INTRODUCTION

The endospore-forming bacteria include Clostridium spp and Bacillus spp.

These bacteria naturally grow in soil, but they have great clinical implications by
causing human bacterial infections eg: Clostridium tetani causes
tetanus, Clostridium botulinum produces botulin toxin which causes
paralysis, Bacillus cereus causing food poisoning and Bacillus anthracis causes
anthrax in cattle and humans.
Spore production is a very important characteristic of some bacteria, allowing
them to resist adverse environmental conditions such as dessication, chemical
exposure, extreme heat, etc. Endospore walls are very resistant to penetration of
ordinary stains and identification of spores can be made using the endospore stain.

Activity 1: Watch the video in the given link below

https://youtu.be/UchEPAJh4cs

EQUIPMENT AND MATERIALS:

1. Water bath
2. Beaker
3. Glass slide
4. Paper towel
5. Malachite green
6. Safranin
7. Pipette
8. Tap water
9. Immersion oil
10. Microscope

PROCEDURE
 1. Set up a hot water bath at 40oC.
2. Air dry and heat fix a smear of bacteria on the glass slide.
3. Cover the smear with a small piece of paper towel.
4. Saturate the paper towel with Malachite Green as primary stain.
5. Put the glass slide on the beaker and steam the slide for 5 minutes with
boiling water as mordant.
6. Add additional stain as it boils off.
7. Allow the slide to cool, remove the paper towel piece and rinse with tap water
for 30 seconds as decolorization.
8. Counterstain with safranin for 20 seconds.
9. Rinse lightly to remove safranin.
10. Let the slide dry and view the slide in immersion oil under the microscope at
1000x magnicificant.
11. Spores stain green while vegetative cells stain red or pink. Spores can survive
difficult environmental conditions like extreme heat, dehydration, low nutrients,
radiation, etc.

RESULT INTERPRETATION

Spores
stain green

Vegetative cells
stain red or pink
PRINCIPLE:

The Malachite Green dye and Safranin are both alkaline with positively charged. The
nature of bacteria cytoplasm is basophilic create attraction of the dyes to be
absorbed by the bacteria cell.

Visualization of the cells under the microscope will show the appearance of pink-red
stain for the vegetative cell forms, which take up the counterstain while the
endospores will appear as green dotted particles (ellipses), due to the colour of
Malachite Green dye taken up by them.
a) Define mordant and why is the mordant used in this staining?
the use of steamed-heat which softens the endospore covering allowing penetration
of the dye into the spore. The malachite green dye binds to the spore mildly and if
washed with water, without fixing, it easily washes away, and that’s why the
application of steamed heat is important to allow the dye to penetrate the
endospore. Water is used as a decolorizing agent, to was away from the malachite
dye from vegetative forms.

b) Why the slide need to be counterstained with safranin?

pink-red stain for the vegetative cell forms, which take up the counterstain

c) Explain the microscopic observation of clostridium spp using this staining in

the figure below (explain in term of bacteria morphology)
Based on the observation of clostridum spp using spore staining, shape of
clostridium is rod or more to spherical shape. It has single flagella. It has develop
the terminal spore.

REFERENCES
1. https://www.asmscience.org
2. https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiolo
gy_Labs/Microbiology_Labs_II/Lab_07%3A_Endospore_Stain_and_Bacterial
_Motility
3. https://youtu.be/UchEPAJh4cs

4. https://iopscience.iop.org/article/10.1088/1742-
6596/812/1/012066/meta#:~:text=A%20method%20of%20endospores
%20staining,solution%20in%20spore%20bacterial%20stain.