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Total Marks / 45
Assignment 1
1. Explain the principle of positive staining.
(3 marks)
Positive staining is done by staining the cell walls of Gram-positive bacteria with crystal violet. The cell wall of Gram-positive bacteria is made up of a thick layer of peptidoglycan which undergoes dehydration during decolourisation causing the pores to shrink. This shrinkage of pores traps CV-I complex and stain the cell purple.
2. Using a flow chart, outline the process of gram staining.
(5 marks)
Flood the slide with crystal violet for 1 minute then rinse with water
Flood the slide with iodine for 1 minute then rinse with water
Flood the slide with solution of ethanol and acetone for 10 to 30 seconds then rinse with water
Flood the slide with safranin for 1 minute then rinse it with water
Blot dry the slide
3. What is the function of iodine when used in gram staining?
(2 marks)
Its function is to form large crystals with crystal violet so that they stick to the bacterial cell wall and are not washed away easily.
4. Attach photos on JPEG format showing a gram positive and gram negative bacterial sample showing their gram staining reaction. Give the name of the bacterial cells, shapes and arrangement.
(9 marks)
Bacillus cereus: Rod shaped and chain arrangement, Gram positive http://aapredbook.aappublications.org/content/1/SEC131/SEC143/F178.large.jpg Neisseria gonorrhoea: Coffee bean shaped and diplococcus arrangement, Gram negative http://upload.wikimedia.org/wikipedia/commons/2/2e/Gonococcal_urethritis_PHIL_4085_lores.jpg 5. Explain the principle of capsule staining using both a basic and acidic dye.
(5 marks)
Capsule staining with basic dye is basically to stain the bacterial cells within the capsule without staining the outer part. Capsule staining with acidic dye is to stain the background without it penetrating into the capsule.
6. Using a flow chart, outline the process of endospore staining.
(5 marks)
Prepare smear of the bacteria to be stained
Heat-fix the smear
Place a piece of filter paper over the smear
Saturate the paper with malachite green
Heat the slide to steaming for 5 minutes to drive the dye into the endospores
Remove the paper and wash the slide with distilled water to remove excess dye
Counterstain the smear with safranin which it will stain the vegetative cells red-pink
Wash the slide with distilled water and blot dry it
7. What will be the appearance of bacteria that possess endospore? Attach a JPEG photo each of cells with and without endospores after endospore staining.
(5 marks)
8. Name two genus of bacteria that possess endospore.
(2 marks)
Bacillus. Clostridium.
9. What is acid-fastness?
(2 marks)
Acid-fastness is a physical property of some bacteria which have resistance to decolourisation by acids during staining.
10. Using a flow chart, outline the process of acid-fast staining.
(5 marks)
Prepare smear of the bacteria to be stained
Heat-fix the smear
Apply carbol fuchsin to the heat-fixed smear
Heat the slide to steaming for 5 minutes to drive the dye into the cell
Wash the slide with distilled water
Treat the slide with acid-alcohol solution to decolourise both non acid-fast cells and background
Counterstain with methylene blue
Wash the slide with distilled water and blot dry it
Acid-fast bacteria will appear pink in colour while non-acid-fast bacteria will appear blue in colour
11. What is the possible reason why bacteria that exhibit acid-fastness stained poorly with gram stains?
(2 marks)
Bacteria that exhibit acid-fastness have high lipid content in their cell wall resulting in a waxy texture. As such, only a small amount of gram stains will be able to enter and stain the cell wall
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