Fwr Bacteriology
Exercise 8-B
Differential Staining
Gram Staining and Acid Fast Staining
Introduction:
Differential Staining, one which facilitates differentiation of various elements in a specimen is a general term that can refer to a number of specific processes. Using multiple stains can better differentiate between different microorganisms or cellular components of a single organism. Gram’s Stain is a widely used method of staining bacteria as an aid to their identification. It was originally devised by Hans Christian Joachim Gram, a Danish physician. Gram’s Stain differentiates between two major cell wall types. Bacterial species with walls containing small amounts of peptidoglycan, and, characteristically, lipopolysaccharide, are gram negative whereas bacteria with cell wall containing relatively large amounts of peptidoglycan and no lipopolysaccharide are gram positive. The acid-fast stain is a differential test to identify the presence of a certain types of bacteria in a given sample. It is a laboratory test to differentiate the components of a substance and to determine if it contains acid fast bacteria such as mycobacterium.
Procedure:
Bacterial Smear Preparation 1. Prepare 2 Bacterial Smears of each broth culture of bacteria labelled A and B
Gram’s Staining 1. Flood the two bacterial smears with crystal violet (primary dye/stain) for 10 seconds. 2. Wash the smears with running water. 3. Flood with gram’s iodine (mordant) and wait for 10 seconds, then wash with running water 4. Decolorize the smear with 95% ethanol until the thinnest parts of smear are colorless then wash with running water. ( Note: This is the most critical and the most affected by technical variations in timing and reagents) 5. Counterstain it using Safranin and wait for 10 seconds. 6. Wash with water then air dry.
Acid Fast Staining 1. Heat the beaker with water using hot plate ( since hot plate is not available, use tripod stand and alcohol lamp) and wait until
Differential Staining
Gram Staining and Acid Fast Staining
Introduction:
Differential Staining, one which facilitates differentiation of various elements in a specimen is a general term that can refer to a number of specific processes. Using multiple stains can better differentiate between different microorganisms or cellular components of a single organism. Gram’s Stain is a widely used method of staining bacteria as an aid to their identification. It was originally devised by Hans Christian Joachim Gram, a Danish physician. Gram’s Stain differentiates between two major cell wall types. Bacterial species with walls containing small amounts of peptidoglycan, and, characteristically, lipopolysaccharide, are gram negative whereas bacteria with cell wall containing relatively large amounts of peptidoglycan and no lipopolysaccharide are gram positive. The acid-fast stain is a differential test to identify the presence of a certain types of bacteria in a given sample. It is a laboratory test to differentiate the components of a substance and to determine if it contains acid fast bacteria such as mycobacterium.
Procedure:
Bacterial Smear Preparation 1. Prepare 2 Bacterial Smears of each broth culture of bacteria labelled A and B
Gram’s Staining 1. Flood the two bacterial smears with crystal violet (primary dye/stain) for 10 seconds. 2. Wash the smears with running water. 3. Flood with gram’s iodine (mordant) and wait for 10 seconds, then wash with running water 4. Decolorize the smear with 95% ethanol until the thinnest parts of smear are colorless then wash with running water. ( Note: This is the most critical and the most affected by technical variations in timing and reagents) 5. Counterstain it using Safranin and wait for 10 seconds. 6. Wash with water then air dry.
Acid Fast Staining 1. Heat the beaker with water using hot plate ( since hot plate is not available, use tripod stand and alcohol lamp) and wait until
References: http://www.amrita.vlab.co.in/?sub=3&brch=73&sim=1338&cnt=1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC273886/ http://www.projects.itn.pt/Luisa%20Alves/Bibliografia%20citada%20na%20proposta/staining%20method%20for%20fluorescence%20viable%20and%20total%20bacteria.pdf