LAB NOTES FOR EXAM 1 SECTION

Transcription

1 LAB NOTES FOR EXAM 1 SECTION EX. 2-1: DIVERSITY AND UBIQUITY OF MICROOGANISMS Purpose: Microorganisms are found everywhere in the environment around us. To demonstrate this and to get a taste of the different types of organisms in our environment, we can culture these microorganisms by collecting them on a swab and transferring them to an agar plate. Media & Materials: 1 Tryptic Soy Agar (TSA) plate, 1 sterile swab and 1 tube of sterile saline per person 1. We will use a simplified version of the lab manual protocol. The lab manual describes several methods for sample collection. Confer with your lab partners at your table so that each person uses a different method. Your table should have a total of 4 plates (5 if you have an extra person at your table). 2. Label the plate with your name, today's date, and the source of inoculum to be collected (for example, desktop, doorknob, faucet, hair, skin, etc.). You may also use any public body surface except for your mouth (this means any areas of skin that you might normally expose in polite company). Transfer any microbes you may have picked up to the agar plate by gently swabbing the surface of the plate with the swab. The instructor will demonstrate the method for spreading the cells on the plate. 3. The plates will be incubated at 37ºC until next lab period. 4. Observe your plates during our next lab meeting. On your data sheet, make note of the various colors, textures, and shapes that are produced when microscopic organisms are allowed to reproduce in large numbers. The individual areas of growth you see are colonies and consist of millions of identical cells that all arose from a single parent cell. 5. Be sure to dispose of your cultures in the Biohazard waste containers when your observations are completed. EX. 1-3: ASEPTIC TRANSFER AND INOCULATION TECHNIQUES Since this is the first time you will be working with bacterial cultures, the procedure for handling and transferring microorganisms will be described in detail. Aseptic technique, the procedure used to prevent contamination, is carried out so that you, your neighbors, and your belongings are not contaminated by the microbial culture and so that microbes from the environment do not contaminate your bacterial culture. Purpose: In this experiment you will be transferring living cells grown on two different types of media: broth and slant. You will use these cells to inoculate a fresh slant and a fresh broth culture. Cultures: a broth culture of Serratia marcescens and an agar slant culture of Serratia marcescens Media: 1 tryptic soy agar (TSA) slant and 1 tryptic soy broth (TSB) per student 1. Before beginning, label all tubes of sterile media with the name of the organism, the date, and your names or initials. ALWAYS label before inoculating. 2. Follow the protocol described in the manual. We will be using only one organism today, Serratia marcescens. This organism produces a red pigment when incubated under the appropriate 1

2 conditions. This red or pink color will help you to determine if your aseptic transfer has been successful when you look for growth next lab period. You should not see any pink or red color in your freshly inoculated tubes today. 3. Use the broth culture of Serratia marcescens to inoculate the slant. Use the slant culture to inoculate the broth. 4. Your instructor may demonstrate some techniques that are slightly different from the ones described in the lab manual. These variations are acceptable as long as they are safe and produce the desired results. If you feel confused by any differences, ask for clarification. 5. After you replace the screw top on a culture tube, back off the top about 1/2 turn before placing the tube in the incubator in order to allow air to circulate. Do not incubate cultures with the top screwed down tightly. Those bugs need to breath, just like you! 6. Incubate all cultures at 37ºC until next lab period. 7. During the next lab meeting, make your observations on the data sheet located near the back of your lab manual. Dispose of your cultures in the appropriate Biohazard container as directed by your instructor. EX. 1-4: STREAK PLATE ISOLATION OF PURE CULTURE (PART 1) Purpose: A pure culture is one that contains a single type of organism. You must first isolate single colonies in order to cultivate a pure culture. Single colonies contain identical cells that have arisen from a single cell and are genetic clones of each other. Single colonies can only be obtained by spreading single cells far apart from each other on the surface of an agar plate, then allowing them to grow. There are several ways to separate single cells, but we will only be using the streak plate method using an inoculating loop. Cultures: a mixed broth culture containing both Serratia marcescens and Staphylococcus aureus and a mixed broth culture containing both Escherichia coli and Micrococcus luteus. Media: 2 TSA plates per pair of students The Streak Plate Method 1. Each student will inoculate a mixed broth culture onto a separate agar plates according to the streak plate procedure described by the lab manual and demonstrated by the instructor. Work with your lab partner so that each of you streaks a different mixed culture. 2. When you make the streaks across the plate, do them gently so that you do not dig into the agar with the loop. This will cause growth to occur in streaks, rather than in single colonies. 3. Incubate your plates upside down (lid on the bottom, agar on top) in a wire basket at 37ºC for until next lab period. Baskets can be shared between lab partners. These plates are considered mixed cultures because they will contain two different types of colonies. 4. Next lab meeting: make your observations on the data sheet pages. Use blank paper if you need additional space. Include a drawing and use the information in Ex. 2-2 to write a description of a colony from each different type of organism. Observe the plates for visible differences in colony morphology (size, color, shape, elevation, margin, texture, optical characteristics). Did you get nice isolated single colonies? They should be spaced far enough apart so that you can pick up an without touching more than one colony using a loop. Serratia marcescens colonies should be pink or reddish. Staphylococcus aureus form smaller, white colonies. Escherichia coli produces beige colonies 2 to 3 mm in diameter, while Micrococcus luteus will produce smaller yellow colonies. 2

3 EX. 1-4 (and EX. 2-2): STREAK PLATE ISOLATION OF PURE CULTURE (PART 2) Purpose: Isolate a pure culture from mixed culture plates by restreaking a fresh streak plate from a single isolated colony Organisms: Last period s mixed culture plates with isolated colonies from Ex Media: 4 TSA plates per pair of students 1. Label fresh plates with the names of the four organisms found on your mixed culture plates, your initials, and the date. Look at the two streak plates that you and your lab partner prepared from mixed cultures the last lab period. Choose single colonies that are well separated from the others so that they are easy to pick up. 2. From a mixed culture plate, aseptically touch the edge of the sterile loop to the colony. It is not necessary to scoop up the entire colony. You should not transfer a visible quantity of inoculum to the fresh plate. Using the streak plate dilution method that you used last week, streak a fresh, labeled plate with the appropriate colony. Remember to flame the loop in between streaking each quadrant of the plate. If the colonies are very small and close together, you can use a needle to pick up cells. Note: Make sure that you pick up your inoculum from only one colony. 3. Use the same method to streak plates of all four organisms. 4. Place the plates inverted in a basket. Incubate the four plates at 37ºC until next lab period. 5. Next lab period: You will use these plates as part of Ex. 2-2, Colony Morphology. Record your observations on your data sheet. Use additional paper for drawings or other information. Each plate should have only one type of colony. EX. 2-2: COLONY MORPHOLOGY Purpose: Observe possible difference in colony morphology between different bacterial species grown on solid culture media. Organisms: broth cultures of Bacillus subtilis and Mycobacterium smegmatis Media: Two TSA plates per pair 1. With a sterile loop, inoculate Bacillus subtilis and Mycobacterium smegmatis onto separate TSA plates using the streak plate method. NOTE: Be careful to avoid cross-contamination between plates. Be especially conscientious in flaming your loop after transferring any Bacillus species, which produces spores. Make sure that the loops are flamed long enough to glow orange. Take the time to flame the loops and go through the procedures slowly and carefully. Report any spills to the instructor so that proper cleanup can occur. 2. You have already streaked plates with the other organisms from Ex Incubate all the plates 37ºC until next lab period. The Mycobacterium and Micrococcus cultures may require additional incubation time. Therefore, if the growth is scant and the colonies are very small, incubate these cultures for 1 to 2 more periods. 3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. There may be demonstration plates of other organisms to view as well. Be sure to include observations on these cultures in your results. 3

4 WHEN YOU ARE FINISHED MAKING ALL YOUR OBSERVATIONS, CULTURES SHOULD BE DISPOSED OF IN YOUR RED AUTOCLAVE BAGS AND THOSE BAGS PLACED IN THE LARGE GRAY BIOHAZARD WASTE CAN. EX. 2-3: GROWTH PATTERNS ON SLANTS Purpose: Observe possible differences in morphology between different bacterial species grown on slant media Organisms: Bacillus subtilis and Mycobacterium smegmatis Last period s mixed culture plates with isolated colonies from Ex. 1-4 Media: 6 TSA slants 1. Label each slant tube with the date, organism name, and your initials. 2. Using the aseptic technique for inoculating slants that we learned last week, inoculate each tube. For Serratia marcescens, Micrococcus luteus, Escherichia coli, Staphylococcus aureus, carefully pick up cells of a single colony from your mixed plates. Normally, pure cultures would be used for transferring organisms to slants or broths, but we are using the mixed plates due to time considerations. If you do not have sufficient single colonies of each organism, there will be some pure culture plates available as well. 3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. For your uninoculated control, use a fresh TSA slant, but return it to the cart when finished. EX. 2-4: GROWTH PATTERNS IN BROTH Purpose: Observe possible differences in morphology between different bacterial species grown in broth media Organisms: Bacillus subtilis and Mycobacterium smegmatis Last period s mixed culture plates with isolated colonies from Ex. 1-4 Media: 6 TSB broths 1. Label each broth tube with the date, organism name, and your initials. 2. Inoculate these tubes with the same cultures used in Ex Again, be especially careful when transferring cells from your mixed culture plates. 3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. For your uninoculated control, use a fresh TSB broth tube, but return it to the cart when finished. EX. 2-7: FLUID THIOGLYCOLLATE: ATMOSPHERIC OXYGEN REQUIREMENTS Purpose: Observe the growth patterns of different organisms according to their oxygen requirements 4

5 Organisms: Broth cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Neisseria sicca, and Clostridium butyricum Media: Four tubes of thioglycollate broths and one tube of supplemented thioglycollate per pair 1. Thioglycollate is a reducing agent that removes oxygen from the broth. Label the tube of SUPPLEMENTED thioglycollate for the organism Clostridium butyricum (don't forget your initials and the date). Label the other tubes for each of the remaining organisms. 2. After observing the instructor's demonstration, inoculate each broth using a sterile disposable bulb pipette: Transfer 0.25 ml (the first mark on the sterile pipette above the joint) of the appropriate culture by gently squeezing the bulb to expel the inoculum into the broth from the bottom of the tube to the top. DO NOT ALLOW ANY AIR TO BUBBLE INTO THE BROTH. 3. Discard the pipette in your autoclave bag. 4. Repeat the procedure for the other four organisms. Incubate the tubes at 37ºC until next lab period. 5. Next lab period: DO NOT SHAKE or disturb the broth cultures before you have the opportunity to make your observations. Make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. EX. 7-3: THE KIRBY-BAUER ANTIBIOTIC SENSITIVITY TEST PROCEDURE Purpose: Observe the effects of a variety of antibiotics on a Gram positive and a Gram negative organism using the Kirby Bauer antibiotic sensitivity test. We will be using a procedure similar to the one described in the lab manual, with some modifications. Follow the procedure described below: Organisms: broth cultures of Escherichia coli and Staphylococcus aureus Media and Materials: 2 tubes sterile TSB, 2 disposable 1 ml bulb pipets, 2 large Mueller-Hinton agar plates, 2 sterile cotton swabs, Antibiotic disks for streptomycin, tetracycline, penicillin, chloramphenicol, cephalothin, erythromycin, novobiocin, vancomycin 1) Dilute each stock culture 1:50 by adding 0.25 ml of culture to 5 ml sterile TSB. Be sure to label the TSB tubes before adding the stock culture. Mix well. 2) Label each plate with name of an organism. Inoculate each plate with a single organism by dipping a sterile swab into the diluted broth culture so that the swab is saturated. Remove excess inoculum by gently rolling the swab against the inner surface of the tube. The swab should be moist, but not dripping. 3) Use the swab to evenly cover the entire surface of the agar in a horizontal direction. Remoisten the swab, turn the plate 90 and streak the surface in the other direction. Make sure that you cover the entire plate and that the bacteria are spread all the way to the plate edge. 4) Dispose of the used swab in the autoclave bag. 5) Allow the plate to absorb the liquid for a few minutes, with the top on and right side up. 6) Inoculate the second plate with the other organism using the same procedure. Make sure to use a fresh swab for the second culture. 7) We will NOT be using a disk dispenser. Instead, use a pair of flamed and cooled of forceps to remove the antibiotic paper disk from the end of the cartridge. Place disks on the agar surface of 5

6 the plate, using the laminated guide to space them evenly around the plate. Gently touch the disk with the tip of forceps so that it adheres to the surface of the agar. Do not push the disk into the agar. Each disk is already marked, so you do not need to label the plate with the antibiotic names. Symbol Amount Antibiotic Intermediate Range (mm) S10 10 µg Streptomycin TE µg Tetracycline P10 10 µg Penicillin C µg Chloramphenicol CF µg Cephalothin E µg Erythromycin NB µg Novobiocin VA µg Vancomycin ) Invert the plates and incubate them 37ºC until next lab period. Since these plates should not incubate more than 48 hours, be sure to view them the next period. IF they are incubated too long, colonies may begin to grow in the zone of inhibition, making the interpretation of your results difficult. 9) Next lab period: examine the plates for the presence or absence of a zone of inhibition around each disk. 10) Measure the diameter of the zone of inhibition around each disk and determine the susceptibility (resistant, intermediate, or sensitive) of the organisms to the antibiotics. Consult the chart in the lab manual for interpretation of zone size. Consult the chart above for the intermediate range for the eight antibiotics. A measurement that is smaller than the intermediate range indicates resistance while a measurement larger than the intermediate range indicates susceptibility. EX. 2-9: TEMPERATURE These experiments will be done on plates, rather than broths, to reduce the amount of media required for each experiment. The instructor will demonstrate the changes in the procedure as written below. Purpose: Determine the optimum growth temperature of different bacterial species Organisms: Escherichia coli, Staphylococcus aureus, Bacillus stearothermophilus, Pseudomonas fluorescens Media: Four TSA plates per pair 1. Draw two perpendicular lines on the bottom of the plate to divide the plates into fourths. Write the incubation temperature on each plate: 4 C, 25 C, 40 C and 60 C. Label each sector of each plate with the name of one of the four organisms listed above. Label the plates with your name and date as usual. 2. Make a straight line inoculation of each organism on each of the four plates. The instructor will demonstrate. 6

7 3. Put your plates in the appropriate baskets on the cart. These baskets will be incubated at the appropriate temperature. The 4 incubator is the refrigerator. The plates are incubated at the appropriate temperature until next lab period. 4. Next lab period: examine the plates for the presence or absence of growth and record your observations. Use a scale from 0-3 (0 being no growth and 3 being heavy growth) to describe the degree of growth. Note the temperature or temperature range that supports the growth of each organism. Is the organism a psychrophile, a mesophile, or a thermophile? In what possible environments would you find these organisms? EX. 2-11: OSMOTIC PRESSURE Purpose: Demonstrate the effect of osmotic pressure on microbial growth Organisms: Escherichia coli, Staphylococcus aureus, Bacillus subtilis Media: 5 beef heart infusion (BHI) plates, one with each of the following salt concentrations: 0.85%, 5%, 7.5%, 10% and 20% NaCl Work in pairs 1. Divide the plates into thirds. Label each sector with the name of one of the three organisms listed above. Label the plates with your name and date as usual. 2. Make a straight line inoculation of each organism on each of the five plates. 3. Incubate your plates at 37ºC until next lab period. 4. Next lab period: examine the plates for the presence or absence of growth and record your observations. Note the salt concentration that supports the growth of each organism. Which organism or organisms are halophiles? In what possible environments would you find these organisms? 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. 7

8 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) 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 8

9 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. 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 9

10 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. 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 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. 10

11 NOTE: 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. 11