Assignment:Staining techniques for Anatomical structures

of plants
Submitted by:Sadaf Jaan Mohammad
Roll No:28
Class:BS.Botany 6th semester
Subject:Plant Anatomy(Labwork)
Submitted to:Dr.Aisha Nazir
Institute of Botany

University of Punjab,Lahore.
 Staining
Staining
Staining is a method of imparting colour to cells, tissues or microscopic components, so they
are highlighted and visualized better under a microscope.Staining is carried out with the help
of a reagent termed as “stain“. This method uses a wide variety of natural and synthetic
stains, which is used to add colour to the colourless specimen to be studied.

Stain
Stains are chemical reagents or dye that imparts colour to cells and tissue sections of the
biological specimens and aids in its visualization under a microscope. Stains work by
increasing the contrast between different cellular components, thereby highlighting specific
cell structures.
Classification
Stains can be classified into the following types, depending upon its chemical nature and the
type of staining methods.

Based on chemical nature: There are three kinds of stain, acidic, basic and neutral,
depending upon the chemical nature of the stain.
Based on the staining method: There are four kinds of stain, viz. direct, indirect,
differential and selective stains.

Purpose
● Enables us to see the organism better: Microorganisms are very minute
creatures as well as appear transparent, so staining makes the specimen 9easy to
identify.
● Helps to differentiate organisms: Staining helps in distinguishing between
the two different groups of organisms, depending upon the colour retaining ability
of the cells (some microbes retain the colour of stain, while some don’t).
● To identify a particular structure: For further study of microorganisms, it
is also important to study the various internal and external structure of organism
like flagella, capsule, nucleus, spores etc.
Mechanism
Stains are organic compound composed of a benzene ring, a chromophore group and an
auxochrome group.Now benzene is a colourless solvent, and the chromophore group is a
molecule that imparts colour to the benzene. As a result, the compound formed is called a
‘chromogen’ and it was put forth by O. N. Witt in 1876. Now, this chromogen is not a stain
in itself, it is just a coloured compound.
The second part of the stain, the auxochrome, is a chemical group that ionizes the
chromogen i.e. it imparts a positive or negative charge to the chromogen group. As a result,
the auxochrome enables the ionized chromogen to bind to cells or tissue fibres of opposite
charge and thereby colour it

Common stains and their uses

● Iodine: Stains carbohydrates in plant and animal specimens brown or blue-black.

Stains glycogen red.
● Methylene blue: Stains acidic cell parts (like nucleus) blue. Use on animal, bacteria
and blood specimens. Can be used as a substitute for Janis B Green.
● Eosin Y: Stains alkaline cell parts (like cytoplasm) pink. Use on plants, animals and
blood. Can be used as a substitute for Congo Red and Carmine.
● Toluidene blue: Stains acidic cell parts (like nucleus) dark blue. Good to show
mitosis in plant cells.
● Wright’s stain: Stains red blood cells pink/red.
● Crystal Violet: Stains bacteria purple.

● Aceto-orcein: Biological stain for chromosomes and connective tissue.

● Sudan III: Biological stain used as a lipid indicator.

Requirements for staining

Stain – Majority of the stains used for staining bacteria are of the basic type as nucleic acid
of bacterial cells attract the positive ions, e.g. methylene blue, crystal violet. Acidic stains are
used for background staining.
Mordant – It is a chemical that forms an insoluble complex with the stain and fixes it or
causes the stain to penetrate more deeply into the cell. These are used in indirect staining. For
example, Gram’s iodine in Gram staining and phenol in Ziehl Neelson’s staining.
Accentuater – It is a chemical which when added to a stain to make the reaction more
selective and intense. For example, potassium hydroxide added in Loeffler’s methylene blue.
Decolorizer – It is a chemical used to remove the excess stain in indirect regressive
staining. For example, ethanol in Gram’s staining.

Types of Staining

Simple Staining
A staining method that uses only a single dye that which does not differentiate between
different types of organismsThere is only a single staining step and everything is stained with
the same color.Simple stains are used to stain whole cells or to stain specific cellular
components.

Types of simple staining:

1. Direct / Positive staining : stain object

2. Indirect / Negative staining: stain background
Direct staining (Positive staining)

A simple staining technique that stains the bacterial cells in a single color.Many of the
bacterial stains are basic chemicals; these basic dyes react with negatively charged bacterial
cytoplasm (opposite charges attract) and the organism becomes directly stained Examples
are methylene blue, crystal violet, and basic fuchsin.
 Fig:Direct staining
Indirect staining (Negative staining)

In this staining process, instead of ells background is stained. Here, an acidic dye like
nigrosin or Indian ink is used. Acidic stain carries a negative charge and repelled
by the bacteria, which also carry a negative charge on their surface. Hence, an acidic dye do
not stain bacteria, Instead, it forms a deposit around the organism, leaving the organism itself
colorless or transparent upon examination.

Fig:Indirect staining

Differential Staining

It differentiates between the physical and chemical properties of two different groups of an
organism, depending on the cell-wall characteristics. It makes the use of multiple or more
than one stains. It can be categorised into two types that are given below:
Gram staining

It provides an important tool to differentiate the two major groups of bacteria, i.e.
gram-positive and gram-negative. Dr Hans Christian Joachim Gram introduced this method
in 1884. It is carried out by the use of differential stain known as Gram’s stain.

This staining procedure defines two bacterial groups: those which retain the primary dyes
(“Positive by Gram’s Method” or “Gram-Positive”) and those which are easily decolorized
(“Negative by Gram’s Method” or “Gram-Negative”). This is the starting point for bacterial
identification procedures.

The Gram Stain

The difference in dye retention is dependent on such physical properties as thickness,

density, porosity, and integrity of the bacterial cell wall, as well as its chemical composition.
Gram-Positive bacteria have thick, dense, relatively non-porous walls, while Gram-Negative
bacteria have thin walls surrounded by lipid-rich membranes. Some non-bacterial organisms
with thick cell walls (e.g., some yeasts) also stain Gram-Positive. Gram-Positive bacteria
which have lost wall integrity through aging or physical or chemical damage may stain
Gram-Negative.

Fig:Gram staining
Acid fast Staining

It differentiates species of mycobacterium from the other groups of bacteria. Paul Ehrlich first
developed it in 1882. And later, this technique was modified by a scientist named Ziehl
Neelson.Acid fast staining property of the genus, Mycobacteria, depends upon their
lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes
are combined with phenol. ➢ The exact method by which the stain is retained is unclear but
it is thought that some of the stain becomes trapped within the cell and some forms a complex
with the mycolic acids. This is supported by the finding that shorter chain mycolic acids or
mycobacterial cells with disrupted cell walls stain weakly acid-fast, e.g. Nocardia

Fig:Acid fast staining

Special Staining

It helps in the identification of particular internal and external structural components of the
specimen. It includes capsule, endospore and flagella staining.

Capsule staining

It differentiates the capsule from the rest of the cell body. This is carried out by the use of
both positive and negative dyes.

Capsule: It can define as the polysaccharide envelope, which surrounds the cell wall.
Capsule performs many functions like cell protection against desiccation, phagocytic actions
and also helps in cell attachment to the host.e bacteria.
 Fig:Capsule Staining

Endospore Staining

It differentiates the endospore from the vegetative cell and makes the use of both acidic and
basic stains.

Endospore: A term itself defines its meaning, in which endo stands for inside and spore
stands for a reproductive structure. Therefore, endospores are the reproductive structures
inherent to the cell. It acts like a dormant spore, which can resist harsh physical and chemical
conditions. Endospores are commonly found in gram-positive bacteria.

Fig:Endospore stain
Haematoxylin and eosin (H & E): Routine stain

This is the most common histologic stain, used to differentiate different tissue structures. It
also plays an important role in the diagnoses of various pathologies. Haematoxylin, is a
naturally occurring dye found in Longwood tree wood in Central America. In the H&E stain,
a mixture of oxidised hematoxylin known as hematein is used. Due to poor affinity of
hematin with tissues, a mordant is incorporated in the H&E stain. Most commonly used
mordants are salts of aluminium, iron and tungsten. This substance is known as hemalum.

Single Staining

Staining is essential because different tissues and other structures can be easily differentiated
by staining. The plant materials, in which there is no differentiation of tissues such as
members of algae, fungi and bryophyta, are stained by a single staining process.

Double staining

But members of pteridophyta, gymnosperms and angiosperms are stained by the double
staining method due to the presence of differentiation of tis­sues.

Some Methods of Double Staining:

Under-mentioned are some of the commonly used methods of double

staining:

1. Safranin-Fast Green Method:

Keep the mate­rial to be stained in safranin for three to five min­utes and then wash it with
water. See under the microscope that only thick-walled cells are stained. Excess of stain is
destained by acid alco­hol. Again wash the material very thoroughly with water so that even
the traces of acid are re­moved.
Now stain the material with few drops of fast green for few seconds. Time for keeping the
material in fast green varies from few seconds to one minute for different materials. Wash the
mate­rial with glycerine and mount in a drop of glycer­ine.

With this method, all thick-walled cells get red stain and all thin-walled cells the green stain.

Entire process can be tabulated as follows:

2. Safranin-Aniline Blue Method:

Follow ex­actly the same procedure as mentioned above ex­cept that in place of fast green use
aniline blue.

3. Haematoxylin-Safranin Method:

Keep the sections in Delafield haematoxylin for four to five minutes and remove the excess
of stain with water. Wash with ammonia. Wash the material very thor­oughly with water. Now
stain with safranin for few minutes. Wash the sections with glycerine for removing excess of
stain and mount in glycerine.
The entire method can be tabulated as follows:

Preparation of Slides

Dry Mount:

The dry mount is the most basic technique: simply position a thinly sliced section on the
center of the slide and place a cover slip over the sample.Dry mounts are ideal for observing
hair, feathers, airborne particles such as pollens and dust as well as dead matter such as insect
and aphid legs or antennae. Opaque specimens require very fine slices for adequate
illumination.Since they are used for primarily inorganic and dead matter, dry mounts can
theoretically last indefinitely.
Wet Mount:

Used for aquatic samples, living organisms and natural observations, wet mounts suspend
specimens in fluids such as water, brine, glycerin and immersion oil. A wet mount requires a
liquid, tweezers, pipette and paper towels.

To prepare the slide:

● Place a drop of fluid in the center of the slide

● Position sample on liquid, using tweezers
● At an angle, place one side of the cover slip against the slide making contact with
outer edge of the liquid drop
● Lower the cover slowly, avoiding air bubbles
● Remove excess water with the paper towel

Temporary slides

Temporary microscope slides are made for the purpose of short time observations. After the
observation session, they are discarded. In most cases the slides use a liquid mounting
medium, such as water. Wet mounts can be temporary slides.

● Take 2-3cm long pieces of the material.

● Hold the material between thumb and first finger of your left hand.
● Hold the razor in the right hand with edge of the blade facing you and handle at right
angle to it.
● Dip the top of the material in water.
● Then start cutting transverse sections as fast as possible in a watch glass containing
water.
● Select the thinnest section of the material with the help of a delicate brush.
● Take a clean watch glass with water, transfer thin sections of the material.
● Put a few drops of saffranin stain in the watch glass with water.
● Leave it for 3-5 minutes.
 ● Drain off stain and wash with water if necessary.
● Put the thinnest section in the centre of the slide.
● Put a drop of glycerine over the material.
● Cover it with a coverslip with the help of needle.
● Observe it under a compound microscope after staining and mounting.
Permanent slide (Mounting)
Preparation of permanent slide (Mounting) Mounting of tissue (section, squash, smear) in a
suitable mounting medium (e.g. Canada balsam, Euparal). After staining, it is necessary to
avoid drying up of tissue and not to render it opaque. The mounting media should have a
refractive index closer to glass to avoid refraction. It should harden quickly in contact with air
and should check de-staining. The principal aims of mounting are:
(i) To render the tissue transparent;
(ii) To increase the visibility of tissue under microscope;
(iii) To hold it with the protecting coverslip firmly in place;
(iv) To preserve it for a long period.
For dehydration, the slide is passed through absolute ethanol, keeping in each for 2 sec. 2.
Differentiation is done by passing the slide through clove oil I for 2-5 min (observation under
microscope is required for satisfactory staining) and then transferred to clove oil II and kept
for 10-15 min. 3. For clearing, the slide is kept in xylol I, II and III for 1 hour in each. 4.
Finally, it is mounted in canada balsam under a coverslip and the slide is allowed to dry
overnight on a hot plate (35-45°C).
 References
● https://www.microbehunter.com/what-are-the-different-kind
s-of-microscope-slides/
● reference.com/science/difference-between-temporary-slide-p
ermanent-slide-using-microscope
● https://brainly.in/question/4603161
● https://en.wikipedia.org/wiki/Microscope_slide
● https://www.microscopemaster.com/microscope-slides.html
● https://www.biologydiscussion.com/zoology/practicals/prepa
ration-of-permanent-slides-zoology/60158