Isolation and Identification of
Bacteria
The most important task of a bacteriology is to identify the
pathogens from the clinical sample so that appropriate treatment
can be instituted.
There are several methods to identified the different type of
bacteria.
They are,
1. Isolation in pure form
2. Staining reaction
3. Morphology of bacterial colony
4. Cultural characteristics
5. Metabolism
6. Biochemical properties
1.Isolation in pure form:
Studies on the biochemical, antigenic and other characters of
bacteria can be done only if the organism available in the pure
form.
Technique:
Plating on solid culture media - clinical sample is streaked onto
a solid medium (like: MacConkey agar, nutrient agar or blood
agar in such a way so as to ensure isolated discrete colonies.
Use of selective growth condition - most important example of
this is the growth of anaerobic bacteria which will not take place
in an environment having oxygen.
,2.Staining reaction:
The age of the culture is important. In older cultures, staining
characteristics either vary or are not brought out well. Simple
stains bring out the best morphology. Differential and special
stains are necessary to bring out characteristics like: gram
negative and gram-positive bacteria, Acid fast and nonacid fast,
spirochetes, capsule and Flagella, etc.
a. Gram stain - Gram stain divides the bacteria into Gram
positive & Gram negative.
The basic procedure is,
i. Take a heat fixed bacterial smear.
ii. Flood the smear with CRYSTAL VIOLET for 1 minute, then
wash with water. [PRIMARY STAIN]
iii. Flood the smear with IODINE for 1 minute, then wash with
water.
iv. Flood the smear with ETHANOL‐ACETONE, quickly, then
wash with water. [DECOLORI
v. Flood the smear with SAFRANIN for 1 minute, then wash with
water. [COUNTERSTAIN]
vi. Blot the smear, air dry and observe.
,Examine under microscope.
i. Gram positive bacteria ‐ violet
ii. Gram negative bacteria ‐ pink
Shapes of Bacteria:
Bacteria display three basic shapes:
i. Round‐ cocci, (from the Greek kokkos ‐ a berry),
ii. Rod shaped – bacilli (from the Latin bacillus ‐ a stick or rod),
iii. Spiral (quelled).
i. Coccus:
Staphylococcus species
Streptococcus species
, ii. Bacillus:
Clostridium species
Listeria species
Bacteria
The most important task of a bacteriology is to identify the
pathogens from the clinical sample so that appropriate treatment
can be instituted.
There are several methods to identified the different type of
bacteria.
They are,
1. Isolation in pure form
2. Staining reaction
3. Morphology of bacterial colony
4. Cultural characteristics
5. Metabolism
6. Biochemical properties
1.Isolation in pure form:
Studies on the biochemical, antigenic and other characters of
bacteria can be done only if the organism available in the pure
form.
Technique:
Plating on solid culture media - clinical sample is streaked onto
a solid medium (like: MacConkey agar, nutrient agar or blood
agar in such a way so as to ensure isolated discrete colonies.
Use of selective growth condition - most important example of
this is the growth of anaerobic bacteria which will not take place
in an environment having oxygen.
,2.Staining reaction:
The age of the culture is important. In older cultures, staining
characteristics either vary or are not brought out well. Simple
stains bring out the best morphology. Differential and special
stains are necessary to bring out characteristics like: gram
negative and gram-positive bacteria, Acid fast and nonacid fast,
spirochetes, capsule and Flagella, etc.
a. Gram stain - Gram stain divides the bacteria into Gram
positive & Gram negative.
The basic procedure is,
i. Take a heat fixed bacterial smear.
ii. Flood the smear with CRYSTAL VIOLET for 1 minute, then
wash with water. [PRIMARY STAIN]
iii. Flood the smear with IODINE for 1 minute, then wash with
water.
iv. Flood the smear with ETHANOL‐ACETONE, quickly, then
wash with water. [DECOLORI
v. Flood the smear with SAFRANIN for 1 minute, then wash with
water. [COUNTERSTAIN]
vi. Blot the smear, air dry and observe.
,Examine under microscope.
i. Gram positive bacteria ‐ violet
ii. Gram negative bacteria ‐ pink
Shapes of Bacteria:
Bacteria display three basic shapes:
i. Round‐ cocci, (from the Greek kokkos ‐ a berry),
ii. Rod shaped – bacilli (from the Latin bacillus ‐ a stick or rod),
iii. Spiral (quelled).
i. Coccus:
Staphylococcus species
Streptococcus species
, ii. Bacillus:
Clostridium species
Listeria species
