Preparing a Bacterial Smear and Gram Stain

  1. What is the first step in performing a Gram stain?
    making a bacterial smear
  2. Gram stain characteristics are used to help identify _____.
  3. What types of cultures can bacterial smears be prepared from?
    broth and solid media cultures
  4. What are the steps to preparing a bacterial smear from a solid media culture?
    • flame the inocculating loop
    • while still hot, submerge loop in beaker of distilled water and remove
    • transfer the water drop to the center of a clean slide
    • flame loop again
    • obtain bacteria from an isolated colony on a streak plate
    • without springing the loop, gently mix the cells in the drop of water
    • air dry the slide - do not fan the slide like you do a blood smear
    • once dried, use forceps and pass the slide through the upper part of the flame two or three times to heat fix the smear
  5. Why do we heat fix the smear?
    • it adheres the bacterial cells to the slide
    • it kills the bacterial cells and makes them stain easier due to the coagulated proteins
  6. How do we prepare a bacterial smear from a broth culture?
    same as from a solid media culture except you do not need to add the sterile distilled water to the slide unless the broth is very thick
  7. Which type of bacterial smear takes longer to dry?
    bacterial smear made from broth culture
  8. How old should the cultures be in order for us to get consistent gram stain results?
    18 - 24 hours old
  9. Stained smears can always be read later, but must be prepared when the culture is younger than _____.
    24 hours old
  10. Which types of smears can be disposed of in the regular trash?
    heat fixed smears
  11. Which types of smears need to be disposed of in a biohazard bag?
    non-heat fixed smears
  12. What are the four steps to a gram stain?
    • crystal violet stain
    • iodine stain
    • decolorizer (alcohol)
    • safranin stain
  13. What is stain uptake related to?
    bacterial cell wall structure
  14. What does the cell wall structure of a gram negative bacteria look like?
    more lipids in the cell wall
  15. What does the cell wall structure of a gram positive bacteria look like?
    more proteins and carbohydrates in cell wall
  16. What does crystal violet do?
    turns bacterial cells blue
  17. What does iodine do?
    makes the crystal violet stick better
  18. What does the decolorizer do?
    • dissolves lipid layer of gram negative bacteria so blue stain can leach out more easily
    • dehydrates and closes pores of gram positive bacteria so blue stain is retained
  19. What does the safranin stain do?
    turns decolorized gram negative bacteria pink
  20. What happens if we over-decolorize?
    removes the blue from gram positive bacteria, turning them pink
  21. What color are gram positive bacteria?
    • purple
    • take up the crystal violet stain and do not decolorize
  22. What color are gram negative bacteria?
    • pink, red
    • lipid cell walls decolorize and safranin stain is retained
  23. What are the steps to doing a gram stain?
    • place an air dried and heat fixed slide on a staining rack
    • flood the smear with crystal violet and let it stand for 1 minute
    • tilt the slid and rinse away the excess crystal violet using distilled water
    • replace the rinsed smear on the rack and cover with iodine solution for 1 minute
    • rinse with distilled water
    • while still tilting the rinsed slide, decolorize by allowing the decolorizing solution to run across the smear, stop when the run-off is clear but no longer than 30 seconds
    • rinse with distilled water
    • cover smear with safranin stain for 1 minute
    • rinse with distilled water
    • gently blot slide dry with a paper towel
  24. What do we need to identify when looking at a gram stain under oil immersion?
    • identify color - purple/blue is gram positive, red/pink is gram negative
    • identify morphology - cocci, rods, spiral, pleomorphic
    • identify cell arrangement - single, pair, cluster, chain
Card Set
Preparing a Bacterial Smear and Gram Stain
Microbiology Two