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Identification Methods for Microorganisms

Morphological characteristics
 Useful for identifying prokaryotes and eukaryotes
Differential staining
 Gram staining, acid-fast staining
 Cell wall structure

Biochemical tests
 Determine presence of bacterial enzymes, metabolic activities
Immunological tests
 Determine antigens
 Toxins
Genetic tests
 Determine presence or structure of genes
 Staining
Even with the microscope, bacteria are difficult to
see unless they are treated in a way that increases
contrast between the organisms and their background.
The most common method to increase contrast is to
stain part or all of the microbe.
Bacteria cells are almost colorless and transparent
A staining technique is often applied to the cells to color
them → Their shape and size can be easily determined
under the microscope.
 Types of staining techniques

Simple staining Differential staining

(use of a single stain) (use of two contrasting stains
separated by a decolorizing agent)

For visualization of
morphological Identification Visualization
shape & arrangement. of structure
Gram Acid fast
stain stain Spore Capsule
stain stain
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 Types of dyes (stain)
Stains are chemicals containing chromophores, (groups that impart color).
Their specificity is determined by their chemical structure and charge they carry.
Accordingly there are 3 types of dyes

1- Basic dye (cationic dye):

is a stain that has positively charged chromophores.
Examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene
blue.

:Acid dye (anionic dye) -2

.is a stain that has a negatively charged chromophores
.Examples of acid dyes are Nigrosine and sodium eosinate

:Neutral dye -3

is a stain that has both a negatively and positively charged chromophores (net charge
.is neutral)
.Example: eosin methylene blue
 Mechanism of staining
 The surface of bacteria is some what negatively charged
 When we use cationic dye (crystal violet) it dissociates in
aqueous solutions into CV+ and chloride (Cl–) ions. These
ions penetrate through the cell wall and cell membrane of
bacterial cell. The CV+ ion interacts with negatively charged
components of bacterial cells and stains the cells purple, while
the background is unstained. This is called Direct simple stain.
 When we use an anionic dye (Nigrosine) it is repelled by the
bacterial surface. It stains the background and leave the
bacteria transparent. This is called Negative 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.

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Smear preparation

S Fixation

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[INSERT FIGURE 4.15]
 Heat fixation
Heat fixation accomplishes three things:
(1) It kills the organisms.
(2) It causes the organisms to adhere to the slide.
(3) It alters the organisms so that they more readily accept
stains (dyes).
 If the slide is not completely dry when you pass it through
the flame, the organism will be boiled and destroyed.
 If you heat-fix too little, the organism may not stick and
will wash off the slide in subsequent steps.
 If you heat-fix too much, the organisms may be
incinerated, and you will see distorted cells and cellular
remains.
 Simple Staining

It is the use of single basic dye to color the bacterial

organism.
e.g. methylene blue,
crystal violet,
Safranin.
All bacteria take the color of the dye.
Objective:-
To show the morphological shapes and
arrangement of bacterial cells.

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 Simple Staining
Procedure:-

MB

1-2 min

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 Basic Shapes of Bacteria

Cocci Bacilli

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 Arrangements

Cocci

Irregular Clusters Tetrads Chains or Pairs

Staphylococci Micrococci Streptococci

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 Results
Type of staining:
Name of stain:

Shape of cells:
Arrangement of cells:
Color:

Name of m.o:

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 Gram Stain
In the late 1800’s, Christian Gram
observed that some genera of bacteria
retained a dye-Iodine complex when
rinsed with alcohol, while other genera
were easily decolorized with alcohol
and could be then visualized by a
contrasting counterstain
It is the most important differential
stain used in bacteriology because, it
classified bacteria into two major
groups:

a) Gram positive b) Gram negative:

Appears violet after Gram’s Appears red after Gram’s stain
stain
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 Principle of Gram Stain
1-The first step involves staining with the basic dye crystal violet.
This is the primary stain. Crystal violet is attracted to both
Gram-positive and Gram-negative microorganisms.
2-Treatment with an iodine solution, which functions as:
A- a mordant; that is, it increases the interaction between the
bacterial cell and the dye so that the dye is more tightly bound or
the cell is more strongly stained.
B- It also stabilizes the Crystal violet into the peptidoglycan layer
of the cell wall.
The peptidoglycan layer is much thicker in Gram-positive bacteria
than in Gram-negative bacteria; hence, the Crystal violet is more
extensively entrapped in the peptidoglycan of Gram positive
bacteria.
3-The smear is then decolorized by washing with an agent such as
95% ethanol or isopropanol-acetone. It dissolves lipids in the
outer membrane of Gram-negative bacteria and removes the
Crystal violet from the peptidoglycan layer.
In contrast, the Crystal violet is relatively inaccessible in Gram-
positive microorganisms and cannot readily be removed by
alcohol in Gram-positive microorganisms.
4-Finally, the smear is counterstained with a basic dye, different in
color than crystal violet. This counter stain is usually safranin.
The safranin will stain the colorless, Gram-negative bacteria pink
but does not alter the dark purple color of the Gram-positive
bacteria.
Crystal violet
↓
Iodine
↓
Alcohol
↓
Safranin

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 Gram +ve Gram –ve
S. aureus E. coli

Step 1: Crystal Violet

Step 2: Gram’s Iodine

Step 3: Decolorization
(Aceton-Alcohol)

Step 4: Safranin Red

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Principle of Gram staining technique

1- Crystal violet: all cells are stained

violet.

2- Iodine acts as a mordant (fixes the

dye) all cells remain violet.

3-Alcohol acetone decolorizes gram

negative cells only: Gram-positive
remains violet , gram-negative
becomes colorless.

4-Counter stain with Safranin: Gram-

positive remains violet while gram
negative becomes red.
 Why do gram-positive cells keep their color while gram- negative cells lose it?

Due to the difference in the structure of the cell wall.

Gram-negative cells have peptidoglycan

Gram-positive cells have peptidoglycan
+ teichoic, no pores are formed by
alcohol and the dye is retained.
+ve: Retention of the crystal violet/
Iodine complex
(less)+ NO teichoic acid+ large amount
of lipids
Alcohol dissolves the lipids , forms
large pores that leak the dye leading to
loss of CV/Iodine complex.
-ve: Loss of the crystal violet/ Iodine
complex
 Gram Stain

Procedure:

CV
safranin
iodine

30
10 sec
30-60 sec
sec
2 min

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 Procedure
1-Shake the suspension very well.
2-Transfere aseptically 3-4 loopfuls to a clean glass slide marked
from below.
3-Leave to air dry.
4- Heat fixation.
5-Stain with Crystal violet 30 sec
6-wash gently with water.
7-Add Iodine (cover the whole slide) for 1 min.
8-Remove Iodine, do not wash add fresh Iodine for 2 min.
9-Remove , decolorize with:
-one drop water
-one drop (alcohol/ acetone) till wash drops are faint violet.
10-Conuter stain with safranin 5 min.
11-Wash with water
12- Blot dry , add oil, examine.
 Results
Shape: Cocci
Arrangment: irregular clusters

Colour: Violet
Gram’s reaction: Gram’s +ve

Name of microorganism:
Staphylococci

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 Results

Shape: Bacilli
Arrangment: Chains
Colour: Violet
Gram’s reaction: Gram’s +ve

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 Results
Shape: Rods
Arrangment: Single

Colour: red
Gram’s reaction: Gram’s –ve

Name of microorganism:
Gram negative bacilli

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