EXPERIMENT # 5

Object: To study about the staining in microbiology.

Microbial Staining
Staining refers to the use of colored dyes/stains to make cells and/or cellular
structures visible or to produce contrast between different types of cells / cellular
components, i.e. it enhances the contrast of a microscopic image. Staining allows
visualization of cells and cellular components, it also allows differentiation of
different cell types or different cellular organelles. It is important in the detection
of microbes in a medium, i.e. blood. It is also very vital in the medical diagnosis of
infections.

Staining techniques may either involve a single stain, intended to point out cells /
cellular components (simple staining) or multiple stains to differentiate between
different cells / cellular components (differential staining).

Stains
Stains are colored organic compounds (salts) used for staining cells, tissues,
microorganisms, etc. Stains contain ions which impart them color, these ions are
called chromophore. If the chromophore is a positive ion, then the dye/stain is
called a basic stain and if it is negative, then the dye/stain is known as acidic stain.
According to pH, stains can be classified into:

Acidic Stains:

These stains have a negative charge, hence they bind to positively charged cellular
structures like some proteins. Acidic dyes are not very often used in a
microbiology lab, except to provide background staining like in negative staining.

Examples include nigrosine, picric acid, eosin, carbol fuchsin, etc.

Basic Stains:

These stains have a positive charge, hence they bind to negatively charged
molecules such as nucleic acids and acids in bacterial cell walls (teichoic acids in
gram-positive cells and phospholipids in gram-negative cells). Since the surface of
bacterial cells are negatively charged, basic dyes are most commonly used in
bacteriology.

Examples include: crystal violet, methylene blue, safranine, basic fuchsin, etc.

Neutral Stains:

These stains are usually formed from precipitation, when aqueous acidic and basic
stains are combined. Neutral dyes stain nucleic acids and cytoplasm.

Examples include: eosinate of methylene blue, Giemsa stain, etc.

Types of Staining
Based on the process, staining is grouped into three types: Simple staining, differential staining,
and special staining.
Simple Staining
Simple staining is quick and is generally used for studying the morphology of cells using a single
stain. In simple staining only single stain is used. Depending on the targeted component, simple
staining can be classified into two types:

 Positive staining: This technique uses simple basic stains like safranin, crystal violet,
etc.
 Negative Staining: This technique uses acidic stains like India ink, nigrosin, etc.

Differential Staining
Differential staining involves multiple stains to distinguish between different types of
microorganisms or structures within a specimen. Some examples include gram staining, acid fast
staining, etc.

 Gram Staining: Gram staining categorizes bacteria into two groups based on their cell
wall composition.
1. Gram-positive bacteria
2. Gram-negative bacteria
 Acid-Fast Staining: Acid fast staining is a staining technique that is used to stain
bacteria with waxy coating on cell walls. This method involves staining with carbol
fuchsin, heat treatment, acid-alcohol wash, and counterstaining with methylene blue.

Special Staining

Some staining techniques utilize specific stains or procedures to highlight particular cellular
structures, substances, or microorganisms. These techniques are used for detailed study of
particular cellular component. Some of these staining methods are discussed below:

 Capsule Staining
 Endospore Staining
 Flagella Staining

Steps Associated With Staining

1. Preparation of Smear
2. Fixation of Smear

Fixation can be done in one of two ways:

 Heat Fixation

 Chemical Fixation

Materials Used In Staining

Some of the materials used in microbial staining techniques are as follows:

 Stains & Reagents

 Staining Rack
 Glass Slides & Cover Slips
 Inoculation Loop
 Dropper
 Wash Bottle
 Bunsen Burner
 Compound Microscope

Precautions
The precautions that should be taken while mounting on a glass slide include;

 You should use a clean and dry glass slide and coverslip. You should be
very careful while putting the coverslip.
 Apply a small amount of mounting medium to the surface of the slide; try to
use an amount that will just fill the space under the coverslip.
 Slowly tip the coverslip onto the mounting medium and avoid creating
bubbles as you lower it into place.
 Avoid the overstraining or under staining of the sample.