Lab Unit 3

Question 1

Mannitol salt agar (MSA)

Answer 1

• isolate Staphylococcus from mixed sample
• selective and differential
• 7% salt- only salt tolerant grow

Mannitol (sugar) is the differential agent

• survivors ferment mannitol or do not

phenol red pH indicator

Question 2

Mannitol salt agar results

Answer 2

yellow = acidic (uses mannitol)

red= does not uses mannitol

Staphylococcus aureus- salt tolerant and uses mannitol (grows, yellow)

Staphylococcus epidermidis- salt tolerant and does not use mannitol (grows, red)

Micrococcus luteus- growth, not yellow

Streptococcus pyogenes- no growth (did not inoculate in lab)

Question 3

Phenylethyl alcohol test (PEA)

Answer 3

selective agar isolates Gram positive species from sample containing Gram negative bacteria

-phenylethyl alcohol inhibits DNA synthesis in Gram negatives

Question 4

Phenylethyl alcohol results

Answer 4

E. coli- negative, doesn’t grow

Staphylococcus aureus- positive, grows

Strepylococcus pyogenes- positive, grow

Question 5

Staphytect Plus agglutination test

Answer 5

replaces the coagulase test

distinguishes Staph aureus from Staph epidermidis and other Gram positives

antibody agglutination reagent (kit) binds Staph aureus antigens (sample)

Question 6

Staphytect Plus agglutiation test results

Answer 6

clumping and formation of blue particles- POSITIVE for Staph aureus

no clumping or blue particles- NEGATIVE for Staph aureus

Question 7

Answer 7

Mannitol Salt Agar

yellow- uses mannitol

growth- salt tolerant

Staphyloccus aureus- salt tolerant and ferments mannitol

Staphyloccus epidermidis- salt tolerant, doesn’t use mannitol

Micrococcus luteus- salt tolerant, doesn’t use mannitol

Question 8

Answer 8

Phenylethyl alcohol agar (PEA)

selective agar

Gram positive- grows

E. coli- negative

Staphylococcus aureus- positive

Streptococcus pyogenes- positive

Question 9

Catalase test

Answer 9

distinguishes Staph from Streptococcus

bacteria mixed with H2O2 on slide

H2O2- catalase substrate

Product- O₂(bubbles) and H₂O when positive

Strep- Negative

All others- positive

Question 10

Coagulase test

Answer 10

Coagulase hydrolyzes blood proteins

Clumping positive

Staph aureus- POSITIVE

All other bacteria NEGATIVE

Question 11

Blood agar

Answer 11

Blood is the differential agent

Hemolysin enzymes- break down red blood cells

1. Beta- clearing in blood (Staph aureus, Strep pyogenes)
2. Alpha- partial clearing in blood (Strep bovis, Enterococcus)
3. Gamma- Staph epidermidis

Question 12

Answer 12

Blood agar

Beta- clearing- Staph aureus

Alpha- partial clearing- Enterococcus, Strep bovis

Gamma- no clearing- Staph epidermidis

Question 13

Bacitracin Sensitivity test

Answer 13

“A” disc has bacitracin

Placed over bacteria on blood agar

No growth around disc- SENSITIVE- Strep pyogenes

Growth around disc- RESISTANT

Strep pyogenes- SENSITIVE

Strep pneumoniae- RESISTANT

Question 14

Optochin sensitivity

Answer 14

“P” disc

Streptococcus pneumoniae- SENSITIVE (clearing)

Streptococcus pyogenes- RESISTANT (no clearing)

Question 15

Answer 15

Bacitracin/Optochin sensitivity

Bacitracin- Strep pyogenes Sensitive

Strep pneumoniae Resistant

Optochin- Strep pneumoniae Sensitive

Strep pyogenes- Resistant

Question 16

Bile Esculin

Answer 16

Selective/Differential Agar

Bile- Selective agent

Esculin- Differential agent (turns BLACK if positive)

Positive- Strep bovis

           Enterococcus faecalis

Negative- Strep pyogenes

Question 17

Answer 17

Bile Esculin test

Positive- Strep bovis

Enterococcus faecalis

Negative- Strep pyogenes

Question 18

6.5% Salt broth

Answer 18

Selective Agar- salt is the selective agent

Turbidity- Positive

Clear- Negative

Positive- Staph, Bacillus, Enterococcus faecalis

Negative- Strep pyogenes

Question 19

Answer 19

6.5% Salt broth

Positive- Staph, Bacillus, Enterococcus

Negative- Strep

Question 20

Oxidase test

Answer 20

Tests for cytochrome oxidase of aerobic pathway.

Apply bacterium to swab, add oxidase reagent.

Blue/Black on swab is positive.

Positive- Pseudomonas

           Neisseria sicca

Question 21

Answer 21

Oxidase test

Positive- Pseudomonas, Neisseria sicca