Pure Culture Techniques

Pure CultureConsists of only a single type of organism.

When working in the laboratory, we need to work with a single species of organism.

And Pure cultures must be obtained artificially

In clinical laboratory used culturing and pure culture techniques for :

-Isolation of bacteria in pure culture and study the colony characteristics. - To demonstrate their properties. -To determine sensitivity to antibiotics. - To diagnose the bacteria by antigen and biochemical tests. - To counting of bacteria.

Pure Culture

Steps in obtaining a pure culture:

1 .Sterilization

2 .Isolation

Sterilization

The process of eliminating all microorganisms from lab, tools and growth medium

when handling microorganisms.All media used for growing organisms is sterilized (autoclaved)All glassware are sterilized in an autoclave at 121o C, at 15 pounds of pressure for about 15 minutesWork area should be treated with a disinfectantInoculation loops and tube lips should flamed

When a bacterium grows on a plate, the number of cells increases until a visible mass of cell, called a colony appears. Usually colony arises from a division of a single bacterial cell.

The colony can be considered a pure culture.

Isolation Microorganisms occur in huge numbers.

Isolation is done by: 1.Streak plate method

2 .Spread plate method

3 .Pour plate method

1.Streak Culture (Surface Plating): this method used for isolation of bacteria in pure

culture and used different methods:

A\ Quadrant Streaking ( Zigzage line) Divide your plate into (4) areas by drawing a (A,B,C,D) on outside bottom of the plate.

1.Transfer bacteria by platinum loop by touching bacterial colony.

2.Make 2-3 of parallel lines on area (A).3.Sterile your loop on flame and left it to cool. 4.Turn your plate 90° and make series parallel

lines on the area (B)

5.Sterile your loop and left it to cool.6.Turn your plate 90° and make series

parallel lines on area (C).7.Sterile your loop.

8.Turn your plate and make series parallel lines on area (D).

9.Sterile your loop.10.Make zigzag line from the end of last

parallel lines.11.Incubate at 37 °C for 24-48 hrs.

12.Observe pure colony culture.

Plate A is a good example of a streak platePlate B is a bad example of a streak plate

A B

B\ T – streak 1.Lable your plate. Divide your plate into (3) areas by drawing a (T) on outside bottom of the plate.2.Inoculate one an area using a single continuous motion.3. Sterile your loop and leave it to cool.4. Cross – streak that area three to four times and continue streaking into second area.5. Repeat the procedure in step 3,4 .6.Incubate at 37°C for 24-48 hrs.7.Observe the pure colony culture.

Triple Streak Method

C/Radiant Streak

1.Inoculate the bacteria with loop and make series parallel lines.

2.Sterile your loop.3.Turn your plate 90° and make series parallel

lines.4.Incubate at 37°C for 24-48 hrs.

5 .Observe the pure colony culture.

D/Continuous Streak

1 .Inoculate the bacteria by loop and make continuous streaking motion.

2 .Incubate at 37°C for 24-48 hrs.3 .Observe the pure colony culture.

There are several methods we can make to ensure the purity of the culture:Re streaking : isolate colony from streak plate should be only a single type of isolated colony by:

1.The colony should be only contain a single morphological colony type.

2.Microscopic examination should be demonstrate only one type of cells.

2 /Spread – plate technique:

Bacteria usually grow together in population containing a number of species. So spread – plate technique is an easy method to isolate bacteria in a single colony.Procedure:

1.Transfer 0.1ml of bacteria to the surface of agar.2.Dip L- shape glass into a beaker of ethanol and then spreaded

through the flame of burner and allow to cool on the agar surface.3.Spread bacteria over the agar surface and ensure the surface of

plate has been covered.4.Invert the plate and incubate at 37 °C for 24 – 28 hrs.5.After incubation observe the morphology of colonies.

3 /Pour plate culture:

1 .15ml of agar melted and left to cool to 45 °C.

2 .Diluting of fraction of culture is series dilution.

3.Adding 1ml of every dilution to the molten agar and mixed well.

4 .Poured into a sterile petri dish and allow to solidify.

5.Incubate at 37 °C for 24-48 hrs and observe the colonies of bacteria on the agar surface.

Pour Plate

Culture characteristics: Description of colonies of bacteria.

1 .Shape : circular, irregular, rhizoid.2 .Size : surface of colony is measured by millimeter range

(2-3 mm )or the colony very small about )0.5-1 mm(.3.Elevation : convex, convex papillae or raised with flat

surface.4.Edge : entire, lobate or fimbriate.

5.Colour : like green color by psendomonas aeruginosa.6.Opacity : transparent or opaque.

7.Consistency : hard, firm, soft or mucoid.

Colony Morphology

Colony morphology

Color

Shape

size

Margin

Elevation

Opacity

Consistency

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