Aim – Culture the bacteria from soil sample by using nutrient agar media.
Material Required – Soil Sample, Inoculation loop, Petri plates, Test Tubes, Bunsen Burner, Alcohol, Beaker.
Principle - Nutrient agar is made with various nutrients which allow the growth of a wide variety of
microorganisms that do not usually require specific nutrients or supplements. The primary constituents of the
media are peptone, beef extract, and agar. Agar is the solidifying agent that provides a stable surface for the
organism to grow.
Composition Gram/litre
Peptone 5.0
Beef Extract 1.5
Sodium Chloride (NaCl) 1.5
Agar 15.0
Distilled Water 1000 ml
Procedure –
1. Measure the composition in the beaker and volume makeup up to 1000 ml.
2. The suspension is then heated to boiling to dissolve the medium completely.
3. The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes.
4. Once the autoclaving process is complete, the beaker is taken out and cooled to a temperature of about
40-45°C.
5. The media is then poured into sterile Petri plates under sterile conditions.
6. Once the media solidifies, the plates can be placed in the incubator over for 48 hours.
Result –
The media forms light yellow coloured clear to slightly opalescent gel (white powder like appearance) on Petri
plates
Aim – To Perform the Streak Plate Technique to culture the bacteria
Principle
• The principle of the streak plate technique is mechanical dilution.
• When an inoculating loop containing the microbial sample is streaked across the surface of a solid agar
medium, the microbial cells are gradually diluted.
• As streaking continues, fewer and fewer microorganisms are deposited from the loop.
• By the final streaks, individual cells are separated far enough apart to grow into isolated colonies.
• Each isolated colony originates from a single cell (clonal growth) and therefore represents a pure culture.
Requirements
• Nutrient agar plate (or any selective/differential medium depending on purpose).
• Inoculating loop (sterilized by flaming).
• Bunsen burner or spirit lamp (for aseptic technique).
• Microbial culture (broth or plate).
• Incubator.
Procedure
1. Preparation
• Sterilize the inoculating loop by heating in the flame until red hot and allow it to cool.
• Hold the agar plate in one hand and the loop in the other under aseptic conditions.
2. Inoculation
• Dip the sterilized loop into the microbial culture (broth or colony).
• Lift the lid of the agar plate slightly (to prevent contamination) and streak the loop lightly over one
quadrant of the agar surface (called the primary inoculum area).
3. First Streak
• Cover about 1/4th of the plate by streaking back and forth closely. This area contains the heaviest
concentration of microorganisms.
4. Flame and Cool
• Sterilize the loop again (flame and cool).
• Rotate the plate slightly (90°).
5. Second Streak
• From the edge of the first streaked area, drag the loop into a fresh area of agar (quadrant 2) and streak
again. This spreads out and dilutes the cells.
6. Third and Fourth Streak
• Repeat flaming and cooling.
• Continue streaking into quadrant 3 and quadrant 4, each time pulling from the previous area into the new
one.
• Each quadrant has fewer cells, leading to isolated colonies.
7. Incubation
• Close and invert the agar plate (to prevent condensation on colonies).
• Incubate at the appropriate temperature (usually 25–37°C, depending on organism).
Result
• The first quadrant shows confluent growth (dense).
• The second and third quadrants show reduced growth.
• The last quadrant shows well-isolated colonies, each arising from a single bacterial cell.