Special stains Flashcards
what species produce endospores?
Bacillus and Clostridium
why are endospores produced?
-they are produce when environments become unfavorable
-depletion of nutrients, change in temperature, lack of water
-when the environment becomes favorable again they will germinate back into vegetative cells
Endospores
-are dormant forms of the bacteria resistant to heat, ultraviolet radiation, toxins, chemical disinfectants, freezing, and desiccation
In normal environmental conditions how do bacteria exists?
-in a vegetative state where they are able to reproduce and grow
What stain is used to identify endospores?
Schaeffer- Fulton stain
What does the Schaeffer- Fulton stain identify?
vegetative and endospore forms of Bacillus and Clostridium
Steps in Endospore stain (Schaeffer-Fulton)
- Place malachite green stain on the slide while holding over steam for 5 minutes (heat is the mordant)
- decolorize the stain with water (vegetative cells: white, endospores: green)
3.Flood the smear with the counterstain safranin for 1 minute, then rinse with water
(vegetative cell: red, endospore: green)
What is the mordant in the Schaffer Fulton stain
heat
what is the primary stain in Schaffer- Fulton stain
malachite green
What is the counterstain in Schaeffer-Fulton stain
safranin
What species contain mycolic acid
mycobacterium
what does mycolic acid do?
-is a waxy outer layer on cells that block primary stains from being absorbed
What can certain species of Mycobacterium cause?
-Leprosy or tuberculosis
What does acid-fast staining (Ziehl-Neelsen) identify?
-bacteria with mycolic acid
Steps in Acid-fast staining (Ziehl-Neelson)
1.Prepare a mixture of S.epidermis and Mycobacterium thin smear
2.Place carbolfuchsin stain on the slide while holding over steam for 5 minutes (heat is the mordant, which softens the waxy outer layer, allows for penetration)
-all bacteria appear magenta
3.Decolorize with acid alcohol dropwise until smear is decolored, rinse with water (removes stain from non-acid fast cells)
4.counterstain with methylene blue, flood the slide for 2 minutes, the rinse off with water
results: mycobacterium (magenta)
s. epidermis: blue
How to create a thin smear?
- create smear of bacteria on slide
- air dry
- heat fix
What is methylene blue
a positively charged, basic stain and will bind to the surface of non-acid fast cells
What is glycocalyx
-is a gelatinous sticky substance surrounding the outside of the cell
-made up of proteins and polysaccharides
capsule
-a glycocalyx that is firmly attached to the cell surface
-this can prevent bacteria from being recognized by the host
-can also camouflage by containing chemicals that are found in the host cell
Negative capsule stain
- use a primary dye such as crystal violet to stain bacteria
- stain the background with an acidic negatively charged dye like Nigrosin or India Ink
Background: India Ink or Nigrosin (pink)
Bacteria: Crystal violet (purple)
capsule: clear, colorless, white halo
What is the mordant in the Acid-fast staining (Ziehl-Neelson)
heat
what is the primary stain in Acid-fast staining (Ziehl-Neelson)
carbolfuchsin
what is the secondary stain in Acid-fast staining (Ziehl-Neelson)
methylene blue
crystal violet
-is positively charged/basic and will stain the negatively charged features on the bacteria besides the capsule
Name two negatively charged dyes
Nigrosin and India Ink
How do negatively charged dyes work?
they repel the cell/capsule that is also negatively charged
Flagellar staining
- primary stain and mordant are mixed and used as the primary stain
-this combination allows the dye to be less soluble and coat the surface of the flagella
-this increases the diameter/size and colorize it so easier to see
Monotrichous
-one flagella only on one side
ex: vibrio cholerae
Amphitrichous
-having a single flagellum on each of the two opposite ends
ex: spirillum volutans
Lophotrichous
multiple flagella at one end or end point
ex: helicobacter pylori
Peritrichous
having flagella all over, covering the entire surface
ex: Proteus vulgaris
Vegetative cell
-is the parent cell of an endospore. the vegetative cell breaks open to release the endospores into the environment
Why can you not see flagella on smears stained with usual technique?
-The flagella are to thin to see without a compound microscope, their width is less than the resolution of microscope
Why is heat used to stain bacterial endospores?
-heat drives the stain into the cell
what are some characteristics of endospores?
-endospores are resilient forms of a bacterial cell that forms within the parental cell. They have an impervious cell wall and are resistant to heat/cold, pathogens, toxic changes and desiccation
What is the difference between central and terminal endospores?
central endospores are located within the middle of the vegetative cell. And terminal endospores are located at the end of vegetative cells.
What is meant by the term vegatative cell?
-is the parent cell of an endospore, it breaks open to release the endospore into the environment
How does the spore stain resemble the acid-fast stain
-both the endospore and acid-fast stain require the use of heat to drive the primary stain through the cell wall
What pathogen can be presumptively identified using the acid fast stain?
mycobacterium species
Why can you not see flagella on smears stained within the usual staining technique?
-the flagella are too thin to see with a compound microscope
What is the principle behind the negative staining technique?
-negative stains, stain the entire background surround the cells. A counter stain allows the cells to be visualized and the capsule is left unstained. This leaves a halo like capsule visible between negative and counter stains
What structures may be observed with a negative staining technique
capsules
what stains endospores?
malachite green
what stains vegetative cells in the endospore stain?
safranin
what stains the background in the negative stain?
India Ink or nigrosin
What is the primary stain for acid fast?
carbol fuchsin
