The Acid-Fast Stain
Name: _____________________________________________________
Course Section: ______________________________________________
The following activity will teach students how to complete an Acid-Fast Stain and identify bacteria based on their different cell wall properties. Read the following procedure carefully before starting the activity.
|
Materials
|
-
2 microscope slides
-
Wax pencil
-
Mycobacterium smegmatis
and
Staphylococcus epidermidis
-
Carbol fuchsin stain
-
Methylene blue stain
-
Acid alcohol
-
DI water
-
Bibulous paper
-
Staining tray and slide stand
-
Inoculation loop
-
Hot plate
-
Clothespin
-
Forceps
-
1000 ml beaker
-
Timer
-
Incinerator
-
Microscopes: lens paper, lens cleaner, immersion oil
|
|
Procedure
|
|
Step 1: Prepare A Bacterial Smear
-
Get into groups of 2.
-
Label the edge of the microscope slide with 1 person's initials and the bacterial species. You may abbreviate the name of the bacteria so it fits on the slide. Try to write small.
-
On the bottom of the slide, you may draw a circle in the middle of the slide with a sharpie or wax pencil so you know where to make your smear. This is optional.
-
Sterilize your inoculation loop and use it to pick up a small amount of DI water. Place the water on top of the slide, in the middle of the circle if you drew one.
-
Sterilize your loop again and pick up a small bacterial colony from the stock plate.
-
Gently, mix the bacteria into the water drop on the slide. You want to spread the water and bacteria out to about the size of a dime.
-
Let the water and bacteria smear completely air dry. The slide should look dry and "crusty" before moving to Step 2.
|
|
Step 2: Heat Fixation
-
Once the bacterial smear is completely dry, use a clothespin to clip the end of your slide.
-
Then, place your slide directly on a hot plate for 30 seconds.
-
After 30 seconds, place your slide onto the slide stand in your staining tray. Don't touch the slide, it will be hot. Don't place the slide on the cold table, the glass slide could crack.
-
Once your slide is completely cool, move on to Step 3.
|
|
Step 3: Acid-Fast Staining
-
Primary Staining
-
Take your heat-fixed slide to the fume hood (the fumes from carbol fuchsin can be toxic).
-
Once the water is boiling, place your slide on the slide rack above the boiling water.
-
Cover the smear area of your slide with a square piece of bibulous paper.
-
Carefully apply the carbol fuchsin stain to the bibulous paper.
-
Steam with the stain on the slide for 5 minutes while continuously applying more stain so the bibulous paper never dries out.
-
After 5 minutes remove the paper with forceps and discard it in the trash bin.
-
Using a clothespin take your slide back to your lab station and rinse with water.
-
Decolorizer, Wash Step
-
Hold the slide at a 45° angle and gently rinse with the acid alcohol until the run-off is almost clear.
-
Over or under-decolorizing will affect your staining results.
-
Wash the decolorizer off the slide with a gentle DI rinse.
-
Counterstain
-
Add a few drops of methylene blue to the smear.
-
Leave the stain for 2 minutes.
-
Hold the slide at a 45° angle and rinse with DI water
-
Place the slide between 2 sheets of bibulous paper and gently pat dry. Do not wipe the slide to dry it.
-
Microscopy
-
When the slide is dry, observe your specimen using oil immersion.
-
Repeat Steps 1 through 3 for the remaining bacterial species.
-
When both slides are done, clean your microscope and place it back in the cabinet.
-
Rinse your staining tray in a sink and put all materials back in their labeled locations.
|
Results
|
Species
|
Color
|
Acid Fast +/-
|
Morphology and Arrangement
|
|
Staphylococcus epidermidis
|
|
|
|
|
Mycobacterium smegmatis
|
|
|
|
General Questions
1. In the acid-fast stain procedure, which step is used to differentiate acid-fast positive from acid-fast negative bacteria? Explain your answer.
2. What is the purpose of steam in the acid-fast staining procedure?
3. Describe the structure of the cell walls of acid-fast positive bacteria. How do their cell walls differ from gram-positive and gram-negative cell walls?
4. What is the medical importance of using the acid-fast stain (i.e., what diseases/infections can it help to identify)?