Acid-fast/ Endospore/ Capsule staining Flashcards
Why are acid-fast bacteria named that?
They will retain the primary stain even after treated with acid alcohol
What do the cell walls of acid fast bacteria contain?
mycolic acid
What is mycolic acid?
a waxy lipid that makes the cell impermeable to most stains
What does mycolic acid do?
protects the bacteria from desiccation and prevents digestion from phagocytes
What two genus’ are involved with acid-fast bacteria?
Mycobacterium and Nocardia
What are two examples of acid-fast bacteria?
Mycobacterium tuberculosis and Mycobacterium leprae
What are the steps of an acid-fast stain?
- normal procedures with heat fix etc. 2. bacteria are stained with carbolfuchsin 2. acid alcohol is then added 3. methylene blue is then used as a counterstain
What happens to non acid-fast bacteria when acid alcohol is added?
they are quickly destained
What happens to acid-fast bacteria when acid alcohol is added?
They hold onto the carbolfuchsin because the carbolfuchsin has phenol in it and phenol is more soluble in in fat vs acid alcohol. The lipid in mycolic acid will retain the dye even after the acid alcohol rinse.
What happens to the non acid fast bacteria when methylene blue is added?
they will be stained blue
What happens to the acid-fast bacteria when methylene blue is added?
Nothing. They will remain red since the mycolic acid is impermeable to methylene blue.
What are vegetative bodies?
bacteria that are actively metabolizing
What genus’ produce endospores?
Clostridium and Bacillus
What are four examples of diseases caused by endospores?
C. tetani, C. botulinum, B. anthracis, C. difficile
What are four characteristics of endospores?
- smaller and more compact than vegetative bodies
- thick outer layer of keratin
- are resistant to heat, drying, detergents, harsh environments, UV
- are not actively metabolizing
Why do endospores form?
Harsh environments make them do this as a survival mechanism
What happens when the environment is more appealing to the endospore?
It will germinate and re-form the vegetative body.
Making an endospore is NOT ______, it is a ______.
reproductive mechanism, survival mechanism
What are the steps of an endospore stain?
A standard fixed smear is prepared and then malachite green is added and gently heated over a flame for 5 minutes. Malachite green is then rinsed off and counterstain safranin is added for 2 minutes and then rinsed and dried.
Why must the stain be gently heated during an endospore stain?
since the endospores have a thick layer of keratin, heating is required to allow for stain penetration
What does safranin stain during an endospore stain?
It stains the vegetative bodies
What are the four characteristics of capsules?
- gelatinous coat surrounding certain bacteria
- composed primarily of uncharged polysaccharides, some polypeptides
- size is influenced by the media the organism is cultured in
- increases bacterial virulence
What are the four ways capsules increase bacterial virulence?
- help evade phagocytosis
- help bacteria attach to cause infection
- function as an osmotic barrier, evade desiccation
- function as a nutrient reserve
What are the steps for a capsule stain?
Prepare a standard negative stain with Congo Red. Then flood the slide with 0.1N HCl as a fixitive. Then add safranin.
What does congo red stain during a capsule stain?
the background only
What does safranin dye during a capsule stain?
it stains the cell body only. This is because the capsule has no charge so a basic dye will not stain it.
How does the capsule appear during a capsule stain?
as a halo around the stained bacterium against the dark background.
