Exercise 2: Morphology of Selected Bacteria Flashcards
Why was agar used in preference to gelatine for making solid media?
- Agar remains solid at the optimum temperature for the growth of most bacteria (37 degrees celcius), while gelatin melts at 35 degrees celcius
- Gelatin can also be digested by gelatinases, a protease produced by some bacteria.
Why do well-separated colonies appear larger than those in areas of heavy growth?
Well separated colonies have greater access to nutrients and less competition than colonies that are close together. They can therefore grow to greater sizes and are less hindered by nutrient depletion.
Explain why one individual colony does not always:
a) consist of one type of bacterium
b) originate from a single parent cell
a) Contaminants may be present under the colony
b) As colonies may arise from a multitude of parent cells. Moreover, multiple individual colonies can fuse to form a larger colony.
Why do you pass the dried smear through the bunsen flame?
- Heat fixing kills the bacteria on the slide without seriously interfering with cell structure
- It prevents the bacteria from washing off the slides during dying process (Ensures that the organism adheres to the slide)
What would happen if you used a whole colony to make a smear?
- On the slide there would be too much overlap between bacteria and it would be difficult to distinguish individual cells.
- Dye would have trouble diffusing across the cell which would mean that it would not effectively de-colourise the bacteria
Is there an association between cell shape and gram stain?
- No, as gram staining depends on the nature of the peptidoglycan layer of the cell
What structure of the bacterial cell determines its gram reaction?
The nature of the peptidoglycan layer
Explain why each step of the gram staining procedure is crucial in determining the outcome?
- Crystal violet - enters all cells
- Gram’s iodine - enters all cells and binds to the crystal violet
- Alcohol - disrupts the wall of gram negative bacteria and causes the crystal violet - iodine complex to leave the cell
- Carbol fuchsin - gram negative bacteria stain pink. This pink colour is not shown in gram positive bacteria which still retain the purple.
Why do some organisms form clusters and others form chains?
Depends on plains that they divide on during binary fission.
Clusters: divide on two plains
Chains: divide on a single plain
Describe the shape and arrangement of endospores. Why are they difficult to stain?
- Endospores involve the formation of a coat and an exosporium surrounding the DNA of a spore-forming micro-organisms
- They are difficult to stain because of the density of their outer coat. They are designed to resist extreme conditions and are highly acid resistant. Most dyes are made of acid, which makes colouring endospores difficult
- The Schaffer and Fulton method is used to stain for spores, with malachite green causing the spores to stain green.
Discuss the gram stain and Kinyoun’s Stain. Describe the cell wall structure of the Mycobacterium sp. and Bacillus sp. and relate this to the use of this particular staining method.
The gram stain is used to stain organisms such as those within the Bacillus sp. which have a gram positive rod shaped nature. This means that they have a thick peptidoglycan layer that can retain the crystal violet-iodine complex during the gram stain.
Mycobacterium sp. on the other hand have a cell wall that primarily consists of lipids such as mycolic acid. It must be stained via the Kinyoun’s stain method which involves carbon fushin and a counterstain. Acid fast bacteria such as Mycobacterium tuberculosis show up red on this stain.
What main advantages do capsules confer? What stain is used to detect them?
- Capsules enable immune evasion and retain cell shape during osmotic pressure.
- Capsules are detected via the Maneval’s Stain which uses a stain called Congo Red. The capsule appears as a red halo around the cell.
What main advantages do capsules confer? What stain is used to detect them?
- Capsules enable immune evasion and retain cell shape during osmotic pressure.
- Capsules are detected via the Maneval’s Stain which uses both Congo red stain and Maneval’s stain. The capsule appears as a clear halo around the organism.
Describe the MAST method and the feature of bacteria that it is used to identify
- It identifies the motility of bacteria
- The MAST method is based on the technique of using a stab-culture of semi-solid medium
- Non-motile organisms -> form a bright red colour restricted to the track of the inoculation stab
- Motile organisms -> produce a diffuse pink colouration throughout the medium
What is the most critical step in Gram staining?
- The decolourising with 95% alcohol
