-
What is the first step in performing a Gram stain?
making a bacterial smear
-
Gram stain characteristics are used to help identify _____.
bacteria
-
What types of cultures can bacterial smears be prepared from?
broth and solid media cultures
-
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
-
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
-
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
-
Which type of bacterial smear takes longer to dry?
bacterial smear made from broth culture
-
How old should the cultures be in order for us to get consistent gram stain results?
18 - 24 hours old
-
Stained smears can always be read later, but must be prepared when the culture is younger than _____.
24 hours old
-
Which types of smears can be disposed of in the regular trash?
heat fixed smears
-
Which types of smears need to be disposed of in a biohazard bag?
non-heat fixed smears
-
What are the four steps to a gram stain?
- crystal violet stain
- iodine stain
- decolorizer (alcohol)
- safranin stain
-
What is stain uptake related to?
bacterial cell wall structure
-
What does the cell wall structure of a gram negative bacteria look like?
more lipids in the cell wall
-
What does the cell wall structure of a gram positive bacteria look like?
more proteins and carbohydrates in cell wall
-
What does crystal violet do?
turns bacterial cells blue
-
What does iodine do?
makes the crystal violet stick better
-
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
-
What does the safranin stain do?
turns decolorized gram negative bacteria pink
-
What happens if we over-decolorize?
removes the blue from gram positive bacteria, turning them pink
-
What color are gram positive bacteria?
- purple
- take up the crystal violet stain and do not decolorize
-
What color are gram negative bacteria?
- pink, red
- lipid cell walls decolorize and safranin stain is retained
-
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
-
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
|
|
