Cell and Molecular Biology Laboratory First Term 2016-2017

Experiment 2
Cell Staining Techniques
Belen, Alexis

Bitera, Danica
Buenaventura, Gelsie Cantre, Godwin
Department of Biological Sciences, College of Science
University of Santo Tomas, Espana Manila 1051

Castillo, Jamila

Date Submitted: October 4, 2016

Introduction
Cell staining is a technique used in microscopy to enhance and better visualize cells and
cell components under the microscope. Stains and dyes are usually used in this technique; these
are substances that adhere to the cell, which gives a contrasting color against a background.
There are two major categories, the direct staining in which the organism is stained and the
background is left unstained; and the indirect staining in which the background is stained and the
organism is left unaltered. There are a lot of cell staining techniques used in the laboratory.
Different classifications are used to determine which type of cell staining technique is to be
utilized. One classification is the mode of the specimen used, either in vivo staining, in vitro
staining or ex vivo staining. In this experiment, in vivo staining is utilized in which the organism
under study, in this case the Paramecium sp., is alive or staining is on a living specimen. The
purpose of this staining technique is to determine and visualize cytological details that are not
apparent when unstained. This technique could also reveal certain chemical reactions that are
happening within the cell. The Paramecium sp., in this experiment is stained with Lugols iodine,
Methyl green and Nigrosine. The objective of this experiment was to compare the structures that
were seen in those given stains and to determine the principle on how the structures were stained.

Procedure
1. 10 L of Paramecium sp. suspension culture was placed in the center of a glass slide and a
cover slip was lowered gently above, avoiding trapped bubbles.
2. A microscope was focused using the scanner (4X) and its light source was adjusted to
reduce glare.

Cell and Molecular Biology Laboratory First Term 2016-2017

3. The objectives were shifted to LPO to see very motile cells.

4. Afterwards, a strip of filter paper was inserted on one side of the cover slip to draw out
some excess liquid.
5. The sides of the cover slip were coated with petroleum jelly using a toothpick to prevent
the slide from drying out.
6. The objectives were then switched to HPO to identify the cell structures that can be seen
in the unstained cells.
7. All of the steps were repeated to add Lugols iodine, methyl green and nigrosine but at
the step where while the water was drawn out from one side with a filter paper slip, a
drop of the chosen stain was placed on the other side.
Results and Discussion
Cell staining is a basic process in studying microscopic organisms. In this process,
specific parts of the cells and other components of the cells are being shown depending on the
stain used and the types of cell staining technique that was employed. Stains have different
mechanisms in marking the cell. It involves specificity and permeability.
Paramecium sp. is a unicellular ciliated protozoan, which is commonly used as
representative of ciliates in showing the movement and mechanism of cilia. They belong to
phylum Alveolata, subphylum Ciliophora and class Nassophorea (Ruppert, et.al., 2004). They
have fixed anterior-posterior polarity. They exhibit two types of cilia, the somatic ciliature and
oral ciliature. The somatic ciliature are found in the surface of their body which aids in their
movement. Through this, ciliate animals are the fastest protozoa. Oral ciliature is associated with
the mouth region. This microorganism can be found in almost all of the bodies of water
especially in freshwater. They are typically elongated and ovoid microorganisms. They have two
types of nucleus, where their genetic material contains. These are the macronucleus and
micronucleus. They have distinct oral groove which the food particles enter the microorganism
and through this, the anterior polarity is being distinguished (Ruppert, et. al., 2004).
Under LPO, unstained Paramecium sp., can be observed as fast dot-like structures
moving around. Under HPO, the inside of an unstained paramecium is composed of a jelly-like

Cell and Molecular Biology Laboratory First Term 2016-2017

fluid called protoplasm. Bits of food vacuoles and other materials float around in the protoplasm.
The outline of the cell membrane can also be identified.

Image 1.0 Unstained Paramecium under LPO

Image2.0 Unstained Paramecium under HPO

Several dyes are being used in vivo important cell components and parts. One of the
stains that are being used in vivo is the Lugols iodine. Lugols iodine or Lugols solution is
commonly used in laboratory in detecting the presence of polysaccharide (Petruzzi, et. al., 2010).
Its chemical name is potassium triiodide.

Figure 1.0. Chemical structure of Lugols iodine or Potassium iodide

The stain is also being used in oral cancer detection. It is composed of iodine and
potassium iodide dissolved in distilled water. In the presence of amylose, a solution colors with
deep blue. While in the presence of amylopectin, it gives a purple color, which is the original
color of iodine. In the presence of glycogen, which is present in the animal cell as storage of

Cell and Molecular Biology Laboratory First Term 2016-2017

glucose, it gives a reddish brown to brown black color. The Lugols solution is being taken up by
the glycogen found in the cell (Basford, et.al., 2013). The iodine forms complexes with the linear
chain of the glycogen (Xia, et.al., 2014).Through this stain, the cilia, which are the locomotory
organelle of the microorganism, became visible and appear as reddish brown color. This is due to
the presence of glycogen that serves as energy storage, providing the required energy for the
locomotory organelle. In the experiment, since the microorganism is surrounded by their cilia,
the whole organism was stained with the Lugols iodine.

Image 3.0 Paramecium cells stained with Lugols iodine under HPO

Methyl green or methylene green is a basic or cationic dye that is being used in staining
chromatin DNA (Umemura, et. al., 2003). It is a heterocyclic aromatic compound that bears a
positive charge in its amino group (Figure 2.0).

Figure 2.0. Chemical Structure of methyl green

Cell and Molecular Biology Laboratory First Term 2016-2017

Figure 3.0. Chemical Structure of DNA backbone

This stain is commonly used with pyronin in differential staining of DNA and RNA. The
phosphate group of the DNA is negatively charged. Due to the cationic nature of the methyl
green due to the presence of the positive charge of the amino group (Figure 2.0) in the structure,
it reacts with the anionic phosphate group of the DNA (Figure 3.0) forming a blue green color. In
the Paramecium sp., the nucleus became visible as a blue green structure bounded by nuclear
membrane prior to the staining of methyl green. This is due to the presence of the DNA in the
nucleus of the microorganism.

Cell and Molecular Biology Laboratory First Term 2016-2017

Image 4.0 Paramecium stained with methyl green under HPO

Nigrosin is a mixture of black dyes such as nitrobenzens, aniline and aniline

hydrochloride, heating together with copper or iron catalyst (Presser, et. al., 2012). This black
dye forms a black background, which enable to observe the presence of the transparent
Paramecium sp. Thus, it reveals the surface details of the microorganism.
Methyl green and Lugols iodine employs positive staining. In positive staining, the cells
were the ones being stained. In contrast, nigrosin relief, which employs negative staining, the
background was the one being stained. This way, the specimen or the cells appear against the
dark background.

Cell and Molecular Biology Laboratory First Term 2016-2017

Image 5.0 Paramecium cells stained with nigrosin under HPO

Conclusion
One stain, Lugols Iodine, allows the cilia of the Paramecium sp. to be better seen under
the microscope, due to the stain forming a complex with the glycogen which is providing energy
for the dynamism of the cilium. Another stain that was used was Methyl Green; it is a positively
charged stain, which would be highly attracted to negatively charged stains such as the nucleus
of the Paramecium sp. The last stain that was used in the experiment was Nigrosin, and however,
unlike the previous stains that were employed the stain doesnt form a complex or react with the
cell. It merely provides a black background against the transparent nature of the organism
providing better visibility.
Therefore cell staining is a paramount laboratory procedural technique in which a specific
part of a microscopic organism, or even the entire microorganism, can be fully seen under the
microscope. With the help of several different stains that react to other parts of the cell, specific
parts of the organism would be enabled to be clearly seen.
Table 1.0 Summary of the stains used and their corresponding effect on the microorganism

Cell and Molecular Biology Laboratory First Term 2016-2017

Unstained

Visible
Parts
Cell
Membrane
and Food
Vacuoles

Mechanism
None

Lugol's Iodine

Glycogen
containing
food
vacuoles
and Cilia

Potassium Triiodide forms a

complex with the glycogen
molecules.

Methyl Green

Nucleus of
the cell

amino group of methyl reacts

with anionic phosphate group
of the DNA

Nigrosin

Better
discern the
Parameciu
m sp. From
the
background

Negatively stains the

background.

Post Laboratory Questions

1. What structures are visible in the unstained Paramecium sp.? With iodine? With methyl
green? With nigrosin?
Unstained Paramecium sp. are colorless and transparent organisms that use cilia
to move around. The most prominent feature in an unstained Paramecium sp. are the food
vacuoles.Iodine is used to stain structures which contain branched sugars. Pure iodine is
black in color but it highly toxic. Iodine is usually combined with Potassium to decrease

Cell and Molecular Biology Laboratory First Term 2016-2017

vapor pressure and make it safer to use. This is why laboratory iodine stains are colored
brown. The iodine stained the cilia and glycogen vacuoles of Paramecium sp. Methyl
green stains DNA which is why the nuclei of Paramecium sp. were stained. Nigrosin is
used as a negative stain. This means that nigrosin stains the surroundings rather than the
specimens. This is useful because living cells do not allow the entry of the stain so they
are easily seen.
2. Do all stains enter the cell or just render more contrast to the background?

The iodine and methyl green enters the cell. These stains allow the close
observation of different structures inside a cell due to their specificity for certain
compounds such as polysaccharides and phosphate groups. Nigrosin does not enter the
cell but stains the surroundings to enable observers to see the cells present in a slide.
3. By providing a dark background, would the cell structure look clearer?
A dark background will provide greater contrast to unstained cells. A dark field
microscope renders the background darker than the specimens. This is ideal for unstained
and living samples. A dark field microscope has a power source under the stage to
illuminate the specimens. A condenser gathers the light in a hollow cone, with the apex
pointed towards the objective lens. The light is directed around the lens but it does not
pass it. This makes the entire slide appear black. When a sample is placed in the
microscope, the apex of the cone hits it. The sample scatters the light and it is gathered by
the objective lens.
4. In your formal report, tabulate the different structures seen under each of the stains used.
Explain the mechanism for each reaction.
Stain
Unstained

Cell structure visible

Cell membrane and food

Iodine

vacuoles
Cilia and glycogen vacuoles

Cell and Molecular Biology Laboratory First Term 2016-2017

Methyl green
Nigrosin

Nuclei
Cell membrane

The stain used is Potassium triiodide. When the stain was added to the slide, the Iodine
dissociates with the Potassium Iodide. The Iodine complexes with the branched or helical
polysaccharides. This produces the brown coloration of the structure inside the cells. There is the
formation of polyiodide chains between Iodine and glycogen which produces the color. Methyl
green stains DNA which enabled the observers to see the nuclei of the Paramecium sp. Methyl
green reacts with phosphate radicals found in DNA which produces the blue-green color.
Nigrosin strains the surroundings of viable cells. This stain is able to enter the cytoplasm of nonviable cells because the plasma membrane ceases being a selectively permeable barrier. This
allows the nigrosin inside the non-viable cells.

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Cell and Molecular Biology Laboratory First Term 2016-2017

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