Lab6

Question 1

What is the first step towards an identification of a bacterium?

Answer 1

Gram stain

Question 2

What is the first step towards an identification of a bacterium?

Answer 2

Gram stain

Question 3

What are gram negative bacteria commonly isolated from?

Answer 3

clinical samples by the microbiology laboratory

Question 4

Which is more common: Gram negative cocci or gram negative rods?

Answer 4

Gram negative RODS are more common than COCCI

Question 5

Despite being not as common, why are gram negative cocci important?

Answer 5

They can cause characteristic or very serious infections

Question 6

Name some clinically relevant gram negative cocci

Answer 6

Neisseria gonorrhoeae,
N. meningitidis,
Moraxella cattarhalis

Question 7

Give some examples of where the identification of gram negative cocci in a clinical sample can be informative

Answer 7

in genital pus for Nisseria gonorrhoeae,

in cerebral spinal fluid for N. meninigitidis

Question 8

What is the basis of a catalase test?

Answer 8

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)

Question 9

What is the purpose of a coagulase test?

Answer 9

It is a rapid method to distinguish staylococci (particular staph aureus) from other species of staphylococci in cultured specimens.

Question 10

What is the basis of the coagulase test?

Answer 10

Staph aureus possesses coagulase clumping factor and/or protein A which is absent in some other species of staphylococci

Question 11

What are the control organisms used in the coagulase test and what control are they for?

Answer 11

Staph aureus (control for coagulase positive)

Staph epidermidis (coagulase negative)

Question 12

What is the purpose of using horse blood agar?

Answer 12

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

Question 13

What does α-haemolysis look like and what organism COULD it indicate?

Answer 13

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

Question 14

What does β-hemolysis look like?

Answer 14

Clear halo is seen around colonies due to the COMPLETE lysis of red blood cells.

Question 15

What does γ-hemolysis look like?

Answer 15

No haemolytic zone or colour change seen on the HBA as no hemolysis occurs.

Often referred to as non haemolytic streptococci

Question 16

What is the purpose and use of mannitol salt agar?

Answer 16

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

Question 17

Why is the optochin test used?

Answer 17

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

Question 18

Why is the bacitracin sensitivity test used?

Answer 18

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)

Question 19

What is the purpose of the novobiocin test?

Answer 19

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)

Question 20

What is the purpose of bile esculin agar test?

Answer 20

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

Question 21

After determination of gram positive bacteria, which test is followed?

Answer 21

Catalase test

Question 22

Following gram negative determination of bacteria, what test should follow?

Answer 22

Oxidase test

Question 23

Why is the oxidase test used?

Answer 23

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

Question 24

What do the results of the oxidase test look like and why?

Answer 24

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.

Question 25

Why would chocolate agar be used ?

Answer 25

It is a non selective medium for growth of very fastidious Bactria like haemophilus and Nisseria. It contains heated lysed blood

Question 26

Why is MacConkey agar used?

Answer 26

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.)

Question 27

Why is CROMagar used?

Answer 27

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

Question 28

Why is Xylose Lysine Deoxycholate agar used?

Answer 28

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

Question 29

Why is eosin methylene blue agar used?

Answer 29

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- )