STAINING

TECHNIQUES
Report by:
Dimapinto
Kulidtod
Padayhag
Velarde
Contents
01 Introduction
02 Objectives
03 Methodology
04 Results and Discussion
05 Conclusion
Introduction
“Staining”
Staining is a technique that can be used to
enhance visualization of structures in
biological tissues under a microscope. By
using different stains, one can preferentially
stain certain tissues, cell components, such as
a nucleus or a cell wall, or the entire cell.

Bacteria for instance are nearly colorless, so

their features are difficult to distinguish when
they are suspended in a fluid and viewed
directly under a microscope.
 “Staining”
The chemical composition of the cell determines which stain is
absorbed.
Acidic parts of a cell absorb stains that are positively charged
Alkaline parts of a cell combine with stains that are acidic or
negatively charged.

The specimen is stained either one of the following categories:

1. Morphological stains - provide information about the cell size,
shape, and arrangement. Examples: simple stain and negative stain.

2. Differential stains - differentiate bacteria based on the chemical

composition of the cell wall.
It requires two stains (primary stain and counterstain) to
be used; one for the positive bacteria, the other for the negative
bacteria. The differential stain that will be used in this activity is the
Gram stain.
 Objectives

01 02 03 04
Determine the Differentiate
Determine Determine
morphology the
the the cell wall
and importance of
structures composition
arrangements different
of a
of a cell. of microbial staining
bacterium.
cells. techniques.
Methodology
01
01 Smear Preparation
02
02 Simple staining 03
Special staining:
03
Gram Staining
 Smear
Preparation
Place a drop of water on a clean glass
slide.
Gently scrape inside of cheek with a
toothpick.
Suspend specimen in water drop and
spread.
Let smear air dry. Fix the slide over
flame, smear side up.
Prepared smear is ready for use.
Simple
Staining
Apply methylene blue to cover the
smear, wait 1 minute.
Rinse with water, avoiding direct flow
on slide.
Examine under microscope from low
to high power.
Record observations through
drawings/photos.
Measure cell size in micrometers
(µm).
Note nucleus size.
 Gram
Staining Prepare smear using material
between teeth or gum line.
Add Gram I (crystal violet), let flow,
wait 1 minute, wash.
Add Gram II (Gram’s iodine), let
flow, wait 1 minute, wash.
Decolorize with Gram III (ethyl
alcohol) for 30 seconds, wash.
Stain with Gram IV (safranin) for 1
minute, wash.
Observe under oil immersion lens.
Gram positive cells:
violet/blue/purple.
Gram negative cells: pink/reddish.
methylene
blue
A simple stain is a basic staining technique used
in microbiology to highlight the entire
microorganism with a single dye. In this method,
a basic dye, such as crystal violet, methylene
blue, or safranin, is applied to a heat-fixed
bacterial smear. The dye stains all cells uniformly,
making them visible under a microscope. Simple
staining is helpful for observing cell shapes, sizes,
and arrangements. However, it does not
differentiate between different types of
microorganisms or their structures.
 Human Cheek Cells stained
with Methylene blue

Nucleus Cell membrane

Gram stain

Gram staining is a more complex staining

technique used to differentiate bacteria into
two major groups: Gram-positive and Gram-
negative. This method is named after Hans
Christian Gram, who developed the
technique.

The Gram stain involves several steps:

Crystal Violet Staining
Iodine Treatment
Decolorization
Counterstaining
 Gram-stained
Human Cheek Cell
Bacteria Cell membrane

Nucleus
 1. WHAT IS THE DIFFERENCE BETWEEN BASIC
AND ACIDIC STAINS? HOW ARE THESE
APPLICABLE IN STUDYING THE CELL?

Microbiology, the study of microscopic organisms, relies heavily on

staining techniques to visualize and understand cellular structures. Two
fundamental types of stains used in this field are basic and acidic stains.
Basic stains, such as methylene blue and crystal violet, carry a positive
charge and readily bind to negatively charged cellular components like
nucleic acids and proteins. These stains highlight overall cell structures,
aiding researchers in observing cell morphology and arrangement. On
the other hand, acidic stains, like eosin and India ink, have a negative
charge. They repel from cellular components and instead stain the
background, making specific structures, such as capsules, visible. These
stains are invaluable in studying distinct cell features under a microscope
2. IDENTIFY THE IMPORTANCE OF EACH STEP
IN GRAM STAINING.

Among the various staining methods, Gram staining is a pivotal

technique in microbiology. It involves a series of steps, each of
which plays a crucial role in differentiating bacterial species. The
primary stain, crystal violet, imparts a purple color to all cells.
Iodine serves as a mordant, forming a stable complex with crystal
violet within the cells. The critical step of alcohol decolorization
dehydrates and shrinks Gram-negative cells, allowing the crystal
violet-iodine complex to wash out. Gram-positive cells, with their
thick peptidoglycan layer, retain the complex. Finally, safranin, the
counterstain, colors Gram-negative cells pink, providing a sharp
contrast to the purple Gram-positive cells.
 3. WHAT ARE THE DIFFERENCES BETWEEN
SIMPLE STAINING AND DIFFERENTIAL
STAINING?

Simple staining is an efficient and easy process. It involves

consistently coloring all bacterial cells with a single stain, such as
methylene blue or crystal violet. This method helps with the
imaging of cell morphology, size, and organization; however, it
only offers scant details on the diversity of bacteria. On the other
hand, differential staining uses a variety of stains to divide
bacteria into groups according to the makeup of their cell walls.
Gram-negative bacteria pick up a counterstain, such as safranin,
making them seem pink, while Gram-positive bacteria retain the
crystal violet stain, making them appear purple.
Conclusions
Staining techniques are essential tools for elucidating the intricate
details of biological and clinical specimens. These methods enable the
visualization of cellular structures, microorganisms, and molecular
components, shedding light on the underlying mechanisms of life and
disease. From the Gram stain's role in bacterial classification to the
versatile use of immunohistochemistry in diagnosing diseases,
staining techniques are invaluable in research and diagnostics.
Moreover, modern advancements, such as the development of
fluorescent markers and nanotechnology-based staining, have
expanded the possibilities for understanding biology at a cellular and
molecular level. Staining techniques continue to be a cornerstone of
scientific discovery and medical diagnosis, advancing our knowledge
and improving patient care.
 THANK YOU
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Hartline, R. (2023, February 20). 1.9: Simple Stain. Biology
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Tripathi, N. (2023, August 14). Gram Staining. StatPearls - NCBI
Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK562156/
Ahern, H. (n.d.). Differential Staining Techniques. Pressbooks.
https://milnepublishing.geneseo.edu/suny-microbiology-
lab/chapter/differential-staining-techniques/