Spore and endospore staining

Bacterial Spores (endospore)

Endospore is dormant stage of some bacterium that allows it to survive under unfavorable
conditions that would normally be lethal such as extreme drought or heat.

- The Vegetative Cell Gives Rise to One Spore.

Bacterial spore
cell Bacterial cell

- The endospore is able to survive for long periods of time until environmental conditions again
become favorable for growth. The endospore then germinates, producing a single vegetative
bacterium.
 Spore formation (sporulation)occurs when nutrients, such as sources of carbon a
nd nitrogen are depleted.
Spores are resistant to heat, dehydration, radiation and chemicals.

Structurally and chemically more complex than the vegetative cell.

The shape and position of spores vary in different species and can be useful for cl
assification and identification purposes.
Endospore formation is not a reproductive process but a process of differention th
at provides the bacteria with a mechanism for survival.
 The shape of the spore is an identifying characteristic

 Swelled vs. Not swelled

spore spore

Bacterial cell
The location of the spore is also an identifying characteristic
 Central, Sub-Terminal, and Terminal spores
 Structure of endospore

1- The core:
innermost area of the spore is the spore protoplast or spore core , consists of a single
copy of chromosome, ribosomes, tRNAs, proteins and enzymes .

2- Germ cell membrane:

the layer immediately surrounding the core (plasma membrane)
Germ cell wall:
- made of peptidoglycan
- provides osmotic stability to the spore protoplast.

3- Spore cortex:
- thick concentric structure outside the cell wall, cortex appears to play a key role in an
energy dependent dehydration of spore during sporulation
4- Spore coat:
- A keratin like thick protein layer situated outside the cortex

5- Exosporium:
- an additional loose layer surrounding the cortex in some bacteria
- consists of lipids and glycoproteins.
Not all bacterial species can form spores

- A few genera of bacteria produce endospore such as Clostridium (gangrene) and

Bacillus (anthrax), both of them are gram + rods.
- Endospore production is associated with Gram Positive bacteria.

- Since not all bacteria form endospores, we can use this as an identification factor.
Some spore forming bacteria are capable of causing disease

• Clostridium botulinum – botulism .

• Clostridium perfingens – gas gangrene
• Clostridium tetani – tetanus
• Bacillus anthracis – anthrax
 Endospores staining

 The Schaeffer-Fulton Stain Procedure is used to differentiate between endospores and vegetative
cells.(Differential staining technique).

 The procedure was designed by Alice B. Schaeffer and MacDoland Fulton, during the 1930s.

 As a spore forms inside of the vagetative cell, the spore wall chemically changes and thicken.
This sporulation process changes the spores stainability, making it increasingly resistant to the
staining dyes, and so gimmick steaming enhances the primary dyes penetration.
 Schaeffer-Fulton Stain Procedure

.Make a smear. Air Dry. Heat fix .1

2. Flood the smear with Malachite Green stain.
3. Cover the flooded smear with a square of filter paper.
Steam slide for 10 minutes (every minute, add a few more drops of .4
.Malachite Green stain)
.Allow slide to cool (after the 10 min. steam process) .5
6. Drain slide and rinse for 30 seconds with DI
water (discard filter paper).
7. Put slide on steam rack.
8. Flood smear with Safranin (counter stain).
This stains the vegetative cell. (Leave for 1
minute)
9. Drain the slide and rinse with DI water.
10. Blot Dry.
11. Use oil immersion objective to view.
Endospores staining under microscope
Thank you