Endospore Stain
Definition, Techniques, Procedures and Significance
Endospores Definition
A vegetative cell (bacteria) is a normal bacterium
that grows and multiplies in favorable environmental conditions. Here, favorable
conditions simply mean that there is sufficient nutrients, optimal temperature
range and moisture etc that support the growth and multiplication of the cells
allowing them to thrive.
As a result of poor/extreme environmental
conditions, vegetative cells undergo a process referred to as sporulation to
form endospores.
Once such bacteria as Bacillus spp and
Clostridium spp find themselves in unfavorable conditions, sporulation begins
with the cells packing everything they need to survive through these extreme
conditions.
Unlike the vegetative cells, endospores are dormant (inactive) and
highly resistant to such extreme conditions as very high temperatures, chemical
changes, enzymatic actions and desiccation among others. This allows the endospores
to protect and preserve the genetic material of the cell and allow it to thrive
once the cell finds itself in favorable conditions again.
* Sporulation is the process through which
endospores are formed. The entire process takes 8 to 10 hours. Whereas
endospores reside inside the original cell, free spores (spores) exist outside
the cell on their own.
- Bacteria chromosome is
replicated
- Plasma membrane separates
the replicated chromosomes to form a forespore
- A second membrane composed
of calcium and dipicolinic acid encloses the forespore
- An external spore coat
encloses the endospore
- The endospore is released
as the vegetative cell it was formed from dies
* Endospores are likely to be found in soil and
aquatic environments.
More on Chromosomes.
Image of an Endospore Diagram
Endospore Stain
Essentially, endospore stain is a differential
stain. As such, it allows for the differentiation of structures and thus the
characterization of a cell based on its physical and chemical nature. In this
case, endospore stain as a differential staining technique is largely used for
the purposes of distinguishing between vegetative cells and endospores.
Endospore Staining Technique/Procedure
For endoscopes, there are two major staining
techniques used. These include:
- Schaeffer-Fulton staining
technique
- Dorner's Method staining
technique
Schaeffer-Dulton Technique
This is the most common technique for endospore staining.
Requirements
- Malachite green 0.5% (this is the primary stain) - It can simply be prepared using 0.5 grams of malachite green with 100 ml of water
- Tap/distilled water (decolorizing agent)
- Safranin (2.5%) - This is the counter stain and can be easily prepared using 2.5 grams of safranin O and 100ml of 95% ethanol
Staining Procedure
- Smear the sample at the
center of a clean microscope slide
- Allow the slide to dry (air
dry) and then heat fix the smear
- Place a blotting paper on
the slide (cut to fit the slide) and saturate it with malachite green stain
solution
- Gently heat the slide until
it starts evaporating (make sure the slide is placed on a staining rack when
heating) - heating can either be done using a Bunsen burner of boiling water.
- Keep heating, removing and
re-heating the slide for between 3 to 5 minutes while adding a few drops of
malachite green to keep the blotting paper moist. However, avoid overheating.
This process is simply aimed at steaming the slide.
- Carefully remove the slide
from the rack and allow it to cool for about a minute or two
- Thoroughly wash the slide
using tap water or distilled water
- Counterstain the slide with
safranin for about a minute
- Rinse the slide and allow
it to dry (air dry)
- Observe under the
microscope
Steps in the Procedure
Unlike the vegetative cells, endospores possess
a permeability barrier. This prevents dyes from entering to stain the
structures of the cell. For this reason, the barrier has to be destroyed, which
is why heat is used. Through heat fixing, the cortex of the endospore is
penetrated allowing for the dye to interact with the petodoglycan and thus
produce desired effects. In this case, heat acts as a mordant.
* a mordant is a substance that is used alongside
the stain/dye to fix it in a given material
After heat fixing, the slide is washed using tap
water or distilled water. Here, water is used as a decolorizer. Because
malachite green binds relatively weakly, it can be washed off easily. However, it cannot be washed off easily once it is locked in the spore wall.
Once they take in the dye, endospores retain the dye and will be
resistant to de-staining. However, vegetative cells will easily lose the stain
when washed with water because they lack the spore wall. After the initial
washing, a counter stain (safranin) is used. The purpose of the counter stain
is to stain the vegetative cells that lost the primary stain.
Here, it is worth
noting that the primary stain and the secondary stain are of different colors.
As such, they allow the technician to differentiate different types of cells
under the microscope. Whereas the counterstain (safranin) is pink/reddish in
color, the primary stain (malachite green) is green in color. Therefore,
endospores will appear green in color while the vegetative cells will
pink/reddish in color under the microscope.
Image of an Endospore under the Microscope
Dorner's Method
Requirements
- Carbolfuchsin stain
- A decolorizing agent (acid-alcohol)
- A counterstain (nigrosin
solution)
Procedure
- Make a smear on a clean
glass slide
- Allow the slide to dry (air
dry) and then heat fix
- Place a blotting paper on
the slide (covering the smear) and saturate with carbolfuschin to steam (for
about 5 minutes) - This should be repeated while adding drops of carbolfuchsin
and avoiding overheating (simply heat to steam)
- Remove the blotting paper
and allow the slide to dry for about a minute
- Wash the slide with
acid-alcohol for about a minute to decolorize and then rinse with tap water
- Add a drop of nigrosine on
the smear to form a thin film
- Allow the slide to dry
- Observe under the
microscope using oil immersion
* While different stains are used in this
technique, the purpose of each step taken here is similar to the one described
above.
When viewed under the microscope, endospores
will be red in colour.
Significance of Endospore Stain
In biology, endospore staining is used for the
purposes of differentiating and classifying bacteria. On the other hand, it is
also very important in medicine and the food industry.
Because they are tough
and hard to destroy it is very important to determine whether they are present
in canned food and thus avoid food poisoning to protect consumers.
Related and Interesting articles:
Cell Staining in Microscopy
Gram Staining - Purpose, Procedure and Preparation
Capsule Stain - Definitions, Methods and Procedures
Learn more about Cell Culture, Cell Division, Cell Differentiation and Cell Staining.
Return from Endospore Stain to MicroscopeMaster Home
References
Jackie Reynolds, Richland College, BIOL 2421. Fall 2011,
Marise A. Hussey, Anne Zayaitz. 2007. Endospore stain protocol.
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