There a large number of staining procedures that are used to enhance the contrast of the whole specimen or parts of the specimen. This module involves performing the Schaeffer-Fulton endospore stain and the Ziehl-Neelson acid-fast stain.
Ziehl-Neelson acid-fast
stain:
This stain may be thought of as a variation of the gram stain as it
uses a similar dye/wash/decolorize procedure and stains the cell wall.
Unlike the Gram stain, where a large number of bacteria were either gram
positive or gram negative, only a few groups of bacteria, due to the specific
makeup of their cell wall (waxy material), will stain acid-fast: the Mycobacterium,
Nocardia, and a few protozoans. Therefore, a positive result narrows down
the possibilities of the type of bacteria it may be.
Procedure:
-Know the details.
-Unlike the Gram stain, the primary dye requires heat to drive the
dye into the cell wall.
-The decolorizer used is an acid-alcohol mixture, which is much stronger
than the Gram decolorizer.
- The heat will be applied using a steam bath.
Tips/Comments:
---The slides with bacteria have already been heat fixed. Just perform
the stain procedure.
---Keep track of your slide.
---Apply a small piece of paper towel over the bacteria, then apply
the stain to the paper towel.
---Do not let the piece of paper hang over the edge of the slide. Otherwise
the dye will run off the slide.
---After removing the slide from the heat, place it on a paper towel
and carry it back to your stain area.
---Acid fast is pink, non acid fast is purple.
??What would it look like if, by accident, you used the Gram decolorizer??
Schaeffer-Fulton endospore
stain:
This stain is used to specifically stain endospores, which are made
by a few groups of bacteria, including Bacillus and Clostridium,
to protect their DNA in times of stress, especially starvation. Therefore,
a finding of endospores narrows down the number of possible bacteria. The
endospore is a tough structure and is difficult to stain. As in the acid-fast
stain, heat is necessary to drive the stain into the spore.
Tips/Comments:
---The slide will need to be made from scratch using bacteria obtained
from a nutrient agar plate.
---Keep track of your slide.
---Apply a small piece of paper towel over the bacteria, then apply
the stain to the paper towel.
---Do not let the piece of paper hang over the edge of the slide. Otherwise
the dye will run off the slide.
---After removing the slide from the heat, place it on a paper towel
and carry it back to your stain area.
---The bacteria stain pink and the spores stain green.
---The spores may be free or may be seen inside the bacteria.