LAB 11 – HAEMOPHILUS, BORDETELLA, PASTEURELLA, BRUCELLA

Question 1

How does a staph streak allow Haemophilus to grow on a BAP?

Answer 1

• S. aureus lyse RBCs to produce hemin (x factor) and NAD (v factor)
• Haemophilus sp. will satellite

NAD - nicotinamide adenine dinucleotide

Question 2

Why does H. influenzae only grow around the disc with both X and V factor?

Answer 2

• requires both exogenous x and v
  (ie. hemin and NAD, respectively)

NAD - nicotinamide adenine dinucleotide

Question 3

Where would you incubate a plate with Bordetella pertussis?

Answer 3

O2; strict aerobe

Question 4

Why does sheep blood not support the growth of Haemophilus sp.? Why can they grow
on CAP using sheep’s blood?

Answer 4

• Haemophilus sp. require both x and v factors
• NADase in BAP breaks down NAD (v factor) needed
• CAP has been heated; RBCs release hemin (x factor) and NADase is inactivated

Question 5

What is special about the staining of Pasteurella multocida?
a. What stain is this most commonly seen with?

Answer 5

bipolar g-cb on Giemsa stain

Question 6

What are X and V factors?

Answer 6

X factor = hemin; an iron porphyrin
V factor = NAD

NAD - nicotinamide adenine dinucleotide

Question 7

What does the ALA test determine? What is it testing for?

Answer 7

delta-AminoLevulinic Acid test:
- determines whether organism requires exogenous x factor
- tests organism’s ability to produce its own hemin

Results: Red fluorescence under UV = (+) porphyrin
No fluorescence = (-) porphyrin

NOTE: also called Porphyrin test

Question 8

Appropriate specimens for Haemophilus sp.?

Answer 8

• blood
• sputum
• CSF
• conjunctival and genital ulcer swabs

Question 9

What cautions have to be taken with Brucella spp.?

Answer 9

• Biosafety level 3 protocols

Question 10

How is brucellosis usually diagnosed?

Answer 10

• based on detection of serum antibodies (1:160 titre), rather than isolation of organism

Question 11

What antibiotic does H. influenzae have a high resistance to? Why?
a. Which specimens are screened for this antibiotic resistance?

Answer 11

• High ampicillin resistance (β-lactamase)
  a. primary cultures from blood and CSF are screened via chromogenic cephalosporin method

Question 12

Specimen type for Bordetella and any special collection instructions?

Answer 12

• nasopharyngeal swabs collected using a tiny calcium alginate swab

Question 13

What effects are B. pertussis highly susceptible to?
a. What media is required to isolate?

Answer 13

• fragile specimens susceptible to toxic unsaturated fatty acids, peroxides and sulfides
  a. requires charcoal or starch and high concentrations (10-20%) of blood for primary isolation

Question 14

What are the following results for Pasteurella multocida?
a. Gram
b. Colonial morph
c. Catalase
d. Oxidase
e. Spot indole
f. Nitrate
g. Ornithine decarboxylase
h. Urease
i. Motility
j. TSI

Answer 14

a. Gram = g-cb, bipolar
b. Colonial morph = muc, gy sm, translucent
c. Catalase = (+)
d. Oxidase = (+)
e. Spot indole = (+)
f. Nitrate = (+)
g. Ornithine decarboxylase = (+)
h. Urease = (-)
i. Motility = (-)
j. TSI = wk A/A

NOTE: no growth on MAC

Question 15

What are the results for the following tests for Brucella abortus?
a. Gram
b. Colonial morphology
c. Urease
d. Oxidase
e. Catalase
f. Nitrate

Answer 15

a. Gram = g-cb
b. Colonial morphology = sm gy, translucent
c. Urease = (+)
d. Oxidase = (+)
e. Catalase = (+)
f. Nitrate = (+)