Lab 10 - Endospore Stain Flashcards
what is a structural stain?
a stain that reveals parts of a cell structure
what are four types of structural/secondary stains?
- Endospore
- Flagella - Leifson’s Technique
- Capsule
- Acid-fast ; Ziehl-Neelsen Method
what are two methods of endospore staining?
a. Schaeffer-Fulton Method
b. Crystal violet method
which of the two endospore staining do we use in lab?
Shaeffer-Fulton Method
endospores are hibernation chambers, what does that mean?
that they are durable structures that resist harsh environments and remain viable for thousands of years (they have a very thick cell wall)
endospores are dormant and desiccated. what does that mean?
dormant: lacking metabolism (sleeping).
desiccated: lacking water (dried out, no chemical reactions occurring).
are endospores are formed inside or outside the cell?
inside the cells
what two bacteria are endospores formed by?
Bacillus and Clostridium
what are two examples of Bacillus and Clostridium and the disease they cause?
Bacillus anthracis causes anthrax.
Clostridium tetani causes tetanus.
what are endospores resistant to?
to heat, drying, chemicals, radiation
are there contamination concerns of endospores? where are the concerns?
yes, there are contamination concerns in labs and hospitals
what kills endospores?
autoclaving or very concentrated bleach
what color is the vegetative cell? what color is the endospore + where is it?
the pink cell is the vegetative cell, with the endospore inside the empty space within. it kind of looks like its glowing inside.
can there be free endospores without being inside vegetative cells?
yes, they can be free
what is a vegetative cell?
a metabolically active cell
when do spores form?
spores form when the nutrient levels drop and the waste levels rise.
what is sporulation?
making an endospore
what is germination?
making a vegetative cell
is forming endospores a form of reproduction?
NO
do endospores help in survival and dispersal?
yes
when spores become airborne and land on a good surface with nutrients, what happens?
they germinate
what are the steps of sporulation?
- Vegetative growth stops and the DNA is replicated.
- A septum forms, dividing the cell asymmetrically.
- The larger compartment engulfs the smaller one, forming a forespore within a mother cell.
- PG-containing material is laid down between the two membranes that now surround the forespore.
- The mother cell usually lyses and releases the endospore. The mother cell is degraded and the endospore is released.
what are fungal spores?
they are not formed inside the vegetative cells
are fungal cells completely dormant?
NO, they have water too
are fungal spores surrounded by a thick coat?
No
do fungal cells give rise to vegetative cells?
yes
Schaeffer-Fulton Method:
what makes the endospore difficult to stain?
their thick coat
Schaeffer-Fulton Method:
what is the primary stain? what allows the primary stain to permeate into the endospore?
The primary stain is malachite green.
The following allows the primary stain to permeate into the endospore’s thick cell wall:
- Using a concentrated stain
- Heating the cells
- Leaving the stain for a long time
Schaeffer-Fulton Method:
After the primary stain, what do we do next?
We rinse with water
Schaeffer-Fulton Method:
What happens when we rinse with water?
The vegetative cells lose the stain and become colorless (because it doesn’t have a thick cell wall coat).
The endospores retain the stain and stay green.
Schaeffer-Fulton Method:
what happens after we rinse with water?
We use a counterstain, safranin, for the vegetative cells
Schaeffer-Fulton Method:
is this a differential stain?
Yes
Schaeffer-Fulton Method:
what colors do the endospores and vegetative cells end up?
The endospores are green.
The vegetative cells are pink.
Schaeffer-Fulton Method:
summarize the 4 steps.
- Cells and endospores are transparent.
- Malachite green that makes the spores and cells green. Heat is used to force the stain into the spores if they are present.
- Decolorization with water removes the stain from the cells, but not the spores.
- Safranin is used to counterstain the cells pink.
what is an example of cells and spores that turn pink and green?
Bacillus subtilis endospores stain as green ovals, and Bacillus subtilis bacterial cells stain as red/pink rods.
what is the crystal-violet method?
it is a similar method to the negative staining technique
crystal-violet method:
what stain do you add to the culture?
crystal violet stain, which is a simple stain
crystal-violet method:
what color do the vegetative cells and endospores become?
The vegetative cells take up the stain and look purple. The endospores do not take up the stain and they remain clear against the purple background.
What is the special stain to observe flagella?
Leifson’s technique
T/F: Flagella are too thin to be resolved with the light microscope.
True
Are flagella fragile? What happens to them during smearing and heat fixing?
Yes, they are fragile. They break off during regular smearing and heat fixing.
Leifson’s technique
What type of slide do we make, and what is it?
We make a “rolling drop slide” which is when we place a drop of culture at one end of the slide and tilt it so that the drop rolls and spreads on the slide.
Leifson’s technique:
What are the steps?
- Rolling drop slide.
- Air dry only - No heat fixing.
- Stain - stain contains a mordant that clumps onto the flagella, making them appear thicker and yellowish-brown.
Leifson’s technique:
When staining Proteus vulgaris, how does it appear?
They have string-like flagella that stain a yellowish-brown color
what are capsules made of that prevents it to take up stains?
They are made of polysaccharides and do not take up the stain.
Capsule stain:
what two steps does it involve?
- Stain the background using a negative stain like India ink - the capsules appear like halos against a dark background.
- Stain the vegetative cells using a simple stain like safranin - the vegetative cells like red/pink
Capsule stain:
When staining Klebsiella pneumoniae, what do the bacterial capsules look like?
The bacterial capsules look clear compared to the dark background and the bacterial cell inside (negative staining)
what do we use acid-fast staining for?
used for Mycobacterium species that contain waxy lipids called mycolic acids in their cell wall, which prevent them from taking up water-based stains.
What are the steps for the Ziehl-Neelson method (acid-fast)?
- Apply phenol-based primary stain (carbolfuchsin) to heated cells.
- Apply an acid-alcohol decolorizer.
- Apply methylene blue counterstain.
Ziehl-Neelson method (acid-fast):
What do acid-fast and non acid-fast bacteria look like at the end?
Acid-fast: retain the primary stain and stain pink.
Non acid-fast: lose primary stain and stain blue.
Ziehl-Neelson method (acid-fast):
When we stain Mycobacterium smergmatis bacteria, what do they look like?
They stain as pink rods which tend to clump together. This is Acid-fast.
Ziehl-Neelson method (acid-fast):
When we stain Staphylococcus epidermis, what do they look like?
They stain as blue cocci in clusters. This is non-acid fast.
Ziehl-Neelson method (acid-fast):
Is it a differential stain?
Yes
what were the steps from the lab?
- We brought a beaker of water to a soft boil.
- We created a heat-fixed bacterial smear slide for both Bacillus subtilis and Bacillus megaterium.
- We put a small piece of paper towel on top of the smear and held it in place using two clothespins.
- We put that slide + paper towel on top of the beaker and saturated the slide + paper towel with malachite green.
- Whenever the paper towel would slightly dry out, we added more malachite green to saturate it once again.
- We kept this slide + paper towel on top of the beaker and left it there for 5 minutes, adding malachite green whenever it dried out.
- We removed the slide and paper towel after 5 minutes. We discarded the paper towel.
- We rinsed the slide thoroughly with water.
- We stain the smear for 30 seconds with safranin dye.
- We dumped the excess safranin dye and rinsed it with water again.
- We dried the slide with the bibulous paper and viewed it at 1000x with oil immersion.
- We did this with both bacterial slides.
