PRACTICAL 4

TITLE: The Staining of Microorganisms - Capsule Staining and Spore Staining.

OBJECTIVE
Explain the rationate and procedure for Capsule Staining and Spore Staining.

INTRODUCTION
It is considered to be a part of the bacterial cell's outer envelope because the cell capsule is a
polysaccharide layer that is not inside the cell envelope. It is a well-organized layer that cannot
be simply wiped away and might be the source of different ailments. The capsule, which is
present in both Gram-negative and Gram-positive bacteria, should not be mistaken with the
second lipid membrane, which is found only in Gram-negative bacteria and includes
lipopolysaccharides and lipoproteins.
Because most capsules are so tightly packed, staining them with standard stains is
challenging because most stains cannot adhere to the capsule. The bacteria and their
surroundings are stained darker for observation under the microscope than the capsule, which
does not stain. Bacterial cells and the surface they are on are stained black when observed,
however the capsule is pale or colorless and appears as a ring around the cell. Bacterial spores
are extremely resistant, dormant structures that grow in response to adversity in the environment.
They aid in the survival of organisms in harsh environmental circumstances. They play no part in
reproduction. When resources, such as carbon and nitrogen sources, are low, sporulation occurs.
Bacterial spores are extremely heat, dehydration, radiation, and chemical resistant. The form and
arrangement of spores vary between species and can help with classification and identification.
Endospores can be spherical or elliptical and can be found in the center of the bacterium
(central), at the end of the bacterium (terminal), or towards the end of the bacteria (subterminal).

HYPOTHESIS
Klebsiella sp. is a bacteria with a capsule and can be identified using indian ink. Bacillus subtilis
is a bacteria that releases spores and can be identified using malachite green.
METHODOLOGY
A. Capsule Staining
1. Several loopful of the klebsiella sp. culture was placed onto a clean glass slide
very near the edge.
2. An equal amount of safranin near the bacterial culture was placed on the slide.
3. By using a loop, it was mixed well and then spread along the slide by using blood
smear technique.
4. The smear was allowed to dry.
5. The smear was then covered with safranin for 10 seconds, rinsed carefully and
blot dry with a paper towel.
6. The smear was observed by using a low and high objective lens.
7. The result was recorded.

B. Spore Staining
1. Several loopful of the Bacillus subtilis culture were placed onto a clean glass slide
near the edge and a smear of the culture was prepared using the blood smear
technique.
2. The smear was then covered with malachite green, then heated with steam for 5
minutes.
3. The slide was then allowed to cool down first.
4. It was then rinsed carefully under slowly running tap water.
5. After that, the slide was counter-stained with safranin for 1 minute, then rinsed
carefully under slowly running tap water again.
6. With a paper towel, it got blotted dry.
7. By using low, high and oil-immersion objective lenses, observations were done.
8. The observation was recorded.
 DATA AND ANALYSIS

Klebsiella sp. after being stained with safranin. 10x magnification (left) and 40x magnification (right)

Bacillus subtilis after being stained with malachite green

DISCUSSION

Based on the results of the experiment for capsule staining, it shows that the staining process of
Klebsiella sp. was successful. The bacteria and the background were stained darker than the
capsule because the capsule does not get stained. We used safranin to stain the bacteria and
background in this experiment. The color of the background appeared to be light pinkish while
the Klebsiella sp. appeared to be red coloured. It appears that the culture was focused on one spot
like most culture bacteria do.
For the spore staining experiment, the spore staining process of Bacillus subtilis was also
successful. We stain the Bacillus subtilis with malachite green. The spore can be seen with green
color while the vegetative cell was stained to red pinkish color. The shape and size of the cells
can easily be observed. The shape of the spores can also be seen easily to confirm that it is
indeed Bacillus subtilis release spores.

CONCLUSION

In conclusion, the experiments were successful and all the results show the exact outcome
expected. The primary application of capsule stain is to differentiate capsular material from
bacterial cells. For spore staining, it was mostly used to detect and identify the presence of
bacterial endospores and vegetative forms in a cell. Hypothesis accepted.
QUESTION
1. What is the importance of the extracellular slime in the food industry?
The importance of the extracellular slime in the food industry is it promotes cell
adherence in the film and shields the bacterial layer from cleansers and sanitizers.

2. What role do capsules play in the infectivity towards organisms?

Host Protection. The major purpose of capsules in pathogenic bacteria is to protect the
bacterial surface from interactions with host immune system components, preventing
either opsonophagocytosis or, in Gram-negative bacteria, complement mediated lysis.

3. What is the difference between spore and endospore?

The primary distinction between spore and endospore is that the former is an active
reproductive structure generated mostly by plants and fungi, whilst the latter is a
dormant, non-reproductive structure produced by bacteria.

REFERENCE

1. 18: Spore stain. (2021, August 1). Biology LibreTexts.

https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs
/Microbiology_Labs_I/18%3A_Spore_Stain
2. Tankeshwar, A. (2022, May 4). Capsule stain: Principle, procedure, results. Microbe
Online. https://microbeonline.com/capsule-stain-principle-procedure-results/