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. IsolationSterilization
The process of eliminating all microorganisms from lab, tools and growth mediumwhen 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 minutes Work area should be treated with a disinfectant Inoculation 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 methodPlate A is a good example of a streak plate Plate B is a bad example of a streak plate
AB
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
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.
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.