IDENTIFICATION OF BACTERIA USING STAINING TECHNIQUES
Staining, in microbiology, can be defined as a technique which is used to enhance and
contrast a biological specimen at the microscopic level. Stains and dyes are used to highlight
the specimen at the microscopic level to study it at higher magnification.
TYPES OF STAIN
1. Basic dye or stain: Basic dyes which are positively charged are preferred because
bacterial cell membrane is negatively charged due to phospholipid that strongly
attracts and binds to each other.
Eg methylene blue and crystal violet
2. Acidic dye or stain: These are negatively charged stains that bind with the positively
charged proteins that are present in the cytoplasm.
Eg: eosin, acid fushsine
DIFFERENT TYPES OF STAINING TECHNIQUES
1. Simple staining:
Simple staining is a technique that uses only one type of stain on a slide at a time.
Because only one stain is being used, the patterns or background will be of one colour.
PRINCIPLE: It works on principle that the bacterial smear is stained with a single reagent,
which produces a distinctive contrast between the organism and its background in order
to study the morphological characteristics.
Basic dyes are used for simple staining.
USE: The purpose of simple staining is to study the morphology and arrangement of
bacterial cells. The most commonly used basic stains are methylene blue and crystal
violet.
, PROCEDURE:
1. Wash the glass slide and air dry it.
2. Sterilize the inoculating loop by dipping in 100% ethanol followed by flame
sterilization on Bunsen burner till it turns hot red.
3. Cool the inoculating loop and pick the bacterial culture using it.
4. Form a smear of bacterial culture over the slide.
5. Fix the smear on a slide by gentle warming over the Bunsen burner (heat fixation).
6. Pour a drop of basic dye over the smear and keep for respective time. The carbol
fuchsin requires 15-20 seconds, methylene blue requires 1-2 minutes and crystal violet
requires 20-60 seconds.
7. Remove the extra dye with the help of distilled water by the use of dropper and air
dry it.
8. Examine the staining smear under the microscope.
2. GRAM STAINING
Gram staining is a type of Differential staining. It is primarily a staining process that uses
more than one chemical stain. Multiple stains can be used to better distinguish between
different microorganisms or structures and cellular components of an organism. Gram
staining is crucial staining technique developed by Danish bacteriologist Hans Christian Gram
in 1882. It is mainly used to categorize bacteria into 2 groups-gram positive bacteria that
retain the colour of the dye and gram-negative bacteria that do not retain the colour of the
dye.
PRINCIPLE: Gram positive bacteria have cell wall made of thick peptidoglycan as compared
to gram negative bacteria. Gram negative bacteria contain an extra layer of thick
lipopolysaccharide which is absent in gram positive bacteria. During gram staining crystal
violet and iodine complex is formed within cell wall where complex binds to magnesium-
ribonucleic acid component of cell wall. When decolourizer is added the lipopolysaccharide
can dissolve in alcohol and dye is released out in gram negative bacteria while in gram
positive bacteria the lipid layer is absent and dye is tightly trapped as complex in thick
peptidoglycan hence colour is retained. Counter stain safranine is used to view the gram-
negative bacteria.
The 4 dyes used here are
1 Crystal violet dropped on smear for 1 minute
, 2 Mordant: Gram's lodine that helps in fixation of crystal violet by forming complex with it.
Dropped on smear for 1 minute.
3 Decolourizer: alcohol that dissolves the lipopolysaccharide layer of gram- negative
bacteria. Dropped for 30secs.
4 Counterstain: safranine to visualize the gram-negative bacteria.
Examples of Gram-positive bacteria are S. aureus (most popular Gram-positive pathogen),
Clostridium difficile, Proteus vulgaris and Proteus mirabilis.
Examples of Gram-negatives include E. coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Salmonella enterica and, Acinetobacter baumannii.
The bacteria that blue violet are gram positive bacteria while bacteria appearing reddish
pink are gram negative bacteria.
3. ACID-FAST STAINING
Acid fast stain is a differential stain used to identify acid-fast organisms such as members of
the genus Mycobacterium. Acid-fast microorganisms are characterized by wax-like, nearly
impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes,
and complex lipids. This type of cell wall is resistant to most compounds, therefore acid-fast
microorganisms require a special staining technique.
The acid-fast bacteria have following layer:
Staining, in microbiology, can be defined as a technique which is used to enhance and
contrast a biological specimen at the microscopic level. Stains and dyes are used to highlight
the specimen at the microscopic level to study it at higher magnification.
TYPES OF STAIN
1. Basic dye or stain: Basic dyes which are positively charged are preferred because
bacterial cell membrane is negatively charged due to phospholipid that strongly
attracts and binds to each other.
Eg methylene blue and crystal violet
2. Acidic dye or stain: These are negatively charged stains that bind with the positively
charged proteins that are present in the cytoplasm.
Eg: eosin, acid fushsine
DIFFERENT TYPES OF STAINING TECHNIQUES
1. Simple staining:
Simple staining is a technique that uses only one type of stain on a slide at a time.
Because only one stain is being used, the patterns or background will be of one colour.
PRINCIPLE: It works on principle that the bacterial smear is stained with a single reagent,
which produces a distinctive contrast between the organism and its background in order
to study the morphological characteristics.
Basic dyes are used for simple staining.
USE: The purpose of simple staining is to study the morphology and arrangement of
bacterial cells. The most commonly used basic stains are methylene blue and crystal
violet.
, PROCEDURE:
1. Wash the glass slide and air dry it.
2. Sterilize the inoculating loop by dipping in 100% ethanol followed by flame
sterilization on Bunsen burner till it turns hot red.
3. Cool the inoculating loop and pick the bacterial culture using it.
4. Form a smear of bacterial culture over the slide.
5. Fix the smear on a slide by gentle warming over the Bunsen burner (heat fixation).
6. Pour a drop of basic dye over the smear and keep for respective time. The carbol
fuchsin requires 15-20 seconds, methylene blue requires 1-2 minutes and crystal violet
requires 20-60 seconds.
7. Remove the extra dye with the help of distilled water by the use of dropper and air
dry it.
8. Examine the staining smear under the microscope.
2. GRAM STAINING
Gram staining is a type of Differential staining. It is primarily a staining process that uses
more than one chemical stain. Multiple stains can be used to better distinguish between
different microorganisms or structures and cellular components of an organism. Gram
staining is crucial staining technique developed by Danish bacteriologist Hans Christian Gram
in 1882. It is mainly used to categorize bacteria into 2 groups-gram positive bacteria that
retain the colour of the dye and gram-negative bacteria that do not retain the colour of the
dye.
PRINCIPLE: Gram positive bacteria have cell wall made of thick peptidoglycan as compared
to gram negative bacteria. Gram negative bacteria contain an extra layer of thick
lipopolysaccharide which is absent in gram positive bacteria. During gram staining crystal
violet and iodine complex is formed within cell wall where complex binds to magnesium-
ribonucleic acid component of cell wall. When decolourizer is added the lipopolysaccharide
can dissolve in alcohol and dye is released out in gram negative bacteria while in gram
positive bacteria the lipid layer is absent and dye is tightly trapped as complex in thick
peptidoglycan hence colour is retained. Counter stain safranine is used to view the gram-
negative bacteria.
The 4 dyes used here are
1 Crystal violet dropped on smear for 1 minute
, 2 Mordant: Gram's lodine that helps in fixation of crystal violet by forming complex with it.
Dropped on smear for 1 minute.
3 Decolourizer: alcohol that dissolves the lipopolysaccharide layer of gram- negative
bacteria. Dropped for 30secs.
4 Counterstain: safranine to visualize the gram-negative bacteria.
Examples of Gram-positive bacteria are S. aureus (most popular Gram-positive pathogen),
Clostridium difficile, Proteus vulgaris and Proteus mirabilis.
Examples of Gram-negatives include E. coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Salmonella enterica and, Acinetobacter baumannii.
The bacteria that blue violet are gram positive bacteria while bacteria appearing reddish
pink are gram negative bacteria.
3. ACID-FAST STAINING
Acid fast stain is a differential stain used to identify acid-fast organisms such as members of
the genus Mycobacterium. Acid-fast microorganisms are characterized by wax-like, nearly
impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes,
and complex lipids. This type of cell wall is resistant to most compounds, therefore acid-fast
microorganisms require a special staining technique.
The acid-fast bacteria have following layer:
