1. Faculty of Medicine and Health Sciences
Microbiology Lab
Experiment 7
Endospore Staining
First Semester

2. Introductions
Some genera of bacteria have the capability of forming endospores (spores) under adverse chemical or physical conditions.
In such conditions, the bacteria can not find all the necessary chemical and physical requirements needed for survival.
Examples of adverse conditions that induce endospore formation by spore-forming bacteria:
Decreased water level and dehydration
Deceased Oxygen level
Decreased nutrients
Inappropriate temperature and pH)
Unless other wise, these bacterial species form endospores, they will die under these adverse Conditions.
Accordingly, the main goal of endospore formation by spore forming bacteria is SURVIVAL.
The is unlike fungal spores, which are formed for the propose of reproduction .

3. Although several genera of bacteria produce endospores under adverse conditions, spore-forming bacteria of medical importance belong to two genera. These are:
The genus Bacillus
The genus Clostridium
The genus Bacillus has two medically important species that cause disease in humans. These are:
Bacillus anthracis
Bacillus subtilis
The genus Clostridia has four medically important species that cause disease in humans. These are:
Clostridium tetani
Clostridium botulinum
Clostridium difficle
Clostridium perferingens

5. Notes:
Bacteria endospores are called (endospores), because they are formed within bacterial cell.
The vegetative bacterial cell that forms the endospore under adverse conditions is called the mother cell. Once the endospore is formed, the mother cell die.
In some cases, the formed endospore remains within the dead bacterial cell. However, the mother cell may completely disintegrate and the formed endospore is released into the surrounding environment.
Definition:
Vegetative bacterial cell: it is the bacterial cell that is active in terms of metabolic activities and replication

6. Bacterial endospores are formed under adverse environmental conditions.
Accordingly, bacterial endospores are expected to be highly resistant to these adverse environmental conditions such as:\
Extreme temperatures (high and low)
Dehydration
Chemicals ( antibiotic and disinfectants)
Radiations
Definitions:
Bacterial Endospore ( or spore): can be defined as a highly resistant, viable but dormant bacterial cells.
Viable: means not dead (alive)
Dormant : means have their metabolic processes and well as replication completely
stopped.

7. Bacteria can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions

8. Once the adverse environmental condition (s) that have led to the formation of endospore is/are over, and the surrounding environmental conditions of the surrounding environment and start disintegrating.
A vegetative bacterial cell is then emerge from the disintegrating endospore.
The process of endospore formation is called Sporulation ( occurs under adverse environmental conditions)
The reverse condition in which the endospore disintegrate sand a vegetative bacterial cell emerges, is called Germination ( occurs once the adverse environmental conditions are over and becomes suitable again for bacterial survival
When endospores are exposed to favorable conditions, they germinate into a vegetative (metabolically active) cells within 90 minutes.

9. The importance endospores in clinical practice: Bacterial Diagnosis:
The detection of bacterial spores /endospores in bacterial cultures of clinical specimens obtained from infected patients implies that the grown bacterium either belong to the genus Bacillus or Clostridium.
This is because of the fact that certain species that belong to these two genera are the only ones that cause disease in humans and are spore-forming. .
Moreover, endospores of different endospore-forming bacteria can be observed to locate in different positions within the mother cell. This can help in bacterial diagnosis
The endospore location within the mother cell can be:
Central
Sub-terminal
Terminal. 
Central endospores are positioned in the center of the mother cell such as in the case of Bacillus anthracis
Sub-terminal endospores are positioned between the center and the end of the mother cell such as in the case of Bacillus subtilis
Terminal endospores are positioned at the end of the mother cell such as in the case of Clostridium tetani

11. The practical part of this lab…………………..
Endospore Staining:
Principle:
It is differential stain used to visualize bacterial endospores/spores. The main goal of endospore staining is a diagnostic purpose
Because bacterial endospores have a very tough protein coat that is made of that is made of a keratin-like protein, bacterial endospores are highly resistant to normal staining procedures, under normal staining conditions, endospore coat is highly impermeable.
The primary stain in the endospore stain procedure is malachite green, which is driven into the bacterial endospore with the aid of heating.
Unlike basic stains, malachite green is water-soluble non-ionic stain that can be easily removed from the bacterial cytoplasm by water. However, once this stain is forced to enter the endospore with the aid of heating, it is possible after this to remove the stain out of the endospore.
Accordingly, during endospore staining, water is not only used to wash off excess stain, but also function as a decolorizer that can decolorize the bacterial cytoplasm but the bacterial endospore.

14. Endospore Staining Procedure:
Note: Use an old culture for endospore-staining. Spores are typically formed when an organism is enduring stressful conditions. If you wait until the culture is old then it is likely that some of the organisms will be experiencing stressful conditions such as lack of nutrients
After taking all safety precautions steps and cleaning the bench
Perform a bacterial smear of Bacillus subtilis.
Perform heat fixation as you learned earlier
Place the slide above boiling water
Place a small piece of filter paper over the smear and quickly saturate the paper with Malachite Green.
Expose the slide to the steam the boiling water for min. Do not allow the stain to dry out. Every time you see that the Malachite Green saturated filter paper covering the smear is about to dry, add more of the stain
Depolarization step: remove the filter paper off the slide using forceps , and wash the slide gently with water. (dispose of the used filter paper in the trash)
Counterstain with Safranin for 2 minutes and then wash the slide gently with water
Carefully blot the slide dry
Examine the slide under the microscope, using proper microscope technique.

15. Expected Results:
In the smear of non-spore-forming bacterium, you will see only red bacterial cells. Nothing will be stained green, since it is a non-spore-forming bacterium.
In the smear of the endospore-forming bacterium, the bacterial cells will show green endospores within the mother cells surrounded by a red-stained cytoplasm (the color of the counter stain, Safranin).