Cell and Molecular Biology Laboratory First Term 2016-2017

Experiment 2
Cell Staining Techniques
Belen, Alexis Bitera, Danica Buenaventura, Gelsie Cantre, Godwin Castillo, Jamila
Department of Biological Sciences, College of Science
University of Santo Tomas, Espana Manila 1051
Date Submitted: October 4, 2016

Introduction

Procedure

10 L of Paramecium sp. suspension culture were placed in the center of a glass slide

and a cover slip was lowered gently above avoiding trapped bubbles. A microscope was focused
using the scanner (4X) and its light source was adjusted to reduce glare. The objectives were
shifted to LPO to see very motile cells. Afterwards, a strip of filter paper was inserted on one
side of the cover slip to draw out some excess liquid. The sides of the cover slip were coated
with petroleum jelly using a toothpick to prevent the slide from drying out. The objective were
then switched to HPO to identify the cell structures that can be seen in the unstained cells. 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. This microorganism
 Cell and Molecular Biology Laboratory First Term 2016-2017
can be found in almost all of the bodies of water especially in freshwater. They are typically
elongated and ovoid microorganisms. Their bodies are covered with cilia, which also acts as the
locomotors organ of the microorganism.
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
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 starch and as an antiseptic of
emergency drinking water (Petruzzi, et. al., 2010). 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 Lugols iodine. In the presence of glycogen,
which is present in the animal cell as storage of glucose, it gives a reddish brown color. The
Lugols solution is being taken up by the glycogen found in the cell (Basford, et.al.,2013). The
 Cell and Molecular Biology Laboratory First Term 2016-2017
iodine forms complexes with the polysaccharide in the cell in the process of complexation
reaction. 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.

Im

Image 3.0 Paramecium cells stained with Lugols iodine under HPO

Methyl green is a basic or cationic dye that is being used in staining chromatin DNA
(Umemura, et. al., 2003). 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 in the
structure, it reacts with the anionic phosphate group of the DNA 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

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. 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.
 Cell and Molecular Biology Laboratory First Term 2016-2017

Image 5.0 Paramecium cells stained with nigrosin under HPO

Conclusion

Post Laboratory Questions

References
Basford, P. J., Benton, A., Jarvis, T., & Bhandari, P. (2013). Indigo carmine or Lugol's iodine? A
beginner's guide to chromoendoscopy and advanced imaging. Gastrointestinal Nursing
Gastrointestinal Nurs, 11(6), 16-23. doi:10.12968/gasn.2013.11.6.16

Castillo, J.R. (2014). Experiment 2: cell staining techniques. Laboratory Manual in Cell and
Molecular Biology, 12-13.

Petruzzi, M., Lucchese, A., Baldoni, E., Grassi, F. R., & Serpico, R. (2010). Use of Lugols
iodine in oral cancer diagnosis: An overview. Oral Oncology, 46(11), 811-813.
doi:10.1016/j.oraloncology.2010.07.013
 Cell and Molecular Biology Laboratory First Term 2016-2017
Presser, C. (2012). Absorption coefficient measurements of particle-laden filters using laser
heating: Validation with nigrosin. Journal of Quantitative Spectroscopy and Radiative Transfer,
113(8), 607-623. doi:10.1016/j.jqsrt.2012.01.009

Umemura, S., Itoh, J., Takekoshi, S., Hasegawa, H., Yasuda, M., Osamura, R. Y., & Watanabe, K.
(2003). Methyl Green Staining and DNA Strands In Vitro: High Affinity of Methyl Green Dye to
Cytosine and Guanine. Acta Histochem. Cytochem. ACTA HISTOCHEMICA ET
CYTOCHEMICA, 36(4), 361-366. doi:10.1267/ahc.36.361