Group members :

Nkosiyethu Diya 223035689

Onke Khiwa 202206767

Simnikiwe Mlakalaka 202220369

Mabinza Asanda Thembisa 202246831

Tlotitso Mphomela 202101291

Sinobom Ntshentshe 203041391

Anathi Onke Nyikiza 202004878

Group number: 2 (session A)

Course code: MIC 211

Title of the practical: Endospore Staining

Date of the practical: 20 March 2024

Title:

Endospore Staining

Introduction:

The microscopic organisms is a living organism that was the foremost various and broad
compared to others. Microscopic organisms are unicellular life forms, prokaryotic, and tiny.
Microscopic organisms moreover have a chromosome, ribosomes, and a few other species
have a granular nourishment, gas vacuoles, and magnestom. A few bacteria can make
themselves into endospores which empower them to outlive in extraordinary situations
(Zubaidah and Elok 2006). Bacterial spores are bacterial component that's intentioned set in
an endeavor to secure themselves to the unfavorable impacts of the external environment.
Bacterial spores have the same usefulness as One-celled critter blisters since the microscopic
organisms within the shape of spores and sores of Single adaptable cell within the frame of a
moment stage in which the microorganisms that changes shape to secure themselves against
unfavorable outside variables eksospora(Waluyo and General L 2004).

In 1922, Dorner published a method for staining endospores. It employed a lengthy heating
step but resulted in differential staining of endospores and vegetative cells in the same
sample. Endospores and free spores appeared green or blue in contrast to red dye taken up by
the vegetative cells. Shaeffer and Fulton modified Dorner’s method in 1933 to make the
process faster, but heating with a Bunsen burner was still messy. As researchers identified
what was required to make spores stainable, additional modifications to the Dorner and
Shaeffer-Fulton methods were made to make staining and viewing spores occur quickly,
easily, with less mess, and with sharp contrast( Dorner W,1926).

A normal bacterial cell is known as a vegetative form of a bacterial cell. It can multiply freely
when provided with conducive environmental conditions like the provision of nutrients,
temperature, water, and oxygen. But some groups of bacteria, when exposed to unfavorable
conditions such us, lack of nutrients, an insufficient supply of oxygen or CO2, lack of water
and moisture, they can form a protective covering to protect themselves when they are
exposed to harsh environmental conditions adapting comfortably to these conditions. This
special structure is known as an endospore. (Oktari A et al., 2017).
An endospore is non-vegetative structure produced by a group of bacteria belonging to the
Firmicute family. They have special characteristics that stabilize them to survive in adverse
conditions for long periods of time. These include; a phase of dormancy, a tough outer
covering, and they are non-reproductive forms called spores, which offer great resistance to
high temperature, radiations, and chemicals like disinfectants and acids. This enables them to
survive thus they are difficult to stain with basic dyes and hence a special stain is applied that
uses a special dye, along with heat-steam. This staining technique is known as the Endospore
stain, also known as the spore stain. It is used majorly to detect and identify the presence of a
bacterial endospore and bacterial vegetative forms in a cell. Examples of these 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.Endospore staining techniques are classified based on the types
of reagents used by Schaeffer Fulton Stain- used Malachite Green dye and safranin and
Dorner method of endospore staining –uses Carbolfuchsin stain, acid alcohol, and Nigrosin
solution),(Marisse A. H., Anna Z., 2012.)

Aim:

The aim of this experiment was to differentiate between bacterial cells that are capable of
forming endospores and those that are not

Methods and Materials:

The inoculation loop was sterilized by placing it in the flame. The loop was cooled down by
inserting it in the agar. The culture was touched with the cool loop and the loop was then
transferred to a drop of water on a slide. The cells onto the slide were heat fixed by passing it
through the flame few times, in precaution that the slide was not overheated. A piece of a
filter paper was cut or paper towel of a size of the slide. The slide was covered with the filter
paper, and it was then placed over the boiling water bath. The slide was flooded with
Malachite green, and it was allowed to stain over the steam for 20 minutes. In precaution that
the slide does not dry out, if necessary, more stain was added. After 20 minutes the slide was
removed, and the stain was gently rinsed with tap water. The slide was allowed to air dry and
counter stain with Safranin for one minute. The Safranin was rinsed off and the slide was
allowed to air dry. The slide was the examined under 100x objective. The observations were
recorded by making the labelled drawing.

Results:

Attached

Discussion:

From the observations obtained under the light microscope using 100x magnification with the
oil immersion staining bacteria Bacillus subtilis spores, it can be concluded that all the
colouring treatment effected the bacterial spores staining results.

As we know that the warming time affect the dye to penetrate the walls of the bacterial
spores.This could be seen as the endospores appeared green while the vegetative cells had a
red stain. When we were using the light microspore under 100x magnification, it was
observed that the attached endospores and vegetative cells had a rod shaped even the isolated
endospores and vegetative cells all had a rod shape.

In an experiment that was done by Oktari 1, Y Supriatin1, M Kamal1and H Syafrullah they

observed that recolouring of bacterial spores is recoloring utilizing Malachite Green
arrangement of 5% and 0.5% Safranin, which comes about within the colouring will show up
green on the spores, as well as ruddy within the vegetative cells. At Schaeffer Fulton strategy
that's broadly utilized in portray endospores, endospore to begin with stained with Malachite
Green by a warming prepare this arrangement could be a powerful dye to enter endospores.
After treatment Malachite Green, the cell culture is washed with water and after that secured
with paint safranin, this procedure will deliver endospores and green in colour pink in
vegetative cells (Sumarsih S 2004).
In endospores recoloring strategies Schaffer Fulton, that utilized an antacid arrangement
(Malachite Green and Safranin). Colour Malachite Green and Safranin can work well in
microscopic organisms since of the soluble(chromophore component emphatically charged),
whereas the bacterial cytoplasm is basophilic so there was the fascination between the
components chromophore in a colour with bacterial cells, it causes microbes can assimilate
colours well(Ruth M, I 2009 P).

In this think about, analysts utilized a color that's damp is Methylene Blue as an elective to
colour Malachite Green. Analysts shifting the arrangement of Methylene Blue with a
concentration of 0.5%,0.7% and 1%, at that point shifting the pH of a arrangement of
Methylene Blue, and longer warming time at 3-5minutes. Based on the comes about of
research and observation and after that analyzed with a measurable calculation Two-Way
ANOVA test done on the variety of Methylene Blue concentration (0.5%,0.7%, 1%), the pH
variety 10, 11, 12, and the variety of time warming 3 minutes, 4 minutes and 5minutes,
appeared a factually critical distinction, where F number > F table. Since of the noteworthy
contrasts in recoloring comes about are at that point taken after a advance test utilizing LSD
test. After encourage test (LSD), the information gotten from each treatment on a adjusted
recoloring bacterial spores Bacillus subtilis is there is a distinction in each treatment.

In Bacillus subtilis bacterial spores recoloring with a assortment of utilized allow diverse
comes about. At a concentration of 0.5% , the same colouring treatment comes about with the
standard is within the variety of pH 10 with variety of time (5 minutes), varieties in pH 12
with variety of time (3 minutes, 4 minutes and 5 minutes). Whereas the comes about of
diverse medications with standard recoloring is within the variety of pH 11 with variety of
time (3 minutes, 4 minutes and 5 minutes). At a concentration of 0.7% results rise to
treatment with standard recoloring is within the variety of pH 10 with variety of time (4
minutes and 5 minutes), and varieties in pH 11 with a variety of the time (3 minutes and 5
minutes). While the comes about of treatment of distinctive recoloring with the standard is
within the variety of pH 10 with a variety of the time (3 minutes), varieties in pH 11 with
variety of time (4 minutes),and at different pH 12 with a variety of the time (3 minutes, 4
minutes and 5 minutes).At a concentration of 1% comes about the same treatment recoloring
with the standard is within the variety of pH 10 with varieties (4 minutes, 5 minutes), and
varieties in pH 11 with variety of time (4 minutes and 5 minutes). Whereas the comes about
of treatment of diverse recoloring with the standard is within the variety of pH 10 with a
variety of the time (3 minutes), varieties in pH 11 with a variety of the time (3 minutes), and
varieties in pH 12 with a variety of the time (3 minutes, 4 minutes and 5 minutes).From
perceptions gotten within the alteration recoloring microbes Bacillus subtilis spores, it can be
concluded that all the colouring treatment impact on bacterial spores recoloring comes about.

As we know that the warming time greatly affect the colour dividers of bacterial spores
(Albert and Bacillus 2008), this could be seen within the comes about of recolouring with
different concentration (0.5%, 0.7% and 1%) at pH 10, demonstrates that the not long warm-
up time 3 minutes and 4 minutes, bacterial spores are not recoloured, whereas within the
longer warming time is 5 minutes bacterial spores recoloured. Usually caused since the
warming time is longer make the pores of the spore divider is open so as to encourage the
colour to urge into the bacterial spores. Although their materials were more advanced
compared to ours we still mananged to get similar results. We can conclude that our
experiment was succesful.
References:

1.Albert ,Bacillus, 2009.Cellular and Molecular Biology (USA : Caisater Academic)

2.Dorner, W. 1926. Un procédé simple pour la colouration des spores. Le Lait 6:8-12.

3. Marisse A. H., Anna Z., 2012.Endospore Stain Protocol; American Society of

Microbiology.

4. Oktari A., Supriatin Y., Kamal M., Syafrullah H.,2017 The bacterial Endospore Stain on
Schaeffer using methylene Blue Solution; Journal of Physics.

5. Ruth M, I 2009.Photodegradation Methylene Blue with UV light and ZnO Catalyst Thesis,
(Bukit Jimbaran : University of Udayana).

6.Waluyo and General L 2004 Mikrobiologi (Malang :UMM Press)

7. Zubaidah and Elok 2006 Mikrobiologi ( Malang :Universitas Brawijaya Press)