Date: _____________

EXPERIMENT NO. 14

OBJECT: To perform culturing of bacteria by streak-plate method: (a) one-way streaking (b)
two-way streaking (c) three-way streaking (d) four-way streaking.

THEORY:
Streaking:
In microbiology, streaking is a technique used to isolate a pure strain from a single species of
microorganism, often bacteria.

Importance of Streak-Plate Method:

The streak-plate method is a microbiological technique used to isolate individual colonies of
bacteria from a mixed culture, typically by spreading the organisms thinly over the surface of an
agar plate in a pattern that dilutes the sample and allows for individual colonies to grow separately.

Bacteria are found everywhere, in water, soil, food, and on our bodies. To study them, it's crucial
to isolate a single species. This helps identify the bacteria causing diseases and understand their
characteristics.

Growth Medium:
Bacteria need different nutrients to grow. This includes water, a source of carbon, sulfur, nitrogen,
phosphorous, certain minerals, vitamins and growth factors. A very common type of media use in
Microbiology labs is known as “Agar”, a gelatinous substance derived from sea weed. It allows a
wide range of microbial growth.

Incubation:
Incubation is the process of keeping a bacterial plate, in a controlled environment (usually warm)
to encourage growth or development.

TIPS BEFORE STREAKING A CULTURE:

• Maintain aseptic conditions to prevent contamination.

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• Ensure the agar medium in the petri dish has solidified properly.
• Use a small amount of inoculum on the wire loop.
• Handle the petri plate and wire loop carefully near the flame.
• Follow a consistent streaking direction.
• Sterilize the wire loop after each streak by heating until red hot.
• Minimize the time the petri plate lid is open.

REQUIREMENTS:
 Mixed culture of bacteria
 Sterile petri dish with appropriate bacterial media (such as tryptic soy agar, nutrient agar).
 Inoculating loop (usually nichrome, a nickel-chromium alloy, or platinum; it may also be a
single-use disposable plastic loop, which would be discarded between sectors rather than re-
sterilized)
 Bunsen burner
 Marking pen

PROCEDURE:
1. Label the bottom of a Petri dish with organism name, agar type, date, and initials. Mentally
divide the plate into four sectors.
2. Flame the wire loop until red-hot. Allow it to cool without waving or blowing on it.
3. Streak the Plate:
a. Open the agar plate lid slightly.
b. Streak the mixed culture on nutrient agar, moving the loop across the first sector in a
single direction.
c. Turn the plate 90 degrees clockwise. Streak the second sector using the loop starting
from the first streak and moving across.
d. Repeat steps b and c for the third and fourth sectors, ensuring each streak overlaps the
previous one.
4. Incubation: Incubate the plate at 35°C for 18-24 hours.
5. Examine isolated colonies the next day for characteristics like shape, size, and color.

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RESULT:

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