practical 2 and 3

GMC KATHUA (Department of microbiology).
Exp No 2: Study of Bacteria and staining procedures.
Bacteria can be studied either in unstained (wet mount) or stained preparation.
■ Wet mount preparation is useful for demonstration of motility of bacteria by light

microscopy or demonstration of spirochetes by dark ground microscopy.
■ Stained preparations are used to demonstrate structural details of the bacteria by producing
color contrast.
Staining methods.
Structural details of bacteria cannot be seen under a light microscope due to lack of contrast.
Hence, it is necessary to use staining methods to produce color contrast and thereby increase the
visibility. Before staining, the smears are fixed so that they will not be displaced during the
staining process.
Fixation also protects the internal structures of Cells in a fixed position.
It is done by two methods.
1. Heat fixation: It is done by gently flame heating an air- dried film, used for bacterial smears
2. Methanol fixation: Used for blood smears.
Common staining techniques

Common staining techniques used in diagnostic bacteriology include:
1. Simple stain: Basic dyes, such as Methylene blue or Basic Fuchsins are used as simple
stains. They provide the color contrast, but impart the same color to all the bacteria in a smear.
simple staining methylene blue

2. Negative stain.
Bacteria (drop of Bacterial suspension) are mixed with dyes, such as Indian –ink or nigrosin
that produces a uniform dark-colored background against which the unstained organisms stand
out in contrast. This is used for demonstration of bacterial capsule that are usually not stained by
simple stains.
India- Ink method is used for demonstration of the fungus
Cryptococcus neoformans is a common example.
Negative stain shown as clear spheres against a black

background.
3. Impregnation stains.
Cells and structures that are too thin to be visualized by the light microscope can be rendered
visible by impregnation of silver salts on their surface to make them visible, e.g. for
demonstration of Bacterial flagella and is a common method used for staining spirochetes, such
as T. palladium and Leptospira.
Differential stains. Here, two stains are used which impart different colors to different bacteria
or bacterial structures, which help in differentiating bacteria. The most commonly employed
differential stains include Gram stain, acid-fast stain (Zn Stain) and Albert’s stain.
Gram stain: It differentiates bacteria into gram positive and gram-negative groups.
• Acid-fast stain: It differentiates bacteria into acid fast and non acid-fast groups
• Albert stain: It differentiates bacteria having Meta -chromatic granules from other bacteria that
do not have them.
Exp No 3: To perform gram stain.
Gram Stain
This staining technique was originally developed by Hans Christian Gram (1884). Even after
more than 130 years of its discovery and even in the presence of newer modern diagnostic
facilities, still Gram stain remains the most widely used test in diagnostic bacteriology.
Procedure.
❖ Fixation: The smear made on a slide from bacterial culture or specimen is air-dried and then
heat-fixed.
Step 1 (Primary stain): The smear is stained with pararosaniline dyes such as crystal violet (or
gentian violet or methyl violet) for one minute. Then the slide is rinsed with water. Crystal violet
stains all the bacteria violet in color (irrespective of whether they are gram-positive or gram
negative).
Step 2 (Mordant): Gram’s iodine (dilute solution of iodine) is poured over the slide for one
minute. Then the slide is rinsed with water. Gram’s iodine acts as a mordant, binds to the dye to
form bigger dyeiodine complexes in the cytoplasm.
Step 3 (Decolorization): Next step is pouring of few drops of decolorizer to the smear, e.g.
acetone (for 1–2sec) or ethyl alcohol (20 30 sec) or acetone alcohol (for 10 sec). Slide is
immediately rinsed with water. Decolorizer removes the primary stain from gram-negative
bacteria while the gram-positive bacteria retain the primary stain.
❖ Note: Decolorization is the most crucial step of Gram staining. If the decolorizer is poured for
more time, even gram-positive bacteria lose color (over decolorization).
❖ If poured for less time, the gram-negative bacteria do not lose the color of primary stain
properly (under decolorization).
Step 4 (Counter stain): Secondary stains such as safranin or carbol fuchsin are added for 30
seconds. It imparts pink or red color to the gram-negative bacteria.
Alternatively, neutral red may also be used as counter stain, especially for gonococci.
The slide is rinsed in tap water, dried, and then examined under oil immersion objective lens.
Result.
▪ Gram-positive bacteria resist decolorization and retain the color of primary stain i.e. violet or
Blue.
▪ Gram-negative bacteria are decolorized and, therefore take counter stain and appear Pink or
red.
Principle of Gram Staining.
Though the exact mechanism is not understood, the following theories have been put forward.
1. PH theory: Cytoplasm of gram-positive bacteria is more acidic, hence, can retain the basic
dye (e.g. crystal violet) for longer time. Iodine serves as mordant, i.e. it combines With the
primary stain to form a dye-iodine complex which gets retained inside the cell.
2. Cell wall theory: This is believed to be the most important postulate to describe the
mechanism of Gram stain.
➢ Gram-positive cell wall has a thick peptidoglycan layer (50–100 layers thick), with tight cross
linkages.The peptidoglycan itself is not stained; instead, it seems to act as a permeability barrier
preventing loss of crystal violet. More so, alcohol is thought to shrink the pores of the thick
peptidoglycan; hence large dyeiodine complexes are not able to penetrate this tightened
peptidoglycan layer in a gram-positive bacteria.
➢ Gram-negative cell wall is more permeable thus allowing the outflow of crystal violet easily.
This is attributed to: The thin peptidoglycan layer in gram-negative cell wall which is
not tightly cross linked.Presence of lipo- polysaccharide layer in the cell wall of gramnegative
bacteria, which gets disrupted easily by the decolorizer; forming larger pores, that allow the dye-
iodine complexes to Escape from the cytoplasm.
Modifications of Gram Staining.

1. Kopeloff and Beerman’s modification: Primary stain and counter stain used
are methyl violet and basic fuchsin respectively.
2. Jensen’s modification: This method involves use of absolute alcohol as decolorizer and
neutral red as counterstain. It is useful for meningococci and gonococci
3. Brown and Brenn modification: This is used for Actinomycetes.
Uses of Gram Stain

❖ To differentiate bacteria into gram-positive and gram-negative.
❖ For identification: Gram staining from bacterial culture gives an idea to put the
corresponding biochemical tests for further identification of bacteria.
❖ To start empirical treatment.
❖ For fastidious organisms, such as Haemophilus which takes time to grow in culture; Gram
stain helps in early presumptive identification.
❖ Anaerobic organisms, such as Clostridium do not grow in routine culture.
❖ Yeasts: In addition to stain the bacteria, Gram stain is useful for staining certain fungi such as
Candida and Cryptococcus appear as gram positive.

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GMC KATHUA (Department of microbiology).

Exp No 2: Study of Bacteria and staining procedures.

Bacteria can be studied either in unstained (wet mount) or stained preparation.

■ Wet mount preparation is useful for demonstration of motility of bacteria by light

Common staining techniques

simple staining methylene blue

Negative stain shown as clear spheres against a black

Modifications of Gram Staining.

Uses of Gram Stain

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