Microbiology Flashcards
(19 cards)
What are the three main shapes of bacteria?
Bacillus (rod shaped)
Coccus (spherical)
Spirrilus (spiral shaped)
How can bacteria be differentiated by the way they are grouped?
They may be single, in pairs, in chains or in clusters.
What are the steps of the Gram stain technique for identifying bacteria?
- Add purple crystal violet stain, which binds to the peptidoglycan layer in the cell wall.
- Add iodine to fix the dye (forms crystal violet/iodine complex).
- Wash with alcohol.
- Add red safranin counterstain.
What is the colour of staining of Gram-positive bacteria and why?
They stain purple as the stain cannot be washed out with alcohol, and so retains the purple crystal violet stain due to the thick peptidoglycan cell wall. The red counterstain isn’t taken up.
What are Gram-positive bacteria vunerable to?
Penicillin, due to the absence of the outer lipopolysaccharide layer.
What is the colour of staining of Gram-negative bacteria and why?
They stain pink as the initial stain is taken up but the alcohol destroys the lipopolysaccharide membrane, causing it to take up the counterstain.
What conditions do micro-organisms require for growth?
-A growth medium containing water (needed for all metabolic functions), a carbon source (usually glucose for respiration), a nitrogen source (to synthesise amino acids, proteins and DNA) and growth factors such as vitamins and mineral salts
-A suitable temperature, optimum for bacteria is 25-45C whereas mammalian pathogens are 37C
-A suitable pH, most bacteria favour slightly alkaline conditions whereas fungi grow better in neutral to acidic conditions
-Oxygen
What are the differing requirements for oxygen among different bacterium?
Obligate aerobes require oxygen for growth and metabolism.
Facultative anaerobes grow best in the presence of oxygen, but can survive in its absence.
Obligate anaerobes cannot grow in the presence of oxygen.
What is the practice of aseptic technique?
Where the apparatus and equipment are kept free of micro-organisms.
Why do we perform aseptic technique?
It prevents contamination of cultures by other microbes and contamination of the environment.
How can we prevent the contamination of pure cultures and apparatus by bacteria in the environment?
-Sterilise all apparatus and media before use to prevent initial contamination
-Handle cultures carefully, flaming the necks of culture vessels before opening and closing and use equipment such as sterile loops to prevent subsequent contamination.
How can we prevent contamination to the environment by the bacteria being used in the experiment?
-Sterilise the work surface before and after an experiment using a disinfectant.
-Use the correct handling techniques to prevent the contamination of personnel and the immediate environment, such as performing inoculation.
-Sterilise using an autoclave.
-After use, disposable materials, such as plastic Petri dishes, can be sealed inside autoclavable plastic bags, autoclaved and put in the bin.
What is an autoclave?
A sealed container in which glass and metal equipment are heated to 121C in steam under pressure for 15mins after the required pressure has been reached.
How is inoculation carried out?
- Hold the culture bottle in one hand, remove the cap with the little finger of the other hand. Do not place the cap on the work surface.
- Flame the mouth of the bottle for 2-3 seconds.
- Pass the inoculating loop through a flame until red hot, and allow to cool in the air.
- Lift the lid of the Petri dish just enough to allow entry of the inoculating loop.
- Secure the lid of the Petri dish with two pieces of adhesive tape. Do not seal all the way around as this could create anaerobic conditions and encourage the growth of pathogens.
- Do not open petri dish lid after inoculation.
What are the two ways we can assess the size of a population of bacteria directly?
-Viable counts, counting colonies of living cells only
-Total counts, using a haemocycler, count living and dead cells.
How can we assess the size of a population of bacteria indirectly?
Using a measure of turbidity of the culture with a colorimeter.
What is the equation to deduce the population size of bacteria?
Population size = (number of colonies x dilution factor) / volume of sample
What happens to the population of bacteria if the dilution factor is too great?
There will be too few colonies on each plate for the count to be statistically sound.
What happens to the population of bacteria if the dilution factor is insufficient?
Colonies clump together so counting may be inaccurate, resulting in an underestimate of numbers.
