Microbiology Flashcards
Describe gram staining
The Gram stain is one of the most important initial steps in identifying the bacterium responsible for infection. It is quick and easy and helps distinguish between two different groups of bacteria (see examples below); Gram-positive which stain PURPLE, and Gram-negative which stain RED.
Describe the gram stain protocol
Transfer a colony from the sample to the slide using a sterile loop and place a drop of water on the slide
Dry gently and then add a few drops of crystal violet, leaving for a minute
Gently rinse before adding lugol’s iodine for a minute before rinsing again
Cover the slide with alcohol before immediately rinsing and staining with safranin
What should you look for when looking at bacteria
Colour of bacteria in the Gram stain
Gram positive (+) = purple
Gram negative (-) = red
Shape of bacteria, are they rod-shaped (bacillus) or spherical (coccus)
Are they grouped together in clusters or chains, or single bacteria?
Describe MacConkey agar plates
If the plate is red/pink then the bacteria are a lactose fermenter.
If the plate is colourless/yellow then the bacteria are a lactose non-fermenter.
What are haemolysins
Some pathogens produce haemolysins, substances that lyse/destroy red blood cells in blood-containing media, in a process called haemolysis.
Describe the different types of haemolysins
There are three different types of haemolysis:
Alpha (α) = very small zone of haemolysis and colonies can appear green
Beta (β) = large clear zone of haemolysis around the bacterial colonies
Gamma (γ) = no haemolysis
Describe the oxidase test
The oxidase test is used to determine if an organism produces the enzyme cytochrome c oxidase. Oxidase-positive bacteria are aerobic and can use oxygen as an electron acceptor during respiration.
The test strip contains tetra methyl-p-phenylenediamine (TMPD) which is a redox indicator (a chemical that changes colour depending on the oxidation state of the molecule) and acts as an artificial electron acceptor in this test. It is colourless when reduced and changes to dark blue/purple when oxidized.
Describe the protocol for the oxidase test
Use the strips provided which test for cytochrome oxidase activity
Rub the paper pad on the strip onto bacterial colonies on the agar plate
Watch for a colour change, if the strip turns purple then the bacteria are oxidase positive
Place all waste in the waste pots provided
Describe the catalase test
Hydrogen peroxide is a toxic end-product of the aerobic breakdown of sugars. Catalase is a common enzyme found in many living organisms and can break down hydrogen peroxide into gaseous oxygen (which produces bubbles) and water
Describe the protocol for the catalase test
You are provided with an aliquot of hydrogen peroxide
Pick a loopful of bacteria using the plastic loop and mix it in the hydrogen peroxide
Do you see bubbles? If so, then the bacteria are catalase positive
Place all waste in the waste pots provided
Describe the coagulase test
his test checks for the presence of the enzyme coagulase (a clumping factor) which reacts with fibrinogen to form clumps or clots. The coagulase test is used to distinguish different types of Staphylococcus.
Describe the protocol for the coagulase test
Shake the test reagent from the kit thoroughly.
Add 1 drop of the Test reagent to the test circle.
Using a plastic loop, pick bacterial colonies (they should be clearly visible on the loop) and mix with the reagent in the test circle.
Do you see clumping/clots? If so, then the bacteria are coagulase positive. This indicates the presence of Staphylococcus aureus.
Place all waste in the waste bags/pots provided.
Describe the ways in which antibiotic sensitivity can be measured
Measuring the zone of inhibition surrounding antibiotic discs on a bacterial plate
Zone of inhibition (diameter in mm)
Using the E-Test to measure the Minimum inhibitory concentration (MIC) for a specific antibiotic (gives the exact concentration of antibiotic needed to kill/inhibit bacterial growth).
How do you decide which antibiotic to use
To determine which antibiotic to use you should use to treat patient B, it is not necessarily the one giving the bigger the zone (as different concentrations of antibiotics are used and they all have different MICs for your test organism), but the one giving the bigger difference between the actual zone of inhibition diameter on your plate and the reference sensitivity number.
Describe antibiotic sensitivity
measuring the size of the clear zone allows the level of resistance to be determined
