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GRAM STAIN: IDENTIFICATION OF BACTERIA STANDARDS • 3.1.10.C, 3.1.12.C • 3.2.10.B, 3.2.12.B • 3.3.10.A, 3.2.12.A • 3.3.10.B, 3.3.12.B Westminster College INTRODUCTION There are numerous varieties of bacteria that exist in nature. Most bacteria can be divided into two classes, gram-negative and gram-positive, based on a differential staining process called the Gram stain. Differences in the cell wall separate Gram-positive bacteria, which retain a crystal violet dye used in the staining process, from Gramnegative bacteria. The differences in the cell wall also play an important role in the types of antibiotics that will be effective against the bacteria. Gram-positive bacteria have a membrane which is composed of two parts, the cell wall and the cytoplasmic membrane (Fig. 1A). The cell wall is composed primarily of peptidoglycan, a complex of linked polysaccharide chains which provide strength and rigidity. The peptidoglycan layer is responsible for the Figure 1. Graphic representation of the differences ability of the cell to retain the between the cell walls of (A) Gram-positive and Gram stain. Gram-positive (B)Gram-negative bacteria. cells also contain lipoteichoic A. acids (LTA) which extend from the cytoplasmic membrane through the peptidoglycan layer. Both the LTA and the peptidoglycan are targets for antibiotics. Gram-negative bacteria (Fig. 1B) have a cell wall which consists of an outer membrane, a periplasmic space and a cytoplasmic membrane. The peptidoglycan layer, found in the periplasmic space, is much smaller, and there is no teichoic acid present. The outer membrane and cytoplasmic membrane are Westminster College SIM B. Page 1 Gram Stain: Identification of Bacteria comprised of phospholipids. The outer membrane also contains lipopolysaccharides (LPS) and porins. The porins allow small molecules, like glucose, to diffuse through the outer membrane. The LPS are antigenic and may be targeted by certain antibiotics. A cell wall of this type does not retain the crystal violet stain. The Gram stain is a basic microbiology tool to visualize and classify unknown bacteria based on cell wall type. The staining process requires a primary stain, a mordant, a decolorizer and a counterstain. Crystal violet, a purple dye, is the primary stain. An iodine solution is the mordant. The iodine reacts with the primary stain creating a dye complex that is larger than the two molecules individually. A decolorizer, 95% ethanol, dehydrates the outer membrane and peptidoglycan layer of the cell wall, creating a matrix. The crystal violet-iodine complex is trapped by the dehydrated peptidoglycan only in Gram-positive bacteria. Gram-negative bacteria will still have pores large enough to release the dye complex and will appear colorless after this step. To visualize gramnegative samples, a counterstain of safranin is used. This dye will bind to the cell wall staining it a light red to pink color. In this experiment you are given two types of bacteria, one Gram-negative and one Gram-positive. You will use the Gram stain technique and subsequent analysis with a compound microscope to distinguish between these two different types of bacteria. GUIDING QUESTIONS • • • • How does the Gram stain identify differences between bacteria? What are the major differences between gram-positive and gram-negative bacteria? What are the components of the Gram stain procedure? How does each work? Are you able to determine which bacterium is gram-positive? Gram-negative? MATERIALS Escherichia coli culture Sterile inoculation loop (2) Microscope slides (2) Distilled water Test tube clamp Paper towels Gram’s Iodine solution Safranin O solution Immersion oil (optional) Bacillus cereus culture Bunsen burner Beaker 95% ethanol Wax pencil Kimwipes Crystal Violet solution Microscope with 100x objective Additional bacterial cultures (optional) SAFETY • • The stains should be handled with care. Gloves and goggles are recommended when performing the staining part of the lab. The glass slides will be extremely hot after heat-fixing the bacteria. Be careful not to touch the slides until they have cooled completely. Westminster College SIM Page 2 Gram Stain: Identification of Bacteria PROCEDURE 1. There are two glass microscope slides at each lab station. Draw a circle in the center of each slide; this is where the bacterial sample will be placed. Using a wax pencil, label one slide E. coli and the other B. cereus, one for each different bacterial culture. 2. Place one drop of distilled water in the center of the circle of the E. coli slide. Use the loop end of one of the sterile inoculation loops to obtain a drop of E. coli from the culture tube. Mix the E. coli with the drop of water on the slide; spread the liquid thinly within the wax circle. 3. Repeat Step 2 to make the second slide with the B. cereus bacteria. Let both slides air dry for at least 5 minutes. 4. Your instructor will give you directions on how to light the Bunsen burner. Once the burner is lit, grasp one end of the E. coli slide with the test tube clamp, keeping the slide culture side up. Gently move the slide back and forth through the top of the flame until the liquid sample is dry. 5. Place the slide to cool on a paper towel on the lab bench. NOTE: Do not touch the slide for at least 5 minutes! It will be very hot. 6. Repeat Steps 4 and 5 for the B. cereus slide. 7. You may want to wear gloves and goggles for the following steps. 8. When both slides have cooled, add 3-4 drops of the Crystal Violet solution (primary stain) to the heat fixed bacteria on both slides (Fig. 2). Let the slides stain for 1 minute. 9. Carefully pour off the stain into the beaker provided at the lab station. Hold the slide at a 45° angle and rinse with distilled water. Squirt the water just above the bacterial smear and let the water flow over the sample into the beaker. 10. Drain off any excess water. Place the slides back on the paper towel and cover the bacterial smear with Iodine solution (mordant). Let the Iodine solution sit for 1 minute. 11. Pour off any excess iodine; rinse gently with distilled water like in Step 9. 12. Still holding the slide at a 45° angle over the beaker, rinse with 95% ethanol, allowing the ethanol to flow across the bacterial smear. Continue to rinse until no more color is removed from the smear. 13. Rinse the slide with water to remove the ethanol. Drain the excess water. 14. Cover both smears with Safranin O solution and let the slides sit for 1 minute. Westminster College SIM Page 3 Gram Stain: Identification of Bacteria Figure 2. Gram stain procedure a. Crystal violet, 1 min b. Distilled water rinse into beaker c. Gram’s iodine, 1 min d. Distilled water rinse into beaker e. 95% ethanol, decolorize f. Distilled water rinse into beaker g. Safronin O counterstain, 1 min h. Distilled water rinse into beaker i. Blot excess liquid and air dry for 5 min 15. Rinse the slide with water and let the slides drain. Gently blot any excess liquid and let the slide air dry for 5 minutes. 16. Use a compound microscope with a 100x objective to examine each sample. Westminster College SIM Page 4 Gram Stain: Identification of Bacteria Figure 3. Examples of gram-positive and gram-negative bacteria. Gram-positive bacteria Gram-negative bacteria REFERENCES Background information from: http://www.cehs.siu.edu/fix/medmicro/genmicr.htm Graphic of Gram-positive and Gram-negative cell walls: http://www.cehs.siu.edu/fix/medmicro/pix/walls.gif Gram-positive and Gram-negative micrograph: http://bioweb.uwlax.edu/bio203/s2008/jaedike_alic/staining.htm Cavanaugh, Danny; Mark G. Keen, Ph.D.; Department of Microbiology, North Carolina State University; The Gram Stain: An Animated Approach: http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/images/gram_stain_11.swf CREDITS Special thanks to Chris Cassano of Wilmington High School, New Wilmington, PA , and Casey Schmidt, Science in Motion lab assistant, for testing, editing and reviewing this protocol. The lab was revised and adapted from the above references by Dr. Stephanie Corrette-Bennett. Westminster College SIM Page 5 Gram Stain: Identification of Bacteria DATA SHEET Name: _______________________ Group: _______________________ Date: _______________________ DATA ANALYSIS Draw a picture of what you observe for each type of bacteria. Identify which you think is Gram-positive and Gram-negative, based on your results. Bacillus cereus Escherichia coli Gram ______ Gram ______ QUESTIONS 1. Describe how the two bacteria look different after the Gram stain (size, shape, stain color). Westminster College SIM Page 6 Gram Stain: Identification of Bacteria 2. List the three differences between the cell walls of Gram-positive and Gram-negative bacterial cells. a. b. c. 3. What are the 4 reagents in the Gram stain procedure? a. b. c. d. Westminster College SIM Page 7