Introduction

In the laboratory, a variety of stains and staining methods are used. Some only require a
single stain and a few steps, while others need the use of many stains and a more involved
method. The cells must be mounted (smeared) and fixed onto a glass slide before the staining
technique can begin.

A bacterial smear is actually a tiny amount of culture spread across the slide's surface in a
very thin layer. The smear may be chemically or physically "fixed" to the slide's surface to
prevent the bacteria from washing away during the staining process. Heat fixing is a simple
and effective method that involves passing the slide through the flame of a Bunsen burner for
a few seconds, causing the biological material to stick to the glass surface more or less
permanently.

Smears that have been heat fixed are now ready to be stained. Dyes that are either attracted
by charge (cationic dyes like methylene blue or crystal violet) or repelled by the charge
(anionic dyes like eosin or India ink) are added to the smear in a simple stain (Parker, 2016).
The bacterial cells are bound by cationic dyes, which can be seen against the clear
background. Because anionic dyes are resisted by cells, the cells stand out against the dyed
background.
As a means of gathering information about bacteria, differential staining techniques are
applied more frequently than basic stains. Differential staining methods are so chosen
because they allow for the separation of cell types or cell structures. They often need more
than one stain and numerous stages. The Gram stain is the most important of these. The
endospore stain is used to distinguish endospore-forming bacteria from other bacteria, the
acid-fast stain is used to distinguish Mycobacterium species from other bacteria, a
metachromatic stain is used to identify phosphate storage granules, and the capsule stain is
used to distinguish encapsulated bacteria.

Gram staining is a popular method for differentiates between different broad groups of
bacteria based on their cell wall constituents. Gram staining is a technique for identifying
Gram positive and Gram negative cells by coloring cells red or violet. Gram-positive bacteria
stain violet because their cell walls include a strong layer of peptidoglycan, which helps to
keep the crystal violet staining. Gram negative bacteria, on the other hand, stain red due to a
weaker peptidoglycan wall that does not hold the crystal violet throughout the decolorization
process (Bruckner, 2021). The ability of some bacterial cells to maintain a primary stain
(crystal violet) by resisting a decolorization process is the basis for the Gram stain's
differential character. There are four processes to gram staining. Crystal violet is used to stain
the cells first, followed by the addition of a stain fixing agent (iodine). The stain is then
carefully removed from only the Gram negative cells using alcohol. Finally, safranin, a
secondary stain, is used to counterstain the decolorized cells pink. The outer membrane (also
known as the envelope) of Gram negative cell walls dissolves during the alcohol wash
(Aryal, 2018). The crystal violet dye is able to escape as a result of this. The pink dye
safranin is only taken up by the decolorized cells, which explains the color difference
between the two types of cells. Gram positive cells have a purple appearance, while Gram
negative cells have a pink appearance.

When bacteria build their cell walls, they produce the waxy chemical mycolic acid.
Mycolic acid works as a barrier, preventing the cells from drying and being phagocytosed by
the host's immune system cells. This waxy layer also inhibits stains from reaching the cell,
which is why the Gram stain is ineffective against pathogenic mycobacteria such as
Mycobacterium (Libretexts, 2021). The acid-fast staining technique is used to identify these
microorganisms. A heat-fixed smear is inundated with the primary stain carbol fuchsin while
the slide is heated over a steaming water bath to execute the acid-fast stain. The heat "melts"
the waxy cell wall, allowing the dye to be absorbed by the cells. The slide is then allowed to
cool before a decolorizer solution of acid and alcohol is added. Because of the mycolic acid
in their cell walls, cells that are "acid-fast" resist decolorization and preserve the primary
stain. The colour of all other cell kinds will be changed. After that, methylene blue is applied
as a counterstain. Acid-fast bacteria (AFB) will be stained bright pink at the conclusion,
whereas all other cell types will be stained blue.

Next, endospores are dormant forms of live bacteria that are not to be mistaken with fungi's
reproductive spores. In response to harsh environmental conditions, a few genera of Gram-
positive bacteria, practically all bacilli, generate these structures. Bacillus and Clostridum are
two common bacteria that create endospores (Libretexts, 4.5A: Endospores, 2021). Both
reside primarily in soil and as plant and animal symbionts, and create endospores to survive
in a constantly changing environment. Endosporulation (the creation of endospores) is a
multi-stage procedure. Layers of peptidoglycan and protein are created to enclose the genetic
material when the bacterial cell copies its DNA. The endospore is discharged from the cell
once fully grown and might remain dormant for days, weeks, or even years. Endospores
germinate and return to active duty as vegetative cells when more favourable environmental
circumstances prevail. Mature endospores are highly resistant to external conditions like heat
and chemicals, allowing the bacterial species to survive for lengthy periods of time.
Endospores that have been dormant for millions of years have been successfully revived by
merely giving them with water and nourishment.

Other than that, because of the endospore coat is so stain-resistant, a specific approach was
created to make them easier to see under a brightfield microscope. Because endospores are
permeable to water, this approach, known as the endospore stain, uses either heat or a long
exposure time to tempt the endospores to pick up the primary stain, which is commonly a
water soluble dye such as malachite green. The counterstain safranin is used to add colour
and contrast after a decolorization phase that eliminates the dye from the vegetative cells in
the smear. The endospores are green and the vegetative cells are pink when stained using this
approach. Despite the fact that endospores themselves are resistant to Gram staining, bacterial
cells acquired during the creation of these structures can be stained. Endospores appear as
clear oval or round spots within the stained cell in this case. Using phase contrast
microscopy, endospores can also be seen directly in cells.

Objectives Lab 2

1. To identify examples of specialised cells. 

In conclusion, from this experiment we could be observed the blood smear by using
compound microscope. The observation of blood smear shows red blood cells, platelets,
neutrophil, eosinophil or acidophil, basophil, monocyte and lymphocyte. The red blood cells
stains rose pink at the periphery, and whitish pink at the centre. Platelets appears as dark
purple stained spots among red blood cells. Neutrophil nucleus stains deep blue. Eosinophil /
acidophil, basophil have the nucleus that usually bilobed and nucleus stains deep purple.
Monocyte appear as largest size white blood cell. Lymphocyte nucleus stains deep purple and
the cytoplasm light purple. Besides, uses immersion oil technique to increase the resolving
power of a microscope. For the clear microscopic observation, leishman stain is used for
staining blood smear to provide excellent stains quality. Other than that, sperm concentration
was determined by using hemocytometer. The sperm can be viewed under 400x
magnification. Onion root tips was observed and there are five stages of mitosis such as
prophase, anaphase, metaphase, interphase and telophase was viewed under 100x objective
lens.
 Reference:

Aryal, S. (2018, June 12). Gram Staining: Principle, Procedure, Interpretation, Examples

and Animation. Microbiology Info.Com. https://microbiologyinfo.com/gram-staining-

principle-procedure-interpretation-examples-and-animation/

Libretexts. (2021a, January 3). 2.3C: The Acid-Fast Cell Wall. Biology LibreTexts.

https://bio.libretexts.org/Bookshelves/Microbiology/Book

%3A_Microbiology_(Kaiser)/

Unit_1%3A_Introduction_to_Microbiology_and_Prokaryotic_Cell_Anatomy/

2%3A_The_Prokaryotic_Cell_-_Bacteria/2.3%3A_The_Peptidoglycan_Cell_Wall/

2.3C%3A_The_Acid-Fast_Cell_Wall

Libretexts. (2021b, January 3). 4.5A: Endospores. Biology LibreTexts.

https://bio.libretexts.org/Bookshelves/Microbiology/Book

%3A_Microbiology_(Boundless)/

4%3A_Cell_Structure_of_Bacteria_Archaea_and_Eukaryotes/

4.5%3A_Specialized_External_Structures_of_Prokaryotes/4.5A%3A_Endospores

Parker, N. (2016, November 1). Staining Microscopic Specimens – Microbiology.

Pressbooks. https://opentextbc.ca/microbiologyopenstax/chapter/staining-

microscopic-specimens/

Z. Bruckner, M. (2021, January 14). Gram Staining. Microscopy.

https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html