Techniques for isolation of pure culture

The following are a viable techniques that used to isolate pure colonies:

1. The streak –plate method:

· In this technique, the bacterial mixture is transferred to the edge of an agar plate with an inoculating
loop and then streaked out over the surface in one of several patterns. At some point on the streaks,
individual cells will be removed from the loop as it glides along the agar surface and will give rise to
separate colonies Again, one assumes that one colony comes from one cell. The key principle of this
method is that by streaking, a dilution gradient is established on the surface of the plate as cells are
deposited on the agar surface.

· Procedure

1. Flame the inoculating loop to red-hot, allow it to cool near burner in air.

2. Hold the your isolated culture ( previous exercise) in left hand near the flame. Open the lid of the
plate just enough to insert wire loop. Aseptically withdraw a loopful culture with loop.

3. Place the inoculum on the agar plate at least 1 cm away from sides. Spread it in two to three square
cm areas.

4. With sterile cool needle streak or spread the culture from one corner to another and rotating the
plate by 90° after streaking 4-5 times in one direction without overlapping previous streak as
demonstrated by the instructor or shown in picture as page 31

Note:Pure culture :mean laboratory culture containing a single species of organism

Isolate colonies:population of millions of cells that are identical

Stock culture:culture that already contain cells or microorganisims and we take it as source to inoculate
new culture
2. Pour plate method

· In this method we will be able to understand the pour plate method in which the bacteria are mixed
evenly distributed and separated evenly in the throughout the liquid. The method agar is then poured
into an empty plate and allowed to solidify. After inoculation discrete bacterial colonies can then be
formed growing both on the agar media and in agar then on the isolated colonies can be a separately
picked of the isolation plate and transfer to new sterile medium. One advantage of this method is that it
requires somewhat less skill than that required for a good streak plate; a disadvantage, however, is that
it requires more media, tubes, and plates
3. Spread plate method

· In the streak plate method of isolating colonies, you diluted the bacterial culture on the agar plate. In
the spread plate method, you would dilute the bacterial culture in tubes and then transfer the diluted
samples to multiple agar plates. In a sample that is adequately diluted, the cells will be spread far
enough apart on the agar that they will grow into individual colonies.

FigurePure culture: laboratory culture containing a single species of organism

Figure 9: A comparison between the pour plate method and the spread plate method

Note : we use the streak method to make isolate colonies or pure colony

And we use pure and spread method to count the colony in sample
Attention: spread method and pour plate method