if indole is present and produce a thin band of magenta color above the agar surface. The absence of a color change (remains yellow) indicates that indole was not produced and can be recorded as a negative result. You must use caution with Kovac s reagent. It is only to be used in the hood. Do not inhale the reagent or get it on your skin. Hands should be washed immediately if you should get some on your skin. Dispose of the tubes in the small red autoclave bags when you have completed this test. 11