American University of Science and Technology

Department of Laboratory Science and Technology

BIO 210L: Cells and Molecules Laboratory

Experiment 3
Cell staining.

Name: Isra’ Malla

Instructor’s Name: Dr. Jeanne Andary
Date Experiment Done: 30 March 2020
A. Introduction
 On a microscopic level, staining is generally used to enhance the contrast and the visual
of the sample. It is used in describing the components and structure of the tissues. They
can be used to identify whether the cell is alive or not, and to stain specific parts such as
 the nucleus or the cell wall. Most stains are either In-vivo, which is the process of dyeing
living tissues or In-vitro, it involves coloring cells that have been removed from their
biological context. Stains and fluorescent dyes have been around since the 1600s, when
Robert Hooke used dyed wool and hair to further observe the sample. But it wasn’t until
the late 1800s and early 1900s, that they discovered many developments of new stains
and synthetic dyes. It was in 1858 that Joseph von Gerlach who used carmine solution
overnight on his sample, he conducted after observing his sample that only the nucleus
was stained and there was little to no staining in the cytoplasm and organelles. He
concluded that only specific cellular elements could absorb the dye and could not be
washed out, so he therefore was the first to recognize the importance of staining and how
we can describe the staining method. The only disadvantage in cell staining is that it will
kill of the cell. There are two general types of staining, simple and differential staining;
Simple staining is when we stain a specimen with a positively charged dye to see the
details, or with a negatively charged dye where the specimen remains unstained against a
dark background. Simple staining dyes are methylene blue, safranin, or crystal violet.
Whereas differential staining is a process where more than one chemical stain is used. It
can be better at differentiating between different microorganisms or the structure of a
single organism. They involve 2 kinds; separation of microbes into groups (gram
staining/acid fast staining) and visualizing of various structures (wall staining/spore
staining/capsule staining/flagella staining/nucleus staining). The dyes are either acidic,
basic or neutral. The dyes could be gotten from a natural source (plant based/nature) or
synthetic.

Fig 1: showing the difference of the simple staining and the differential staining.

 The purpose of this session is to become familiar with the staining techniques whether
it’s simple or differential. These staining techniques are used in various fields:
1. Histology
Histology is the study of the macro-anatomy of organs, tissues and cells in animals and plants
seen through a microscope. They show the cell structure and how the cells are organized to form
different tissues and organs how cells are different each cells get displayed according to their
different functional characteristics. This field is very important in helping out with the diagnosis
of diseases like cancer.
The most common staining in histology are hematoxylin and
eosin stains (H&E stain). Hematoxylin is a natural stain
extracted form a tree found only in Mexico and the West
Indies. These extracted samples are oxidized beforehand to
make hematein (active staining component) in order to be
used. This stain alone can target the nucleus in the cell and
produces a dark blue color (it needs mordant). Eosin is used
with hematoxylin because it stains the organelles in the
cytoplasm different shades of pink, orange or red. These
stains used together, will bring a lot of information needed
about the sample/specimen that’s being examined.
Fig 2: microscopic image of liver
2. Hematology cells with H&E staining
This field consists of the study of diagnosis, treatment and
prognosis of blood diseases. It is also the study of blood, blood-
forming organs and blood diseases. The traditional used test in
hematology is the nuclear dye and it’s called the May-
Grunwarld Giemsa. This method contains 2 dyes, methylene
blue and eosin Y. Methylene blue becomes blue violet and
stains acidic cell components upon oxidation.so the nuclei will
be stained different shades of purple while the eosin stains the
other basic structures and the color it puts is pink. Fig 3: microscopic image of normal
blood cells 3.
withCell
thestructure
May-Grunwarld
It is the study of culturing cells; they refer to culturing Giemsa.
eukaryotic cells. They are grown under the appropriate
conditions in an incubator to stay alive. They add dye to know
if the cell is alive or not. In the living cells there are some cell
membranes that are so intact that they don’t let in certain dyes
like eosin or trypan blue. But in the non-viable cells, they
absorb the dye and get stained in blue. This test is made to
figure out if the cell will absorb the Trypan blue or exclude it.

Fig 4; microscopic view of dead and

living cells stained with trypan blue.
4. Bacteriology
It is the study of bacterial microorganisms. It helps with the
identification, classification, and characterization of different
bacterial species. In this field, the technique used is the
Gram staining, it differentiates bacteria into two main
classes, gram-positive and gram-negative. We need methyl
violet, with 3% iodine solution, washed with alcohol and
counterstained to observe the bacterial cell.
 Fig 5: microscopic image of E. coli
stained with gram-staining

5.
This field in biology studies the structure Fig 6:
and composition of internal molecules in DNA
the cells such as proteins and nucleic staine
acids that are the main components of the d by
cells function process. The most common EtBr
used staining is Ethidium Bromide
(EtBr).
Molecular biology

6. Parasitology
This is the field of biology that focuses on
parasitic organisms. The main dye used in this
branch is iodine. The parasite will absorb the
iodine and give off a light yellow-gold color.

Fig 7:

B. Material Endamoeba
 For onion staining: coli parasite
- Glass slide. stained by
- Acetocarmine stain. iodine.
- Onion epidermis.
- Coverslip.
 For cheek staining:
- Tooth pick.
- Inside of cheek.
- Glass slide.
- Cover slip.

C. Procedure
 For onion staining:
- The internal part of an onion epidermis.
- Place it on a glass slide. Fig .: showing the preparation of both the onion
- Put one drop of acetocarmine. and cheek staining.
- Cover it gently with a cover slip.
- Observe it under the microscope.

 For cheek staining:

- Take a tooth pick and scratch the internal surface of your cheek.
- Spread it gently on a glass slide.
- Add one drop of methalyn blue or any other dye.
- Cover it with a cover slip.
- Observe it under the microscope.

D. Results
In the onion epidermis staining at 40x in the microscope we were able to observe first the
external cell membrane then the internal cell wall, the cytoplasm, the vacuole and the nucleus.
In the cheek cell staining at 40 x in the microscope we were able to observe the cell membrane,
cytoplasm, and the nucleus.

E. Conclusion
Staining helps and aids in visualizing the microbes easier as they appear colored against a while
background. They help classify cells into different types and helps with the observance of
structure and composition of the cell whether it’s a bacterium, parasites or animal/plant cells.

Stain Type Specific Dyes Purpose Outcome

Basic stains Methylene blue, crystal This stain, stains Positive stain
violet, malachite green, negatively charged
basic fuchsine, molecules .
carbolfuschsin, safranin
Acidic stains Eosin, acid fuchsine, This stain, stains Negative or positive;
rose Bengal, Congo red positively charged depends on the cell’s
molecules. chemistry.

Negative India ink, nigrosine Stains background, not Dark background

stains specimen with a light
specimen
Table 1: summarizing the main types simple staining as well as their specific usage.

Stain Type Specific Dyes Purpose Outcome

Gram stain Uses crystal violet, Distinguishes cells Gram-positive cells stain
Gram’s iodine, by cell-wall type purple/violet. Gram-
ethanol (decolorizer), (gram-positive, negative cells stain pink
and safranin gram-negative)
 Acid-fast stain After staining with Distinguishes acid- Acid-fast bacteria are red;
basic fuchsine, acid- fast bacteria, from non-acid-fast cells are blue.
fast bacteria resist non-acid-fast cells.
decolonization by
acid-alcohol. Non-
acid-fast bacteria are
counterstained with
methylene blue.
Endospore Schaeffer-Fulton Distinguishes Other than the endospore
stain procedure, then cell organisms with appear pink to red shades;
is washed and endospores from endospores appear bluish-
counterstained with those without green.
safranin. endospores.
Flagella stain Flagella are coated Used to observe Flagella are visible if
with potassium alum and examine present
mordant, then stained flagellum in the
using basic fuchsine bacterium that have
it.
Capsule stain Negative staining Distinguishes cells Capsules appear clear or as
with nigrosine is without capsules halos.
used to stain the from cells with
background so the capsules.
cell and capsule
appear on a stained
wall.
Table 2: summarizing the main differential staining as well as their specific usage.

F. Acknowledgment
I thank my instructor for her help with me understanding all the different types of staining and
my lab partners for helping me understand how this experience may work.

G. Literature
https://www.nature.com/milestones/milelight/full/milelight02.html
https://en.wikipedia.org/wiki/Staining
https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/9780471729259.mca03
es15
http://www.biologydiscussion.com/bacteria/staining-procedures-for-detecting-
bacteria/55104