GEBZE TECHNICAL UNIVERSITY

MICROSCOPIC PREPARATORY PREPARATION

TECHNIQUES-I

Ebru AKHARMAN
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MICROSCOPIC PREPARATORY PREPARATION

TECHNIQUES-I
AIM
In their natural state, most of the cells and microorganisms that we observe under the microscope lack
color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and
their distinguishing characteristics without artificially treating specimens. We will examine to certain
techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for
sample preparation in greater detail. In this experiment, preparate preparation, Preparation of bacterial
smear, direct staining with E. coli and indirect staining with B. cereus are used.

INTRODUCTION

Direct
Simple Staining
Staining Indirect
Staining
Staining
Differantial
Differantial Staining
Staining Structural
Staining
Microbiologists study the characteristics of microorganisms such as algae, protozoa, bacteria, fungi and
viruses using a microscope. While some organisms such as protozoa and yeast cells are easy to observe using
a wet mount, bacterial cells require staining. Scientists developed several methods such as Gram staining,
acid-fast staining and fluorescent staining for better visualization of bacterial cells and cellular structures.
Using such staining methods, it is possible to identify structural features that help classify bacteria. Staining is
technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently
used in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define
and examine bulk tissues, cell populations, or organelles within individual cells. Bacteria have nearly the same
refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly
invisible to the naked eye. Different types of staining methods are used to make the cells and their internal
structures more visible under the light microscope. Microscopes are of little use unless the specimens for
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viewing are prepared properly. Microorganisms must be fixed & stained to increase visibility, accentuate
specific morphological features, and preserve them for future use.

1. The basic idea behind stains is that they facilitate viewing and examination by providing contrast.

2. Stains also have other uses, such as to distinguish organisms amongst each other. You could also do
viability stains which is typically an oxymoron because when you stain it, you kill the organism. Viability stains
differentiate between live and dead bacterial cells in a sample (before they all die).

3. Stains can also provide structural details and composition and in respect to viability, some status on the
organism such as metabolism.

4. Stains are critical because they provide data on morphology so you could tell the shape, size, and
identification but of course there’s a limit. On a gram stain you can’t specifically get the ID but you could do a
presumptive identification.

Stain: A stain is a substance that adheres to a cell, giving the cell color. The presence of color gives the cells
significant contrast so they are much more visible. Different stains have different affinities for different
organisms, or different parts of organisms. They are used to differentiate different types of organisms or to
view specific parts of organisms

Staining: Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image.
Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for
viewing, often with the aid of different microscopes.

Fixation: Fixation by itself consists of several steps–aims to preserve the shape of the cells or tissue involved
as much as possible. Sometimes heat fixation is used to kill, adhere, and makes them permeable so it will
accept stains

What can be used as stain ?: The substance be used as a stain must be colored or it should react in the system
to give a colored product, because of which some portion of the system becomes colored and the rest
remains colorless. Staining renders the organism more visible, it displays the structure and finer details of
bacteria and it helps to differentiate between organisms.

Image 1: Negative Charged Bacterial Cell Wall

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Types of stains: Based on the nature of chromogen, there are three types of stain.

1. Acidic stain (Anionic stain)

2. Basic stain (Cationic stain)

3. Neutral stain

Acidic stain (Anionic stain): Chromogen of acidic stain is negatively charged. so, it is also known as Anionic
stain. Acidic stain are used to stain the positively charged components such as background staining. Histone
protein is positively charged so it can be stained by acidic stain. Acidic stain can not stain bacterial cell due to
repulsion of same charge.

Examples: Eosin, Nigrosin, India ink

Basic stain (Cationic stain):

Chromogen or coloured part of basic stain is positively charged. so, it is also known as cationic stain. Basic
stain are used to stain negatively charged components such as bacterial cell.

Examples: Methylene blue, Safranin, Malachite green,Basic fuschin, Crystal violet

Neutral stain: In neutral stain, both caation and anion are coloured, such that net charge is neutral. Neutral
stain are actually is a salt of acidic and basic stain.

Examples: Giemsa stain.

Mechanism of Staining:

The process of staining involves ion exchange reaction between the stain and component to be stained. For
example, bacterial cell is a negatively charged due to large number of protein having COO- group. This
negative charged is balanced by positive charged ion presentoutside the cell wall. Therefore a bacterial cell is
represented as (BACTERIAL CELL -) 𝑁𝑎+. To stain the bacterial cell, cationic dye are used having positively
charged chromogen. eg. Methylene blue, which is represented as 𝑀𝑔+ 𝐶𝑙 −. During staining, bacteria cell is
flooded with methylene blue and due to ion exchange mechanism acidic component of bacterial ie bacterial
cell wall become stained.

The reaction occurs as follows;

(BACTERIAL CELL-)𝑁𝑎+ + 𝑀𝑔+ 𝐶𝑙 −======NaCl + (BACTERAIL CELL -)𝑀𝑏 +.

Smear: Smear are made for preparing slides for staining which are used in microscopy. The main purpose of
smear is to seprate cluster of microbial cells so that we can see them seprately which is helpfull in studying
there morphology, and arrangement in colony.

Simple Staining: The simple stain can be used as a quick and easy way to determine cell shape, size and
arrangements of bacteria. True to its name, the simple stain is a very simple staining procedure involving
single solution of stain. Any basic dye such as methylene blue, safranin, or crystal violet can be used to color
the bacterial cells. These stains will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the

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stain positively charged. Since the surface of most bacterial cells and cytoplasm is negatively charged, these
positively charged stains adhere readily to the cell surface. After staining, bacterial cell morphology (shape
and arrangements) can be appreciated.

MATERIAL AND METHOD

Application 1. Preparate Preparation:

Image 3: Preparate Preparation

Application 2. Bacterial Smear Preparation:

Image 4: Bacterial Smear Preparation

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Application 3. Direct Simple Staining:

Image 5: Direct Simple Staining

Application 4. indirect Simple (negative) Staining

Image 6: Negative Staining

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 RESULTS 06.04.2018
Application.1: Application.2:

Image 7: Streptomyces coelicolor bacteria Image 9: E.coli

Image 8: Streptomyses coelicolor bacteria microscopy image Image 10: E.coli microscopy image

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Application.3: Application.4:

Image 11: Bacillus cereus Image 13: Bacillus cereus

Image 12: Bacillus cereus microscopy image Image 14: Bacillus cereus

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Streptomyses coelicolor bacteria naturally have a pink color and can be observed directly with a microscope
without the need for staining. The E. coli bacteria were stained pink with a crystal violet dye. Bacillus cereus
bacteria are observed colorless on the blue background with nigrosine staining. All microscope images are
40X.

DISCUSSION
In this experiment smear and preparation were prepared, also direct and indirect dyeing techniques were
applied. These techniques have made important steps for bacterial identification. Streptomyses coelicolor,
Bacillus cereus and E. coli bacteria were used as examples.

Streptomyses coelicolor bacteria naturally have a pink color and can be observed directly with a microscope
without the need for staining. The E. coli bacteria were stained pink with a crystal violet dye. Bacillus cereus
bacteria are observed colorless on the blue background with nigrosine staining.

In the stained colors, it is observed that E. coli contained crystal violet. However, Bacillus cereus was not
stained with nigrosine dye. The main reason for this is the chemical interaction between the bacteria cell and
the environment. In addition, while E. coli is round in shape, Bacillus cereus is rod-shaped and has a flaccid
structure of Streptomyses coelicolor. In some areas in the painting areas, quite intense cells were observed.
This may be because the smear process is not effective.

RESOURCES

1) www.microbiologyinfo.com
2) www.researchgate.net
3) www.asmscience.org
4) www.yourarticlelibrary.com
5) www.onlinebiologynotes.com

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