Group number: 10A

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Title: Endospore staining
Introduction
The endospore staining is the technique used in microbiology to differentiate between
endospores and vegetative cell in a bacterial sample. An endospore is a dormant, tough and non-
reproductive structure produced by some bacteria in phylum Bacillota. Bacillus and a few other
bacteria genera produce endospores. Bacteria may live in adverse environments by generating
endospores. Endospores are heat, desiccation, chemical, and radiation resistant. Bacteria can
produce endospores within 6 to 8 hours after being exposed to harmful circumstances such high
temperature, freezing or dryness hence bacteria grow best at 37◦C. Endospores are dehydrated
and biologically inactive. They have the potential to last for thousands of years. When exposed
to appropriate conditions, spores can germinate into a vegetative cell in as little as 90 minutes
(Hussey et al, 2007).
germination of endospores occurs as follows. The earliest event after the finishing part of
vegetative cell growth and division is asymmetric septation, a membrane forms towards the end
of the vegetative cell to create two compartments of unequal size. The next stage is engulfment,
during which the larger compartment grows around the smaller one. This small compartment will
later become the central protoplast of the spore, but at this stage it is separated from the
environment by three membranes, the middle one of which is topological and functional of the
front endosespore. The vegetative cell then usually lyses and releases the fully formed
endospore. The heat resistance of the developing spore increases most dramatically during the
later stages of dehydration of the protoplast and the synthesis of the cortex around it (Abel-
Santos 2015).
According to Schaeffer et al (2013) endospores can arise in a variety of locations within the
vegetative cell. They come in three varieties: central, subterminal, and terminal. Endospores in
the centre are called central endospores. The centre of the endospore exists in dehydrated state
and houses the cell’s DNA, ribosomes and large amount of dipicolinic acid. This endospore
specific chemical can comprise 10% of the spore’s dry weight and appears to play a role in
maintaining spore dormancy. Small acid soluble proteins are also found in endospores, these
proteins tightly bind and condense the DNA and are in part responsible for resistance to ultra
violet light and DNA damaging chemicals. To increase penetration of the impermeable spore
cores, an aqueous primary stain (Malachite Green) is applied and steamed. Endospores are not
easily penetrated by stains because of their great resistance as a result steaming the stain is
required.
The Schaeffer-Fulton method is a technique designed to isolate endospores by staining any
present endospore green and any other bacterial bodies red, the technique employs Malachite
green as the principal stain and safranin as counterstain. The endospore is resistant to
decolorization once it has absorbed the stain, whereas the vegetative cell is quickly decolorized
with water leaving the vegetative cells colorless. Finally, to improve visibility, the vegetative
cells are counterstained with Safranin. The endospores look green under a microscope, whereas
the vegetative cells appear red or pink (Hyndman,2018)
Aim
The aim of the experiment was to differentiate endospores from vegetative cells.
Methods and Materials
The benches were sterilized with 70% ethanol because all microorganisms are treated as
potential pathogens to avoid any infections. The Bunsen burner was switched on. Everyone was
required to wear protective clothing (i.e. Gloves and a lab coat) to protect against skin absorption
of the chemicals (malachite green and safranin) and chemical burn as well as protection against
the culture of Bacillus subtilis (which was treated as pathogenic). The inoculating loop was
sterilized by placing it in the Bunsen burner flame until it was red. The inoculating loop was then
allowed to cool down by placing it in agar. The B. subtilis culture was touched with the cooled
loop and then the loop was transferred to a drop of water on a slide to create a smear. The smear
was heat fixed on the slide by passing it through the Bunsen burner a few times to ensure that the
slide was not overheated (overheating would break the slide or kill the organism). A piece of
paper towel was placed on top of the heat fixed smear of B. subtilis culture and the slide placed
on a boiling water bath. The slide was then flooded with malachite green and allowed to stain
over the steam for 8 minutes, more malachite green was added frequently to make sure the stain
does not dry out. After 8 minutes the slide was removed and rinsed gently with tap water and
allowed to air dry. The slide was counterstained with safranin for one minute, then gently rinsed
with water. The slide was allowed to air dry before immersion oil was applied to it. Thereafter,
the slide was examined under a light microscope at 100× magnification.
Discussion
The Endospore staining technique is a differential type of stain since it differentiates between
spore forming cells and non-spore forming cells, as well as the spore part of the cell from the
vegetative part of a cell. A cell will have a green-coloured circular spot when an endospore is
formed in the cell. Endospores can also be seen outside the cell. A vegetative cell will be pink or
red in colour.
As shown in figure 1, the stained B. subtilis culture was observed under a light microscope at
100× magnification, to be a rod-like red cell with a circular green structure in it. The circular
green structures were also found outside the rod-like cells. The endospores outside the vegetative
cell are called free endospores, they are usually lysed and released when are fully formed. This
means that B. subtilis is an endospore forming bacteria. According to Aryal (2018), malachite
green is water soluble and has a low affinity for cellular material, so vegetative cells may be
decolourized with water, safranin is then applied to counterstain any cells which have been
decolorized. At the end of the staining process, vegetative cells will be pink, and endospores will
be dark green.
This would then explain the colours of the stained B. subtilis culture. The endospore retains the
colour of the first dye (malachite green) because the stain cannot be washed off easily when it
has been absorbed by the spore wall. Their coats are very tough and they are resistant to staining,
which is why the primary stain in the procedure is driven into the cells using heat. Once the dye
has been absorbed, the endospores retain the dye and their wall will be resistant to de-staining.
The vegetative part of the cell, on the other hand, retains the colour of the last dye (safranin).
This is because the initial stain is washed off easily because of its properties (it is water
References
Abel-Santos, E. (2015). Endospores, sporulation and germination in Molecular medical
microbiology 
Aryal, S. (2018). Endospore Staining - Principle, Reagents, Procedure and Results.
Hussey M.A. and Zayaitz A. (2007). Endospore stain protocol. American Society of
Microbiology.
Hyndman, R.J. and Athanasopoulos, G. (2018). Forecasting: principles and practice of
endospore staining
Schaeffer, A.B. and Fulton, M.D., 2013. A simplified method of staining
endospores. Science, 77(1990), pp.194-194.