Practicals Flashcards
To prepare the practical test
What is the procedure for a Gram stain?
- prepare and fix the film
- place the slide om rack in sink
- flood slide with methyl violet; allow to act for 1 minute
- wash with water
- flood the slide with iodine solution; allow to act for 1 minute
- drain of excess iodine and decolourise with acetone
- wash quickly under tap
- counter-stain with basic fuchsin; allow to act for 30 seconds
- blot and dry
gram +ve bacteria stain purple, due to thick layer of peptidoglycan
gram -ve bacteria stain pink, du to thin layer of peptidoglycan
What is the catalase test? Why is this test useful in clinical microbiology?
used to detect the presence of catalase, an enzyme which converts hydrogen peroxide to oxygen and water.
carried out by taking a colony and dipping it into 3% hydrogen peroxide solution. A positive reaction is indicated by the presence of oxygen bubbles
the catalase test is an important test for identifying and distinguishing pathogens.
As hydrogen peroxide is an ROS, a microbe with catalase is more likely to be pathogenic as it is able to defend itself from host defences.
What is the oxidase test? Why is this test useful in clinical microbiology?
the oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain.
this involves dropping a specific reagent onto a swab and touching target colony, a positive reaction is indicated by a significant colour change
All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration.
bacteria with this enzyme have more metabolic flexibility as they produce a higher amount of ATP than anaerobes
Many clinically significant bacteria that are oxidase-positive are also common opportunistic pathogens in clinical settings
What is the motility test? Why is this test useful in clinical microbiology?
used to detect the presence of flagella
involves picking a colony on the end of a needle and stabbing it into almost liquid media
after incubation, motile bacteria will have spread out from the insertion point
clinically significant bacteria usually possess flagella
What is the oxidation/fermentation test? Why is this test useful in clinical microbiology?
tests whether a bacteria is oxidative or fermentative.
by inoculating bacteria in medium, leaving one tube exposed to air and another tube overlayed with mineral oil, it can be seen whether a bacteria is oxidative or fermentative
can be used to determine where a bacteria resides within the host eg fermentative may live in the gut where oxygen is not present
What indicates the % ID obtained when using the API test?
The % ID obtained from an API test represents the degree of match between the tested isolate’s profile and the closest matching profile(s) in the API database. A higher % ID indicates a closer match and higher confidence in the identification result. Conversely, a lower % ID suggests a less precise match or a higher degree of uncertainty in the identification.
An API test can be read off by adding up the number of positive reactions in each group and inputting these values into their website.
What is meant by alpha-, beta-, and non-haemolytic Streptococcus?
alpha-haemolysis: partial lysis/damage of the red blood cells w greenish discolouration around the colonies
beta-haemolysis: clearing (w no greenish discolouration) of the red blood agar around the colonies, in streptococci typically a wide transparent halo due to complete haemolysis of the RBCs
non-haemolysis: no lysis of RBCs in agar
What enzyme distinguishes pathogenic Staphylococcus from other non-pathogenic strains?
coagulase: an enzyme complex produced by S. aureus. The cell-associated (bound) from react w fibrinogen in plasma and results in clumping of the blood
the free form activates prothrombin causing clotting of plasma
although commensal strains may not possess coagulase, it has emerged as significant opportunistic pathogens.
What surface protein distinguishes pathogenic Staphylococcus from other non-pathogenic strains?
Protein A: this binds to the Fc region of IgG. This allows S. aureus to evade immune recognition by coating itself in antibodies
What is a COBA plate?
a blood agar plate that is supplemented with colistin and oxolinic acid
both effective antibiotics against gram -ve bacteria
What is a common method to detect for the presence of coagulase and/or protein A?
through the use of the Staphaurex reagent.
this consists of polystyrene latex particles coated w fibrinogen and IgG. when mixed in a suspension of organisms, reaction of cell-bound coagulase w the fibrinogen and/or protein A causes rapid, strong agglutination of the latex particles.
this is performed on a reaction card, a positive test is indicated by patterns of agglutination.
What are COBA plates used for?
COBA plates are selective for Gram-positive bacteria, particularly staphylococci and streptococci, while inhibiting the growth of most Gram-negative bacteria. The selective agents colistin and nalidixic acid inhibit the growth of Gram-negative organisms
They can also be used to differentiate between different types of Gram-positive cocci based on their hemolytic activity, colony morphology, and other biochemical characteristics
What are PABA plates used for?
the detection of bacterial resistance to sulfonamides, a class of antibiotics that inhibit the synthesis of folic acid in bacteria
Bacteria that are susceptible to sulfonamide antibiotics require exogenous PABA for the synthesis of folic acid, an essential metabolite for their growth. Therefore, the addition of PABA to the growth medium allows for the detection of sulfonamide resistance. Bacterial isolates that are resistant to sulfonamides can synthesize folic acid in the presence of sulfonamide antibiotics, bypassing the need for exogenous PABA
What is MIC?
minimum inhibitory concentration
the MIC is the lowest concentration of antibiotc which inhibits visible growth
What are the advantages of using an FTA card?
- ease of use (blood, saliva, etc)
- sample stability
- room temp storage
- versatility (LAMP, PCR)
