Culturing methods Bacterial staining LAB 5 A L
Culturing methods & Bacterial staining LAB. 5 A. L. NOOR AMEER
Purpose of culturing is: 1 - Isolation of microorganisms from a sample. 2 - For counting the microorganism in the sample. 3 - Obtain pure cultures. 4 - To test for antibiotic sensitivity.
Culture methods include: 1 - Streaking plates: Used for the isolation of bacteria in pure culture from clinical specimens (to obtain pure colonies), include: a- ABCD method: (by using loop). b- Continuous streaking (by using loop).
2 - Streaking of the slant: (by using loop). 3 - Stab culture (by using needle). 4 - Lawn culture: (by using swab). 5 - Pour plate method. 6 - Spread plate method (glass spreading rod)
Bacterial staining The bacteria are stained for the following reasons: 1 - To study their shapes 2 - To differentiate the species of bacteria by using differential stain 3 - To study the internal components of the bacterial cell
Based on the function of stain is divided into: 1 - Simple stain: using of a single dye to staining the bacteria as methylene blue, safranin, and crystal violet can be used to determine cell shape, size, and arrangement. 2 - Differential stain: using of more than one dye used to differentiate between different bacteria 3 - Selective stain: using of more than one dye to determine the special structure such as spore, capsule, flagella, cell wall & nucleic acid staining.
Differential stain 1 - Gram staining: used to differentiate bacteria into 2 large groups Gram positive which are blue- purple in color and Gram negative bacteria are pink- red in color - Include stains: 1 - Primary stain (crystal violet). 2 - A mordant: (iodine solution). 3 - A decolorizing agents: ( alcohol, acetone). 4 - Counter stain: (safranin)
2 - Acid- fast stain (Ziehl- Neelson stain): used to determine acid fast organisms that causes tuberculosis (Mycobacterium tuberculosis) which appear as pink bacilli - Include stains: 1 - Primary stain: (carbol fuchsin). 2 - A decolorizing agents: ( acid alcohol). 3 - Counter stain: (methylene blue)
Selective stain 1 - Endospore stain: used to distinguish between the vegetative cells and the endospores. A primary stain (malachite green) is used to stain the endospores while the safranin used as counterstain, the vegetative cells will appear pink and the spores will appear green. Ex: Bacillus & Clostridium. 2 - Capsule staining: used to observe bacterial capsule of Klebsiella pneumonia or any bacteria have a capsule by using primary stain (crystal violet for 2 minutes), the encapsulsated cells will have a halo appearance under the microscop
3 - Flagella stain: flagella are fine, threadlike organelles and usually invisible under light microscope to observe it must be bind with chemical substances such as tannic acid & potassium alum. to increasing the thickness of flagella and then staining with basic fuchsin or silver nitrate.
- Slides: 10