Fwr Bacteriology

Topics: Staining, Bacteria, Bacterial cell structure Pages: 21 (6737 words) Published: July 8, 2013
Exercise 8-B
Differential Staining
Gram Staining and Acid Fast Staining
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 steam is coming up and the water is barely boiling. 2. Place the staining rack over the beaker.

3. Place the prepared slides given (Mycobacterium tuberculosis smear) over the staining rack and put a small piece of paper towel on top of the smear on the slide. 4. Flood the smear with carbol fuschin (primary dye) and leave for 5 minutes. (Keep the paper towel moist with carbol fuschin) 5. Remove and discard the paper towel in the garbage, wash the slide with water. 6. Add acid alcohol (1%HCl + Ethanol) and decolorize for 15-20 seconds. 7. Wash with water.

8. Flood the smear with methylene blue (counterstain) and leave for 1 minute. 9. Wash with water and air dry.
Microscopic Examination
1. Examine the stained bacterial smears under Oil Immersion Objective of the bright field light microscope. 2. Draw and write down observations.
Group Results:
Bacterial Smear A| Bacterial Smear B|
S. aureus| E.coli|
Gram Reaction: Gram Positive (Purple)Morphology:Round/ Cocci in Cluster| Gram Reaction: Gram Negative (Pink)Morphology: Rod shape | Bacterial Smear A| Bacterial Smear B|
M. tuberculosis| |
Acid Fast Reaction: Negative (Blue)Morphology: Rod shape| Acid Fast Reaction:Morphology:| Discussion and Conclusion:
Differential Staining is a staining procedure which differentiates or distinguishes between types of bacteria, methods for simple staining impart same color to all bacteria and other biological material may be slight variation in shade. On the other hand, differential staining methods impart distinctive color only to certain types of bacteria. The basic principle underlying this differentiation is due to the different chemical and physical properties of cell and as a result, they react differently with the staining reagents. Differential staining procedure utilizes more than one stain. In some techniques the stains are applied separately, while in other as combination. There are two most important differential stains,...

References: http://www.amrita.vlab.co.in/?sub=3&brch=73&sim=1338&cnt=1
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