3.9 Methodology of Bacterial Identification
51
distilled water each and marked as 10
−1 –10
−9 were taken. 1 mL of sample solution
was taken out using pipette from the conical flask and was mixed with 10-1 test tube.
Again 1 mL diluted sample was taken from 10-1 marked test tube and was mixed
with 10-2 marked test tube. Like this, serial dilution process for 10
−1 to 10
−9 dilution
in nine (9) test tubes was performed.
3.9.1.3 Preparation of Agar Plate and Spreading
Composition of Agar Medium
• Beef extract—1.5 gm/500 mL
• Peptone—2.5 gm/500 mL
• Sodium chloride—2.5 gm/500 mL
• Distilled water—500 mL
• Agar—8 gm/500 mL
• pH—7.0–7.2.
All compositions were mixed well in a 1000-mL beaker and were sterilized in
an auto-clave under 15 lb/sq. inch pressure at saturated steam condition and 121 °C
temperature for 20–25 min. 10 mL of sterile agar medium was poured into each 9
number of sterile Petri dishes. After solidifying, the plates were marked as 10
−1 –
10
−9 , respectively. Then, 100 micro-liters of each sample dilution from test tubes was
poured and spread over the respective dilution marked Petri dish. Thus, the dilution
sample was poured and spread over 10
−1 to 10
−9 marked Petri dishes. Then, the Petri
dishes were incubated at 37 °C for 24 h.
3.9.1.4 Observations
Colonies were developed upon the Petri dishes within the incubation period. The
colonies were counted and marked in corresponding Petri dishes.
3.9.1.5 Gram Staining of Bacteria
Gram Staining Theory
One of the most important and widely used differential staining techniques in microbiology is gram staining. This technique was introduced by Christian Gram in 1884.
In this process, the fixed bacterial smear is subjected to the following staining reagents
in the order listed: crystal violet, iodine solution, alcohol (decolorizing agent) and
staining. Gram-positive bacteria retain the crystal violet and hence appear deep violet
in color, and Gram-negative bacteria, which loss the crystal violet, are counterstained
by the safranin and hence appear red in color.
51
distilled water each and marked as 10
−1 –10
−9 were taken. 1 mL of sample solution
was taken out using pipette from the conical flask and was mixed with 10-1 test tube.
Again 1 mL diluted sample was taken from 10-1 marked test tube and was mixed
with 10-2 marked test tube. Like this, serial dilution process for 10
−1 to 10
−9 dilution
in nine (9) test tubes was performed.
3.9.1.3 Preparation of Agar Plate and Spreading
Composition of Agar Medium
• Beef extract—1.5 gm/500 mL
• Peptone—2.5 gm/500 mL
• Sodium chloride—2.5 gm/500 mL
• Distilled water—500 mL
• Agar—8 gm/500 mL
• pH—7.0–7.2.
All compositions were mixed well in a 1000-mL beaker and were sterilized in
an auto-clave under 15 lb/sq. inch pressure at saturated steam condition and 121 °C
temperature for 20–25 min. 10 mL of sterile agar medium was poured into each 9
number of sterile Petri dishes. After solidifying, the plates were marked as 10
−1 –
10
−9 , respectively. Then, 100 micro-liters of each sample dilution from test tubes was
poured and spread over the respective dilution marked Petri dish. Thus, the dilution
sample was poured and spread over 10
−1 to 10
−9 marked Petri dishes. Then, the Petri
dishes were incubated at 37 °C for 24 h.
3.9.1.4 Observations
Colonies were developed upon the Petri dishes within the incubation period. The
colonies were counted and marked in corresponding Petri dishes.
3.9.1.5 Gram Staining of Bacteria
Gram Staining Theory
One of the most important and widely used differential staining techniques in microbiology is gram staining. This technique was introduced by Christian Gram in 1884.
In this process, the fixed bacterial smear is subjected to the following staining reagents
in the order listed: crystal violet, iodine solution, alcohol (decolorizing agent) and
staining. Gram-positive bacteria retain the crystal violet and hence appear deep violet
in color, and Gram-negative bacteria, which loss the crystal violet, are counterstained
by the safranin and hence appear red in color.