Endospores. Endospores are dormant structures that surround the DNA and allow resistance to heat,
chemicals, and drying. Endospores are composed of exosporium, a protein covering, spore coat,
layers of protein, cortex, peptidoglycan and core or spore protoplast consisting of
the core wall cytoplasm, nucleoid, and ribosomes. Shown here is a bacterial endospore.
Notice the spore coat, cortex, exosporium, core wall, and DNA.
Here is an electron micrograph of an endospore inside of the sporangium.
Endospore formation - sporulation. Nutrient depletion is a major stimulus for endospore
formation. Once this happens the cell is committed to forming an endospore. The cell
is now called a sporulating cell or sporangium. Endospore formation takes about 6 to 10 hours.
Steps in endospore formation. Number one - the DNA is duplicated and becomes more dense.
Number two - the cell is separated into a sporangium and a forespore. This will become
the endospore. Number three - the sporangium engulfs the forespore and begins to produce
spore layers around the forespore. Number four - the cortex and outer coat layers are developed.
And number five - the mature spore is completely formed. Here we see
a vegetative cell forming an endospore and a complete endospore with remaining sporangium.
Heat resistance. The heat resistance of the endospore is due to these factors: the presence
of dipolic acid that combines with calcium ions. This combination helps dehydrate the endospore.
The core contains small proteins that help protect the DNA from damage and drying. Temperatures of at
least 121 degrees celsius are necessary to destroy an endospore. This temperature can be achieved in
the autoclave. Endospores are also resistant to drying, ultraviolet light, and strong acids
and bases. Endospores must be destroyed before materials are sterile. Germination. To break the
dormancy of the endospore conditions for good growth of the cell are necessary. There has to
be a specific chemical or environmental stimulus along with the presence of water. The stimulus for
germination varies among different species but it is typically an amino acid or an inorganic salt.
During germination enzymes are produced that degrade the cortex and expose the core to water.
Calcium dipicolonate is lost during this process. The core rehydrates and the cell
begins to grow out of the endospore coats. The entire process takes about one and a half hours.
Endospore stain. Because of its high resistance to chemicals the endospore
does not stain using routine staining procedures. In a regular stain endospores appear colorless.
A special staining technique is required to stain the endospore. The two medically significant
endospore formers are clostridium species and bacillus species. The endospore stain allows
you to view the endospore to determine its size, shape, and location inside of the cell.
Let's watch the spore stain procedure: the first step in the endospore stain is to make the smear,
air dry, and heat fix the slide. You place the slide over steaming water
and then you put malachite green on the smear right over where your
smear is on the slide. Leave the malachite green on the slide to steam for one minute.
The malachite green will steam into the endospores and stain them a blue-green color.
After one minute you will take the slide and rinse it with distilled water.
And then continue the stain by using safranin as a counter stain.
This the safranin will stain the vegetative cells red and the endospores will
remain a blue green color. Leave the saffronin on for 30 seconds...
and pour it off.
Rinse, then you will clean your dryer slide to use under the microscope.
The slide is now ready to view under the microscope. Shown here are the endospores.
This concludes our activity: Endospores.