Laboratory Diagnosis of Bacterial

Causes of Pneumonia
(Demonstration)

Presented by
Amira Abbas ElGhazaly
Ass. Lec. of Microbiology & Immunology .FOM.SCU
 Bacteria Causing Pneumonia:

Streptococcus pneumoniae

Haemophilus influenza

Klebsiella pneumoniae

Mycoplasma pneumoniae
Demonstrate:
 1- Microscopic appearance of Strept. pneumoniae
 2- Microscopic appearance of Haemophilus influenza
 3- Microscopic appearance of Klebsiella pneumoniae
 4-Blood agar showing alpha hemolysis
 5- MacConey agar showing lactose fermenting colonies
 6- Colony morphology of Mycoplasma pneumniae
 7- Chocolate agar
 8- Optochin sensitivity test
 9- Quellung reaction
 10- X-V factor test
 11- Blood culture bottle
 Streptococcus pneumoniae
 Microscopic Appearance:
Gram-positive, lancet-shaped, Diplococci (arranged in pairs), but they may also
occur in short chains
 Cultural Characters:
Streptococcus pneumoniae on blood agar appears as alpha hemolytic colonies
(greenish discolouration) similar to Viridans streptococci which are non pathogenic

Streptococcus pneumoniae grows best in 5% carbon dioxide

Alpha-Hemolysis
Strept. pneumoniae Viridans streptococci
Optochin Sensitivity test
 Quellung test
(capsule swelling test)
This method involves testing a pneumococcal cell suspension with specific antisera directed against
the capsular polysaccharide

The antigen-antibody reactions are observed microscopically

A positive quellung reaction is the result of the binding of the capsular polysaccharide of pneumococci
with type specific antibody contained in the typing antiserum (Strept.pneumoniae)

A positive quellung reaction is observed when the capsule appears as a halo (swollen capsule) around
the bacterial cell
Some cases of pneumococcal infections
are positive in blood culture
 Haemophilus influenza
 Microscopic Appearance:
H. influenza in a Gram stain of a sputum sample appears as Gram-negative coccobacilli.
 Cultural Characters:
• H.influenza is a fastidious, facultative anaerobic organism which grows best at
35-37°C with ~5% CO2 (or in a candle-jar) and requires hemin (X factor) and
nicotinamide-adenine-dinucleotide (NAD, also known as V factor) for growth.

• The standard medium used for growth of H. influenzae is a chocolate agar plate
which can be prepared with heat-lysed horse blood, a good source of both hemin
and NAD.

• Growth occurs on a chocolate agar because NAD is released from the blood
during the heating process of chocolate agar preparation (the heating process also
inactivates growth inhibitors) and hemin is available from non-hemolyzed as well
as hemolyzed blood cells.
H.influenza requires both X and V factors for growth.

 In this culture, H.influenza has only

grown around the paper disc that has
been impregnated with both
X and V factors.

There is no bacterial growth

around the discs that only
contain either X or V factor
 Klebsiella pneumoniae
 Microscopic Appearance:
Klebsiella pneumoniae is a Gram negative, encapsulated, rod-shape bacterium
 Cultural Characters:
Klebsiella pneumoniae produce mucoid, pink lactose fermenting colonies on
MacConkey agar (37°C, 24 hours)
 Mycoplasma pneumoniae
Typical fried-egg colony morphology of Mycoplasma pneumoniae