Isolation of Microorganisms
Isolated colony
How isolated colonies on late look like?
Pour plate method for enumeration of bacteria
How to make serial dilutions?
1 ml of original culture is added into 9 ml of sterile distilled water to make initial 1:10 dilution using sterile pipette under aseptic conditions. 1 ml of freshly prepared culture (1:10) is added into 9 ml of sterile distilled water to make initial 1:100 dilution. These steps are repeated for number of times.
One can make double serial dilution by adding 0.1 ml of original culture into 9.9 ml of sterile distilled water (1:100). Repeating this step from newly prepared dilution will give you 1:10000 dilution.
You can play with number tubes and volumes:
To make 1:1000 dilution: initial double dilution (1:100) followed by single dilution (1:10). this will save time and materials.
How to perform Pour plate method?
One has to make serial dilutions of original culture suspension as required. 0.1 ml of diluted culture is inoculated into sterile melted agar in a tubes cooled to a temperature that does not harm microorganisms under aseptic conditions. The content is mixed gently and poured into either basal agar plate or empty sterile petriplate. After solidification of agar, plates are incubated.
Staining techniques and results
Gram's Staining
What are the steps involved in Gram's Staining?
Prepare a thin ans uniform smear of bacterial suspension on clean and grease free slide. Allow the smear to air dry and heat fix it by passing it through flame twice or thrice. Cover this smear with Gention violet for 1 min and then rinse with water. Apply Gram's Iodine for 1 min and then rinse with water. Decolorize the smear with 70% ethanol till blue color disappear from slide. Wash it with water and apply saffranin for 1 min. After rinsing with water and dry in air.
Note: stained slide should appear clean without any precipitates on its surface.
Endospore staining
Endospore staining
4 days old bacterial culture is mixed with an equal volume of ZNCF (Zeil Nelson Carbol Fuchsin) stain. This mixture is kept in boiling water bath for 10 mins. Loop full of this mixture is mixed with a loop full of negrosine dye. A thin and uniform smear is prepared and allowed to dry.