The document discusses otitis media and the identification of Haemophilus influenzae through laboratory methods, including Gram staining and culture on chocolate agar plates. It highlights the growth requirements of H. influenzae and contrasts it with H. haemolyticus, noting their differing reactions in hemolysis tests on blood agar. Essential diagnostic tests like the Kovac's oxidase test are also described for differentiating between various Haemophilus species.

1. 1
OTITIS MEDIA
PROBLEM BASED LEARNING (PBL)
PREPARED BY: MUHAMMAD ARIFF B. MAHDZUB
BACHELOR MEDICINE AND SURGERY (MBBS)
UNIVERSITY COLLEGE SHAHPUTRA, KUANTAN

2. LAB TEST
-OTITIS MEDIA-

3. Otitis media is inflammation of the
middle ear

4. Haemophilus influenzae
• Small
• Gram negative
• Coccobacilli
• Fastidious organism
• Required hemin (iron containing porphyrin)
for growth

5. Gram staining
• As we know that H. influenzae is gram
negative bacteria
• Thus by performing gram staining method, we
can determine whether the fluid from the
middle ear is gram + or gram – bacteria
• If the result show pink,thus it is gram negative
bacteria

6. Gram staining result
• Showing gram (-) H. influenzae (pink)

7. CHOCOLATE AGAR PLATE
• The standard medium used for growth of
Haemphilus influenzae is a chocolate agar
plate (CAP), which can be prepared with heat-
lysed horse blood
• good source of both hemin and NAD because
NAD is released from the blood during the
heating process of chocolate agar preparation
• Examination of growth on CAP shows
large,round,colorless and opaque colonies

8. CHOCOLATE AGAR PLATE
Haemphilus influenzae colonies on a
chocolate agar plate

9. KOVAC’S OXIDASE TEST
• Determines the presence of cytochrome oxidase
• By using filter paper method, the filter paper will turn purple
if the organisms containing cytochrome c as part of their
respiratory chain
• Positive reactions will develop within 10 seconds
• Negative reactions will not produce a colour change on the
treated filter paper.

10. H. Influenzae vs H. haemolyticus
• H. influenzae is a fastidious organism and can be identified on
the basis of growth requirements for hemin and NAD
• But there is also another haemophilus species that required
both hemin and NAD for growth
• It is H. haemolyticus, but this species differ from H. influenzae
by producing beta-haemolyticus
• To differentiate between the two species, hemolysis must be
checked on the blood agar

11. OTHER HAEMOPHILUS SPECIES
H. haemolyticus usually causes hemolysis on these media (blood
agar media) while H. influenzae does not.
Organisms Required hemin Required NAD Beta-haemolysis
H. influenzae + + -
H. haemolyticus + + +
H. aphrophilus + - -
H. parainfluenzae - + -
H. parahaemolyticus - + +
H. paraphrophilus - + -