Staining Techniques for Microbial
Examination
2
INSTRUCTOR INFORMATION
Contact Official email
niveen10@gmail.com
Prof. Niveen Adel Mohamed El-wakeel
Dr.Amany Elmatbouly Elsayed
amanielmatbouly@gmail.com
Dr.Aya Ahmad Elnegery
ayaelnegery@mans.edu.eg
Dr. Nada Hamid Qandeel nadahamid@mans.edu.eg
Dr. Lamis Mohamed Taha Lamis_mohamed@mans.edu.eg
Dr. Aya Gamal Borham ayagamalborham@mans.edu.eg
Dr. Azza Mohamed Mamon Azzam2010@mans.edu.eg
LEARNING OUTCOMES
Determine Determine the steps of performing dry fixed film
Perform Perform the proper procedure for simple stain.
Perform Perform a Gram Stain on a variety of bacterial cultures.
Explain Explain the principles of how the Gram Stain works.
Define and distinguish between Gram positive and negative
Define
bacteria by light microscope
Evaluate Evaluate and interpret your Gram Stain results correctly.
PRACTICE OUTLINE
• Types of different techniques of staining for bacteria.
• Gram staining principle.
• Steps of Gram staining preparation and aim of each
step.
Staining of Bacteria
Bacteria cells are almost colorless and transparent
A staining technique is often applied to the cells to
color them →
Their shape, size and arrangement can be easily
determined under the microscope.
Types of Stain
1- Simple Stain.
- One dye.
-Study morphology & arrangement of organisms.
2- Differential Stain.
- More than 2 dyes.
- Differentiate the organisms.
- Gram stain.
3- Special Stain.
- Capsular Stain.
- flagellar stain.
MAIN LINES
Making Making of a film (Film preparation).
Staining of a film by:
Staining • Simple stain.
• Differential stain.
• Special stain.
Smear Preparation:
Preparation and Fixation of Bacteria for Staining.
Objective:
To kill the microorganism & fix them to the slide to
prevent them from being washed out during the
process of staining.
FILM (SMEAR) PREPARATION
1. Sterilization of the bacteriological loop.
2. Pick a pure colony in a drop of water or bacterial culture drop.
3. Spread on a slide.
4. Drying of the smear.
5. Fixation of the smear.
6. Staining.
Use a single dye to color the bacteria.
e.g. methylene blue, Dil. carbol fuchsin,
All bacteria take the color of the dye.
Show the morphological shapes and
arrangement.
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4
SIMPLE STAINING
- Prepare dried and fixed film.
- Cover the preparation with methylene blue (or diluted
carbolfuchsin) for 1-2 minute.
- Remove excess stain and wash with water gently until
the color is faint.
- Dry the preparation either in air or on the flame.
- Examine with oil immersion lens.
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Simple Staining
Procedure:
MB
1-2 min
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6
SHAPES & ARRANGEMENTS OF THE
BACTERIA
Simple Staining
Name of dye:- methylene blue
Shape of cells:- cocci
Arrangement of cells: clusters
Color:- blue
Name of m.o:- Staphylococci
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8
CHOOSE:
1. Simple stain gives an idea about:
a. Shape and arrangement of bacteria.
b. Gram reaction of bacteria.
c. Presence of bacterial capsule.
d. Motility of bacteria.
e. Genus of bacteria
Gram Stain: Gram
negative
It is the most important
differential stain used in
bacteriology Gram
it classified bacteria into positive
two major groups:
a)Gram positive: b) Gram negative:
Appears violet after Appears red after Gram’s
Gram’s stain stain
Gram Stain
Danish physician,
Composition. Christian Gram
1884.
1- Methyl (crystal) violet 1%, 1ry stain.
2- Iodine solution, mordant.
3-Alcohol 95%, decolorizer,
4- Diluted carbol fuchsin 0.1%/ Safranin
counter basic stain.
Crystal violet
↓
Iodine
↓
Alcohol
↓
Dil carbol fuchsin
(Safranin)
Gram +ve Gram –ve
S.aureus E.coli
Step 1: Crystal Violet
Step 2: Iodine
Step 3: Decolorization
(Alcohol)
Step 4: diluted carbol
Fuschin (saframin)
Gram Stain, cont.,
Theory.
Alcohol
dissolves lipids
in OM of Gram-
ve cells allowing
dye crystals to
be removed
from thinner PG
layer.
CELL WALL IN
GRAM POSITIVE
& NEGATIVE
ORGANISMS
GRAM-POSITIVE
BACTERIA
Have a thick peptidoglycan layer surrounds the cell.
The stain gets trapped into this layer and the bacteria turned
purple.
Retain the color of the primary stain (crystal violet) after
decolorization with alcohol
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Gram-negative bacteria
have a thin peptidoglycan layer that does not retain
crystal violet stain.
Instead, it has a thick lipid layer which dissolved easily
upon decoulorization with Alcohol.
Therefore, cells will be counterstained with diluted
carbol fuchsin and turned red.
CHOOSE:
2. The counterstain in Gram stain is:
a. Methyl violet 1%.
b. Iodine solution.
c. Alcohol 95%.
d. Diluted carbolfuchsin 0.1%.
e. Methylene blue.
CHOOSE:
3. Decolourization in Gram stain is
done by:
a. Sulphuric acid 20%
b. Sulphuric acid 95%.
c. Alcohol 20 %.
d. Alcohol 95 %.
e. Iodine solution.
RESULTS:
Shape: Cocci
Arrangment: irregular clusters
Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism:
Staphylococci
RESULTS:
Shape: Bacilli
Arrangment: Chains
Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism:
Bacillus
RESULTS:
Shape: Rods
Arrangment: Single, pairs,
clusters
Colour: red
Gram’s reaction: Gram’s –ve
Name of microorganism:
Gram negative bacilli
REFERENCES
• Cheesbrough M. (2000): Microbiological tests.
Cited by Cheesbrough, M., (ed.) District
Laboratory Practice in Tropical Countries, Part
2, Microscopical techniques used in
microbiology, Cambridge University Press, UK
- Useful websites:
https://www.youtube.com/watch?v=JvN6t8-
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