Bacterial Culturing Methods & Staining Techniques
Culturing methods
&
Bacterial staining
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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.
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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)
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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
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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.
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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)
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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 .
Learn about culturing methods for isolating microorganisms and staining techniques to study bacteria's shapes, species, and internal components. Explore differential, selective, and simple staining methods like Gram staining and Acid-fast stain.
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Presentation Transcript
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 .
