Lesson 02 PDF
Lesson 02 PDF
Lesson 02 PDF
Microbiology
2
Notes
COMMON STAINING
TECHNIQUE
2.1 INTRODUCTION
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 (highlighting, for example, muscle fibers or connective
tissue), cell populations (classifying different blood cells, for instance), 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 viewing are prepared
properly. Microorganisms must be fixed & stained to increase visibility,
accentuate specific morphological features, and preserve them for future use
OBJECTIVES
After reading this lesson, you will be able to:
z describe the need for staining techniques
z explain terms related to staining techniques
z discuss the substances used as stain
z enlist various staining techniques
z classify & explain various stains
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Common Staining Technique MODULE
Microbiology
2.2 TERMS RELATED TO STAINING
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 Notes
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 stepsaims 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
Staining techniques
Direct staining - The organism is stained and background is left unstained
Negative staining - The background is stained and the organism is left unaltered
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MODULE Common Staining Technique
Microbiology
2.3 KINDS OF STAINS
Stains are classified as
z Simple stain
z Differential stain
z Structural or special stains
Notes
Simple Staining
The staining process involves immersing the sample (before or after fixation and
mounting) in dye solution, followed by rinsing and observation. Many dyes,
however, require the use of a mordant, a chemical compound that reacts with
the stain to form an insoluble, coloured precipitate. When excess dye solution
is washed away, the mordanted stain remains. Simple staining is one step method
using only one dye. Basic dyes are used in direct stain and acidic dye is used
in negative stain. Simple staining techniques is used to study the morphology
better, to show the nature of the cellular contents of the exudates and also to study
the intracellular location of the bacteria
Commonly used simple stains are
z Methylene blue
z Dilute carbol fuchsin
z Polychrome methylene blue
Loefflers Methylene Blue
Method of Staining
Flood the smear with methylene blue, allow for 2 minutes, pour off the stain and
allow the air to dry by keeping in a slanting position and by this the organism
will retain the methylene blue stain
Use
Methylene blue staining is used to make out clearly the morphology of the
organisms eg. H.influenzae in CSF, Gonococci in urethral pus
Polychrome Methylene Blue
Preparation
Allow Loefflers Methylene blue to ripen slowly. Methylene blue stain is kept
in half filled bottles, aerate the content by shaking at intervals, Slow oxidation
of methylene blue forms a violet compound and Stain gets polychrome property.
The ripening nearly takes 12 months and this is hastened by addition of 1%
potassium carbonate
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Common Staining Technique MODULE
Use Microbiology
Use
To stain throat swab from patients of suspected Vincents angina, (Borrelia are
better stained), it is used as a counter stain in Gram stain and to demonstrate the
morphology of Vibrio cholerae (comma shaped)
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MODULE Common Staining Technique
Microbiology indeterminate groups as well. The word Gram is always spelled with a capital,
referring to Hans Christian Gram, the inventor of Gram staining
Gram Reaction
Gram-positive bacteria are those that are stained dark blue or violet by Gram
staining. This is in contrast to Gram-negative bacteria, which cannot retain the
crystal violet stain, instead taking up the counter stain (safranin or fuchsine) and
appearing red or pink. Gram-positive organisms are able to retain the crystal
violet stain because of the high amount of peptidoglycan in the cell wall. Gram-
positive cell walls typically lack the outer membrane found in Gram-negative
bacteria.
Gram-negative bacteria are those bacteria that do not retain crystal violet dye
in the Gram staining protocol. In a Gram stain test, a counter stain (commonly
safranin) is added after the crystal violet, coloring all Gram-negative bacteria
with a red or pink color. The test itself is useful in classifying two distinct types
of bacteria based on the structural differences of their cell walls. On the other
hand, Gram-positive bacteria will retain the crystal violet dye when washed in
a decolorizing solution.
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Common Staining Technique MODULE
Microbiology
Notes
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MODULE Common Staining Technique
Microbiology
Principle
Mycobacterial cell walls contain a waxy substance composed of mycolic acids.
These are -hydroxy carboxylic acids with chain lengths of up to 90 carbon
atoms. The property of acid fastness is related to the carbon chain length of the
mycolic acid found in any particular species
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Common Staining Technique MODULE
7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol contains Microbiology
3% HCl and 95% ethanol, or you can declorase with 20% H2SO4
8. Tilt slide 45 degrees over the sink and add acid alcohol drop wise (drop
by drop) until the red color stops streaming from the smear
9. Rinse with DI water
10. Add Loefflers Methylene Blue stain (counter stain). This stain adds blue
color to non-acid fast cells. Leave Loefflers Blue stain on smear for 1 Notes
minute
11. Rinse slide. Blot dry.
12. Use oil immersion objective to view.
Fig. 2.3
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MODULE Common Staining Technique
Microbiology
Albert staining
Albert stain I
z Toluidine blue 0.15 gm
z Malachite green 0.20 gm
z Glacial acetic acid 1.0 ml
z Alcohol(95%) 2.0 ml
z Distilled water 100 ml
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Common Staining Technique MODULE
Albert stain II Microbiology
z Iodine 2.0 gm
z Potassium iodide 3.0 gm
z Distilled water 300 ml
Capsule staining
The purpose of the capsule stain is to reveal the presence of the bacterial capsule,
the water-soluble capsule of some bacterial cells is often difficult to see by
standard simple staining procedures or after the Gram stain. The capsule staining
methods were developed to visualize capsules and yield consistent and reliable
results
Capsule may appear as clear halo when a fresh sample is stained by Grams or
Leishman stain, Negative staining- using - India ink, Nigrosin
India ink
Commercially available India ink is used undiluted
Procedure
z Place a loop full of India ink on the slide
z A small portion of the culture is emulsified in the drop of ink
z Place a clean cover slip over the preparation without bubbles. Press down
gently
z Examine under dry objective
Uses
India ink is used to demonstrate capsule which is seen as unstained halo around
the organisms distributed in a black background eg. Cryptococcus
Endospore Staining
Bacterial endospores are metabolically inactive, highly resistant structures
produced by some bacteria as a defensive strategy against unfavorable
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MODULE Common Staining Technique
Components
1. 1% Osmic acid
2. Mordant
10% Tannic acid
Sat.potassium alum
10% Ferric chloride
3. Fontanas silver solution
Use
This is used to demonstrate the flagella and the organisms stain black and
flagella appear light brown
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Common Staining Technique MODULE
Microbiology
TERMINAL QUESTIONS
1. List staining techniques
2. Describe different kinds of stains
3. Explain gram staining
4. Explain Acid fast staining
2.1
1. Contrast
2. Stain
3. Fixation
4. Direct
5. Negative
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MODULE Common Staining Technique
Microbiology 2.2
1. Mordant
2. Basic
3. Acidic
4. Methylene blue, Polychrome methylene blue & Dilute carbol fuchsin
Notes
2.3
1. Crystal violet, Iodine & Fuchsin
2. Dark blue or violet
3. Red or pink
4. Peptidoglycan
2.4
1. Acid fast organism
2. Acid fast staining
3. Carbon chain
4. Acid fast and Non acid fast
2.5
1. Alberts
2. Indian ink
3. Malachite green
4. Safranin
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