Gram Staining of Bacteria

Name ______Class ______Date ______Skills Practice Lab Gram Staining of Bacteria

A stain gives color to cells that are colorless, such as bacterial cells. In 1884 the Danish physician Hans Christian Gram developed the Gram stain, which is a differential stain. Differential stains react differently to different types of bacteria. The Gram stain is used to classify bacteria into two large groups, Gram positive and Gram negative. After the Gram-staining procedure, Gram-positive bacteria appear purple and Gram-negative bacteria appear pink. To perform the Gram stain on bacteria, you must first prepare a smear, which is a thin film of bacteria placed on a glass slide and air-dried. The slide is passed through a Bunsen burner flame to fix, or attach, the smear to the slide. A stain called crystal violet stains the bacterial cells purple. Gram’s iodine, which is an iodine solution, is then applied. The iodine acts as a mordant, which is a substance that intensifies the color of crystal violet and helps it adhere to the cells. Next, the smear is washed with ethanol. The ethanol decolorizes, or removes the color from, the cells of Gram-negative bacteria. A counterstain, Safranin O, which is pink in color, is then applied. It is called a counterstain because its color contrasts with that of crystal violet. After the smear is stained with Safranin O, Gram-negative bacteria appear pink. Gram-positive and Gram-negative bacteria have differences in the chemical makeup of their cell walls, which determines how they are stained. Gram-positive bacterial cell walls contain a thick layer of peptidoglycan. Crystal violet and iodine combine to form a large-molecule precipitate within the cell. The ethanol decolorizing wash dehydrates the thick peptidoglycan layer, which traps the crystal violet-iodine precipitate. Gram-negative bacterial cell walls have a thin layer of peptidoglycan and a protective layer of lipopolysaccharide. When Gram- negative cells are decolorized, they do not retain the crystal violet because the lipopolysaccharide layer is dissolved by the alcohol, and the thin peptidoglycan layer cannot retain the crystal violet-iodine complex. Identification of bacteria by Gram staining helps determine which drugs will be the most effective in the treatment of disease. Therefore, Gram staining is an important technique in medicine. Gram-positive bacteria tend to be killed by antibiotics such as penicillin and erythromycin. Gram-negative bacteria are resistant to these drugs but are sensitive to streptomycin and tetracycline. Your goal is to work in the medical field. You just landed a position as a part- time lab technician in a doctor’s office to see if you enjoy the work. You have been directed to determine if cultures of bacteria are Gram positive or Gram negative. Your first job is to learn the Gram-stain technique by testing known samples of Gram-positive and Gram-negative bacteria. Then you will have the skills necessary to perform the Gram stain on cultures of unknown bacteria.

OBJECTIVES Compare and contrast Gram-positive and Gram-negative bacteria. Relate Gram staining to the selection of antibiotics for the treatment of disease. Identify an unknown bacterium as either Gram positive or Gram negative. Original content Copyright © by Holt, Rinehart and Winston. Additions and changes to the original content are the responsibility of the instructor. Name ______Class ______Date ______Gram Staining of Bacteria continued

MATERIALS  250-mL beakers (2)  Bunsen burner  clock or watch with a second hand  compound microscope  coverslips (3)  crystal violet stain  culture of Gram-negative bacteria, Aquaspirillum serpens  culture of Gram-positive bacteria, Bacillus megaterium  culture of unknown bacteria  disinfectant solution  distilled water  ethanol, 95%  eyedropper  gloves  lab apron  lens paper  inoculating loop  Gram’s iodine stain  mounting medium  absorbent paper towels  slide holder  staining tray  Safranin O stain  safety goggles  glass slides (3)  striker  wax pencil

Procedure 1. Put on safety goggles, gloves, and a lab apron. 2. Use aseptic technique throughout this lab. Clean the lab-table surface with disinfectant solution and paper towels. 3. Label one glass slide “Gram +” and one “Gram −” using a wax pencil. Leave the third glass slide unlabeled. 4. Place one drop of distilled water in the center of the slide labeled “Gram −.” Original content Copyright © by Holt, Rinehart and Winston. Additions and changes to the original content are the responsibility of the instructor. 5. Sterilize an inoculating loop by holding the wire in the flame of a Bunsen burner until the wire glows red. CAUTION: Do not touch hot objects. Open the lid of the B. megaterium culture. Note: Remember to slowly lift one side of the lid to a 45° angle. Cool the loop by touching it on the agar of the stock culture in an area of no growth, and then transfer a colony of B. megaterium from the culture to the water on the slide. 6. Stir the bacteria in the drop of distilled water using the inoculating loop, as shown in Figure 1. Allow the smear to air-dry. Resterilize the inoculating loop.

Original content Copyright © by Holt, Rinehart and Winston. Additions and changes to the original content are the responsibility of the instructor. Name ______Class ______Date ______Gram Staining of Bacteria continued

FIGURE 1 STIR BACTERIA WITH LOOP

FIGURE 2 PASS SLIDE THROUGH FLAME

7. After the smear has dried completely, pick up the slide with a slide holder, bacteria side up, and pass the slide quickly through the Bunsen burner flame three times, as shown in Figure 2. CAUTION: Use a slide holder to hold hot slides. Do not hold the slide still in the flame. When finished, turn off the Bunsen burner. Place the slide into a staining tray, and allow it to cool. 8. Cover the smear with 10 drops of crystal violet for 60 seconds. CAUTION: Crystal violet will stain your skin and clothing. Promptly wash off spills to minimize staining. 9. After 60 seconds, pour off the excess stain. Gently rinse the smear in a beaker filled with distilled water, as shown in Figure 3. Tilt the slide to remove any remaining water droplets. Note: Be careful not to disturb the area where the smear is located. 10. Place the slide back into the staining tray, and cover it with 10 drops of Gram’s iodine for 60 seconds. CAUTION: Gram’s iodine will stain your skin and clothing. Promptly wash off spills to minimize staining. 11. While holding the slide at an angle over an empty beaker, use an eyedropper filled with ethanol to gently rinse the smear, as shown in Figure 4, until no stain rinses off. CAUTION: Do not use alcohol when flames are present in the room. Do not light burners when others are using alcohol. 12. Rinse the slide in a beaker filled with clean distilled water. Shake off the excess water.

FIGURE 3

FIGURE 4 RINSE THE SMEAR WITH ETHANOL

13. Cover the smear with 10 drops of Safranin O for 15 seconds. CAUTION: Safranin O will stain your skin and clothing. Promptly wash off spills to minimize staining. 14. Rinse the slide in a beaker filled with clean distilled water, and then carefully blot the slide dry using an absorbent paper towel. Note: Be careful not to disturb the smear. 15. Repeat steps 4–14 using the slide labeled “Gram –” and A. serpens. 16. Repeat steps 4–14 using the unlabeled slide and the unknown bacteria. 17. Add a drop of mounting medium to the smear, and then place a coverslip on each slide. Using the microscope, view each slide under low power, and then switch to high power. Observe the bacteria, and record your observations in the table below.

OBSERVATIONS

Bacterium Description Drawing B. megaterium A. serpens Unknown bacteria

18. Dispose of your materials according to the directions from your teacher. 19. Clean the lab-table surface with disinfectant solution and paper towels. Clean up your work area and wash your hands before leaving the laboratory. Analysis 1. Analyzing Data You prepared slides using bacteria classified as Gram positive and Gram negative. What color should your bacteria be? Do your results agree?

2. Explaining Events Why is the Gram stain useful in classifying bacteria?

3. Explaining Events Why is the inoculating loop heated in a flame and then placed on agar in an area of no growth?

4. Analyzing Data Why did you use ethanol and a counterstain during the procedure?

Conclusions 1. Drawing Conclusions Was the unknown bacteria Gram positive or Gram negative? How do you know?

2. Applying Conclusions If you were a doctor, and a lab technician told you that the results of a patient’s lab test were Gram positive, what antibiotic would you prescribe for the patient? Why?

3. Making Predictions What would happen if a doctor prescribed penicillin for a Gram-negative bacterial infection? Why?

4. Making Inferences Review and compare the structural components of the cell walls of Gram-positive and Gram-negative bacteria. Why do you think Gram-negative bacteria are less sensitive to antibiotics?

Extensions 1. Clinical laboratory microbiologists identify pathogens, or disease-causing organisms, present in the specimens sent to them by doctors. After isolating the microbes, microbiologists run tests to determine the proper antibiotics to kill the pathogens. Find out about the training and skills required to become a clinical laboratory microbiologist. 2. Do research to determine whether bacterial shape is related to the results of Gram staining.