Exercise 2: Cell

Staining Techniques
Group 4
Estrella
Gamboa
Gaticales
Gonzales, Alyzza

INTRODUCTION
Staining technique that is commonly
applied in microscopy to enhance contrast in
a microscopic image
Stains and dyes are used oftentimes to
highlight structures for better viewing,
often with the aid of different microscopes
Stains are molecules that give color by
binding to the cellular structures

Acidic tissues and cells have greater

affinity for basic dyes (ex. crystal violet,
methylene blue, safranin , basic fuschin)

Basic tissues and cells have higher

affinity for acidic dyes (ex. nigrosine,
picric acid, eosin, acid fuschin, india ink)

Bright-field microscope
is a type of light microscope that
illuminates specimen with a bright
background against the specimen
efficient with specimens of high contrast

Tetrahymena sp.
Free-living ciliated protozoa
Cilia help Tetrahymena sp.to move and to gather
food
Ovoid or pyriform body shape which narrowed in
anterior portion
It has central macronucleus and micronucleus,
subterminal contractile vacuole; they live in
freshwater

Methyl green
-Nucleus will be visible

Nigrosin
- Surface detail will be visible
Lugols iodine
- nucleus and locomotory organelles (cilia)
will be visible

Objectives
To identify what parts of the cell are
made visible with the stains used

Explain how the nigrosin relief method

helps elucidate the cells overall structure

Methodology

10L of Tetrahymena sp.

Placed on the center of the glass slide.

Focused using the scanner of the microscope, shifted to LPO.
Inserted a strip of filter paper on one side of the cover slip to
draw liquid from the slide.
The sides of the cover slip was coated with petroleum jelly.
Identified the structures of unstained cells.

Identified structures

Repeated steps 1-5. While water was drawn from one side of the
slide with filter paper strip, a drop of iodine was placed on the
other side
Observed what cellular structure(s) became visible.
Repeat all procedures replacing Lugols iodine separately with
Methyl green and Nigrosine.
Tabulated the difference in cellular structures seen under
different stains.
DATA

Lugols Iodine

Repeated steps 1-5 on 3

different slides. While water
was drawn from one side of
the slide with filter paper
strip, a drop of iodine was
placed on one side

Methyl green

Nigrosine

Results Group 1

Fig. 1.1 Tetrahymena sp., unstained

Fig. 1.3 Tetrahymena sp., stained with methyl green

Fig. 1.2 Tetrahymena sp., stained with Lugols iodine

Fig. 1.4 Tetrahymena sp., stained with nigrosin

Results & Discussion

Results Group 1

Fig. 1.1 Tetrahymena sp., unstained

Fig. 1.3 Tetrahymena sp., stained with methyl green

Fig. 1.2 Tetrahymena sp., stained with Lugols iodine

Fig. 1.4 Tetrahymena sp., stained with nigrosin

Results Group 2

Fig. 2.1 Tetrahymena sp., unstained

Fig. 2.3 Tetrahymena sp., stained with methyl green

Fig. 2.2 Tetrahymena sp., stained with Lugols iodine

Fig. 2.4 Tetrahymena sp., stained with nigrosin

Results Group 3

Fig. 3.1 Tetrahymena sp., unstained

Fig. 3.3 Tetrahymena sp., stained with methyl green

Fig. 3.2 Tetrahymena sp., stained with Lugols iodine

Fig. 3.4 Tetrahymena sp., stained with nigrosin

Results Group 4

Fig. 4.1 Tetrahymena sp., unstained

Fig. 4.2 Tetrahymena sp., stained with Lugols iodine

Fig. 4.3 Tetrahymena sp., stained with methyl green

Fig. 4.4 Tetrahymena sp., stained with nigrosin

Results Group 5

Fig. 5.1 Tetrahymena sp., unstained

Fig. 5.3 Tetrahymena sp., stained with methyl green

Fig. 5.2 Tetrahymena sp., stained with Lugols iodine

Fig. 5.4 Tetrahymena sp., stained with nigrosin

Results Group 6

Fig. 6.1 Tetrahymena sp., unstained

Fig. 6.2 Tetrahymena sp., stained with Lugols iodine

Fig. 6.3 Tetrahymena sp., stained with methyl green

Fig. 6.4 Tetrahymena sp., stained with nigrosin

Results Group 7

Fig. 7.1 Tetrahymena sp., unstained

Fig. 7.3 Tetrahymena sp., stained with methyl green

Fig. 7.2 Tetrahymena sp., stained with Lugols iodine

Fig. 7.4 Tetrahymena sp., stained with nigrosin

Results Group 8

Fig. 8.1 Tetrahymena sp., unstained

Fig. 8.3 Tetrahymena sp., stained with methyl green

Fig. 8.2 Tetrahymena sp., stained with Lugols iodine

Fig. 8.4 Tetrahymena sp., stained with nigrosin

Results Group 9

Fig. 9.1 Tetrahymena sp., unstained

Fig. 9.3 Tetrahymena sp., stained with methyl green

Fig. 9.2 Tetrahymena sp., stained with Lugols iodine

Fig. 9.4 Tetrahymena sp., stained with nigrosin

Results Group 10

Fig. 10.1 Tetrahymena sp., unstained

Fig. 10.3 Tetrahymena sp., stained with methyl green

Fig. 10.2 Tetrahymena sp., stained with Lugols iodine

Fig. 10.4 Tetrahymena sp., stained with nigrosin

CONDITION OF Tetrahymena sp. OBSERVABLE STRUCTURES

Unstained

Vacuoles, Cell membrane

Stained with nigrosin

Vacuoles, Cell membrane

Stained with Lugols iodine

Cilia (locomotory organelle),

Nucleus, Starch Granules (Food
Vacuoles)

Stained with methyl green

Nucleus

Discussion
Unstained Tetrahymena
small circular organisms
moving in fast speeds
transparent with
colorless structure
vacuoles & cell
membrane

Nigrosin Relief Method

surface detail
cells transparent;
background darker
(contrast)
negative staining
outline of cell visible
did not enter cell

Lugols Iodine
nuclei
starch indicator
helical structure of DNA
traps Iodine -> iodine/
starch complex
dark color
cilia
yellow to brown

Methyl Green
macro/micronucleus
methyl cation has high
affinity and specificity
with deoxyribonucleic acid
methyl green stain
chromatin thus making
nuclei visible
light green

stain only the surface of the cell to make

a contrast to the background (Nigrosin)
enter the cell membrane to stain the
structures inside the cell (Iodine and
Methyl Green)

dark background would make the cell

structures -> clearer
cell appears as a bright object against a
black background
show the structures that are hard to
locate in bright background

nigrosin relief help elucidate the surface

detail structure through negative
staining technique
colorless and brighter structures