Question

1. Explain how the TSI tests helps distinguish between lactose fermenters and non-lactose fermenters.

2. What test is used to identify delayed lactose fermenters?

3. What are the following tests detecting from the bacteria and which species are known to test positive?

Catalase:

Coagulase:

Modified Oxidase:

Novobiocin:

Bacitracin:

CAMP:

Hippurate Hydrolysis:

Bile-esculin:

Optochin Susceptibility:

Bile Solubility:

PYR:

4. Sketch a flow chart used to identify the different staphylococci species.

Answer 1

Tsi test triple sugar iron test

Principle

To determine the ability of an organism to attack specific carbohydrates incorporated in a growth medium, with or without production of gas, along with determination of possible hydrogen sulphide production.

Tsi contains three carbohydrates glucose, lactose, sucrose and ferric salts for testing hydrogen sulphide production. Medium is inoculated with bacterial culture at 37 degree overnight.

Yellow color indicates fermentation of carbohydrates

Red color indicates no fermentation

Bubbles in the butt indicate gas also produced along with fermentation

Blackening of medium indicated hydrogen sulphide production.

Various other combinations of tsi reactions are seen.

Enteric bacteria like shigella and salmonella will be able to

Reduce sulphur and to ferment carbohydrates.

2- test for delayed lactose fermenters

ONPG TEST

Lactose fermenting bacteria contain both permease and beta galactosidase enzymes. Permease is required by lactose to penetrate the cell wall and beta galactosidase for breaking galactosidase bond to produce glucose and galactose

Here ONPG an artificial substrate is incorporated as a substrate for beta galactosidase.

Delayed lactose fermenter appear to lack Permease enzyme.

ONPG is structurally similar to lactose except that glucose is substituted with o- nitro phenyl group. ONPG is able to penetrate cell wall as its not dependant on Permease unlike lactose. If the organism contains beta galactosidase the substrate is split releasing o- nitro phenol which is yellow in color.

Catalase test- differentiates between staph n strep. positive for staphylococcus and negative for streptococcus. This test detects the presence of catalase enzyme which breaks down hydrogen peroxide into hydrogen and oxygen.

Novobiocin test. Used for coagulase negative staphylococcus. Positive test indicates, S. Epidermidis or s. Hemolyticus which is Novobiocin( broad spectrum antibiotic) positive.,whereas negative test indicates saprophyticus which is Novobiocin resistant.

Bacitracin

Bacitracin again is a broad spectrum antibiotic which is used to differentiate between Bacitracin sensitive organism such as group A streptococci (positive for Bacitracin) from other groups which are negative.

Camp reaction

Used to identify beta hemolytic streptococci when group B strep are inoculated perpendicular to a streak of staph aureus grown on blood agar. Appears as a accentuated zone of hemolysis as beta hemolysis of staph aureus enhances lysis of streptococci.

Hippurate hydrolysis

Used to identify group B streptococci i. e, streptococcus agalactiae based on their ability to hydrolyse hippurate. Also used for campylobactor sos, listeria monocytogenes etc.

Bile esculin test

Used to differentiate between enterococci and group D streptococcus . eg, strep bovis. Bacteria able to grow in the presence of esculin mixed in 4% bile are positive for the test such as enterococci.

Optochin susceptibility

Used to differentiate between alpha hemolytic organisms that is step. Peneumoniae and strep viridans. Test is positive for strep pneumoniae as it is sensitive to Optochin chemical.

Bile solubility test again used to identify between strep. Pneumoniae and strep viridans determines the ability of bacteria to lyse in the presence of bile salts for a specific temperature n time. Positive for strep peneumoniae.

PYR test

Used to differentiate between gamma hemolytic strains such as enterococci and non enterococci i. e streptococcus bovis. The test is positive for enterococcus and negative for grp D streptococci. It is based on detection of pyrrolidonyl arylamidase activity in bacteria.