October 6

ACID-FAST STAIN

Purpose: To distinguish between acid-fast bacteria and non-acid-fast bacteria. 
 The difference in lipid content of the bacteria's cell wall is what makes the bacteria acid-fast or not

Materials:

1 slide with a fixed smear of unknown sample
Ziehl-Neelsen stain
Acid-alcohol
methylene blue
Hot plate
100 mL beaker with deionized water
strip of filter paper that will cover slide
was bottle with distilled water
bibulous paper
forceps of some kind


Procedure:
1. Put the 100 mL beaker with deionized water on the hot plate and bring to a boil.
2. Place the slide so that it is resting on the top of the beaker (fixed smear side facing up).
3. Put filter paper on the slide and saturate the paper with Ziehl-Neelsen carbolfuchsin.
4. Stain for 3-5 minutes. Continue to add stain as it evaporates; do not allow the stain to dry.
5. Using the forceps, remove the slide from the heat. Remove the paper and throw it away. Allow the slide to cool.
6. Rinse the slide with distilled water to remove any excess stain.
7. Holding the slide at a 45 degree angle decolorize with acid-alcohol. Add the alcohol drop by drop until the color stops running.
8. Immediately rinse the slide off with distilled water.
9. cover the smear with methylene blue for 2 minutes.
10. Rinse the slide with distilled water to remove any excess methylene blue.
11. Blot the water off the slide with bibulous paper.
12. Examine slide under the microscope with oil immersion lens.

Non-Acid-Fast stain

Conclusion: I had a non-acid-fast stain. You can tell because all of my bacteria were stained from the methylene blue and not from the Ziehl-Neelsen.

ENDOSPORE STAIN

Purpose:
To view bacterial endospores under the microscope and to observe the location of an endospore in a sporulating cell.

Materials:
 Side with fixed smear on unknown bacteria
 Malachite green stain
Safranin stain
 staining rack over a sink
Steaming deionized water in a 100 mL beaker
Hot plate
piece of filter paper
distilled water
bilbulous paper
Forceps

Procedure:
1. Fix bacteria to slide
2. Place the slide on a beaker with boiling water.
3. Place filter paper on the slide and saturate the paper with malachite green
4. Stain for 5-6 minutes after the malachite green begins to steam. Add stain as it evaporates. Do not allow the stain to dry.
5. Use forceps to remove the slide from the heat. Remove the paper and throw it away.
6. Allow the slide to cool.
7. Rinse the slide with distilled water for about 30 seconds to remove any excess malachite green stain.
8. Cover the smear with safranin for 60-90 seconds.
9. Rinse the slide with distilled water to remove excess safranin.
10. Blot water off the slide with bibulous paper.
11. Using the oil immersion lens to look at bacteria under microscope.

Non-Endospore Stain

Conclusion: My bacteria do not have endospores. If it did have endospores, then the nucleus' would have been stained green.