Lab6 Flashcards
What is the first step towards an identification of a bacterium?
Gram stain
What is the first step towards an identification of a bacterium?
Gram stain
What are gram negative bacteria commonly isolated from?
clinical samples by the microbiology laboratory
Which is more common: Gram negative cocci or gram negative rods?
Gram negative RODS are more common than COCCI
Despite being not as common, why are gram negative cocci important?
They can cause characteristic or very serious infections
Name some clinically relevant gram negative cocci
Neisseria gonorrhoeae,
N. meningitidis,
Moraxella cattarhalis
Give some examples of where the identification of gram negative cocci in a clinical sample can be informative
in genital pus for Nisseria gonorrhoeae,
in cerebral spinal fluid for N. meninigitidis
What is the basis of a catalase test?
Catalase is an enzyme which breaks down hydrogen peroxide to produce water and oxygen
Catalase production is an important test to distinguish between staphylococci (catalase +ve) and streptococci (catalase -ve)
What is the purpose of a coagulase test?
It is a rapid method to distinguish staylococci (particular staph aureus) from other species of staphylococci in cultured specimens.
What is the basis of the coagulase test?
Staph aureus possesses coagulase clumping factor and/or protein A which is absent in some other species of staphylococci
What are the control organisms used in the coagulase test and what control are they for?
Staph aureus (control for coagulase positive)
Staph epidermidis (coagulase negative)
What is the purpose of using horse blood agar?
HBA is a non selective medium for the growth of many fastidious bacteria
Indicates types of haemolysis (differential) for α, β, and γ haemolytic streptococci
What does α-haemolysis look like and what organism COULD it indicate?
GREEN product.
this is due ti Red blood cells being partially lysed and hydrogen peroxide produced which oxidises the heme iron in the RBC from Fe3+ to Fe2+
COULD indicate Streptococcus pneumoniae or viridans streptococci
What does β-hemolysis look like?
Clear halo is seen around colonies due to the COMPLETE lysis of red blood cells.
What does γ-hemolysis look like?
No haemolytic zone or colour change seen on the HBA as no hemolysis occurs.
Often referred to as non haemolytic streptococci
What is the purpose and use of mannitol salt agar?
SELECTIVE medium for isolation of staphylococci (as high salt content prevents growth of streptococci
DIFFERENTIAL for staph aureus (as S. aureus can ferment mannitol to produce acid that lowers the pH of medium turning it yellow) Other staphylococci like CONS cannot ferment mannitol so it won’t turn yellow
GROWTH indicates staphylococci and YELLOW indicates aureus
Why is the optochin test used?
As a rapid test to distinguish between streptococcus pneumoniae from other α-haemolytic streptococci
Often conducts in HBA
Zone of inhibition ~15mm of growth around disc indicates S. pneumoniae.
Growth up to edge of disc indicates other streptococcal species which are resistant to optochin
Why is the bacitracin sensitivity test used?
A rapid test to distinguish between β-haemolytic streptococci belong to lancefield group A (streptococcus pyogenes) from other β-hemolytic streptococci.
This is a presumptive test. Positive cultures should be confirmed by lancefield typing
Positive = inhibition growth of 10mm in diameter apparent. > CONFIRM BY LANCEFIELD TYPING
(Other streptococci resistant to bacitracin will grow up to or almost there of the disc)
What is the purpose of the novobiocin test?
Rapid test to distinguish between staphylococcus saprophyticus from other coagulase negative staphylococci.
Positive = growth of Bactria up to edge of disc
(Most other coagulase negative staphylococci are sensitive and have a zone of inhibition ~20-30mm growth around disc)
What is the purpose of bile esculin agar test?
BE agar is
SELECTIVE for the isolation of enteric Bacteria
- bile salts inhibit non enteric bacterial growth
DIFFERENTIAL for Bacteria that can hydrolyse esculin
Hydrolysis results in produce that react with ferric filtration in Medium to produce insoluble iron salts which causes blackening of medium.
GROWTH of BE agar with blackening could be:
Enterococci (group D streptococci), klebsiella pneumoniae, or serratia marcescens
After determination of gram positive bacteria, which test is followed?
Catalase test
Following gram negative determination of bacteria, what test should follow?
Oxidase test
Why is the oxidase test used?
It is a qualitative procedure to determine the presence or absence of an intracellular cytochrome c oxidase system in bacteria as part of their respiratory chain. I.e, distinguishes between aerobic and anaerobic bacteria
Useful in the first identification of gram negative rods
Oxidase positive = aerobic = either pseudomonas or vibrio
Oxidase negative = anaerobic = the enterobacteriaceae which are facultatively anaerobic
What do the results of the oxidase test look like and why?
Positive result = turns blue within time limit of test (e.g, 10seconds)
Negative result = does NOT TURN BLUE within time limit of test
This is due to:
- Cytochrome oxidase produced by micro organism doesn’t directly oxidise p-phenylediamine reagent in the test.
- It oxidises cytochrome c which then oxidises the reagent to form a purple coloured compound.
- Oxidase Negative organisms lack cytochrome C, so leaves it colourless within time limit of test.
Why would chocolate agar be used ?
It is a non selective medium for growth of very fastidious Bactria like haemophilus and Nisseria. It contains heated lysed blood
Why is MacConkey agar used?
It is SELECTIVE for growth of gram negative intestinal pathogens and enterococci (as bile salts inhibit growth of non-enteric bacteria)
DIFFERENTIAL for lactose fermenting bacteria.
By using lactose in the medium lac+ bacteria produce acid which lowers pH of agar below 6.8 resulting in red/pink colonies and pink agar (e.g. Escherichia coli, klebsiella sp, enterobacter sp.)
Non lactose fermenting bacteria utilise peptone in the media resulting in the formation of ammonia which raises the pH. The formation of white/colourless colonies forms. (E.g. Salmonella sp., shigella sp., proteus sp., serratia sp., yersinia sp., citrobacter sp.)
Why is CROMagar used?
It is a SELECTIVE and DIFFERENTIAL agar for identification of pseudomonas
Agar contains chromagens which will give blue green coloured colonies of pseudomonas
Other bacteria= colourless colonies
Why is Xylose Lysine Deoxycholate agar used?
It is SELECTIVE and DIFFERENTIAL used in the isolation of salmonella and shigella
Sodium deoxycholate inhibits growth of gram+ bacteria
Agar is bright red/pink due to neutral pH
Sugar fermentation lowers the pH changing it to yellow.
Most gut bacteria except shigella can utilise Xylose to produce acid forming yellow colonies. After using choose, salmonella decarboxylates lysine which increases pH again to mimic red shigella colonies. Salmonella metabolises thiosulfate to produce hydrogen suffice resulting in colonies with black centres.
Other enterobacteria like E. coli extensively ferment lactose and sucrose forming yellow colonies and yellow agar
RED colonies - shigella
BLACK CENTRE colonies - salmonella
YELLOW colonies and YELLOW agar- other enterobacteria like E. coli
Why is eosin methylene blue agar used?
SELECTIVE growth of gram - enteric rods.
Eosin Y and methylene blue inhibit Gram+ bacterial growth to limited degree.
DIFFERENTIAL for lactose fermenting bacteria.
lac+ bacteria produce blue black colonies due to uptake of eosin methylene blue dye complex when pH drops
GREEN METALLIC SHEEN ( E. coli) due to RAPID fermentation of lactose
COLOURLESS/ TRANSPARENT AMBER COLOUR (Lac- )
