Regrine B.

Lagarteja, RMT MICRO 203

2013-88648
IDENTIFICATION OF BACTERIAL UNKNOWNS

SPECIMEN NO.: 11
SPECIMEN: CEREBROSPINAL FLUID

COMMON ISOLATES FOR CEREBROSPINAL FLUID (CSF) SPECIMEN
Adapted from: Lippincotts Microcards: Microbiology Flash Cards, 3
rd
Edition

MORPHOLOGY
Grams stain: Gram (-) coccobacilli

Diagram for Differential Diagnosis of Pathogenic Gram (-) cocco and pleomorphic bacilli
Adapted from: Lippincotts Microcards: Microbiology Flash Cards, 3
rd
Edition

CULTURAL CHARACTERISTICS
Blood Agar Plate: no growth (thrice inoculated)
MacConkey Agar: no growth (twice inoculated)
Chocolate Agar: large, round, smooth, convex, colorless-to-grey, opaque colonies on CAP
Eosin-Methylene Blue Agar: no growth

Regrine B. Lagarteja, RMT MICRO 203
2013-88648

BIOCHEMICAL CHARACTERISTICS

Flow chart for identification and characterization of an H. influenzae isolate
Adapted from: www.cdc.gov

Not performed: serotyping, slide agglutination, quad plate

Suspected isolate: Haemophilus influenzae
PATHOGEN? YES

Haemophilus influenzae

Haemophilus influenzae is a small, nonmotile Gram-negative bacterium in the
family Pasteurellaceae. The organism may appear Gram-positive unless the Gram stain procedure
is very carefully carried out. Furthermore, elongated forms from sputum may exhibit bipolar staining,
leading to an erroneous diagnosis of Streptococcus pneumoniae (Todar, 2012)

Most strains of H. influenzae are opportunistic pathogens; that is, they usually live in their host
without causing disease, but cause problems only when other factors (such as a viral infection,
reduced immune function or chronically inflamed tissues, e.g. from allergies) create an opportunity.
They infect the host by sticking to the host cell using Trimeric Autotransporter Adhesins (TAA).

Regrine B. Lagarteja, RMT MICRO 203
2013-88648



Naturally acquired disease caused by H. influenzae seems to occur in humans only. In infants and
young children, H. influenzae type b (Hib) causes bacteremia, pneumonia, epiglottitis and acute
bacterial meningitis. On occasion, it causes cellulitis, osteomyelitis, and infectious arthritis (Puri J,
1999) (John, Cherian, Steinhoff, Simoes, & John, 1991)

H. influenzae is highly adapted to its human host. It is present in the nasopharynx of approximately
75 percent of healthy children and adults. It is rarely encountered in the oral cavity, and it has not
been detected in any other animal species. It is usually the non encapsulated strains that are
harbored as normal flora, but a minority of healthy individuals (3-7 percent) intermittently harbor H.
influenzae type b (Hib) encapsulated strains in the upper respiratory tract. (Todar, 2012)

Haemophilus influenzae requires two growth factor which are X (hemin) and V (nicotinamide-
adenine-dinucleotide) (Devajaran, Emmons, Talavera, Sanders, Mylonakis, & Cunha, 2007). Its
specific requirement for both hemin and NAD for growth can differentiate it from most other species
of Haemophilus (Centers for Disease Control and Prevention, 2013). Identification of organisms for
X and V factors can be done using X and V paper test. Positive result is growth of the organism.














Identification of hemin (X factor) and NAD (V factor) as growth requirements using paper disks. The top strain is only growing around the disk
containing both hemin and NAD (black arrow), and is presumptively identified as H. influenzae. H. haemolyticus will also only grow around
the paper disk containing both hemin and NAD. To differentiate between the two species, hemolysis must be checked on horse or
rabbit blood agar by inoculating the cell suspension mentioned above on heart infusion agar with 5% rabbit blood (or agar infusion
base containing horse blood). Alternatively, a Haemophilus ID Quad plate can be used (American Society of Microbiology, 2010).

Alternatively, H. influenzae can be identified using Haemophilus ID Quad Plates. They can test for
-hemolysis (clear) on horse blood and assist in differentiatingH. haemolyticus from H. influenzae.
The Quad plate is divided into four compartments. One quadrant includes medium containing hemin
only, the second quadrant with NAD, third with both hemin and NAD. The fourth quadrant contains
heart infusion agar or blood agar base with 5% horse blood used to check for hemolysis and for
differentiating H. haemolyticus from H. influenzae (Centers for Disease Control and Prevention,
2013).


.



Regrine B. Lagarteja, RMT MICRO 203
2013-88648



















Growth pattern for H. influenzae on a Haemophilus ID Quad plate (www.cdc.gov)

Haemophilus influenzae can be encapsulated with one of six types of antigenically distinct
capsules, which can be serotyped using antisera to each capsule (serotypes a-f). H. influenzae may
also be non-encapsulated and such strains that cannot be serotyped are called H.
influenzae nontypeable (NT). These serotypes can be identified using Slide Agglutination
Serotyping Test (Centers for Disease Control and Prevention, 2013).

Bibliography
Todar, K. (2012). Haemophilus influenzae and Hib Meningitis . Retrieved Feb 8, 2013, from Todar's
Online Textbook of Bacteriology: http://textbookofbacteriology.net/haemophilus.html

Devajaran, V., Emmons, W., Talavera, F., Sanders, C., Mylonakis, E., & Cunha, B. (2007, January
16). Haemophilus Influenzae Infections. Retrieved February 8, 2013, from eMedicine from WebMD:
http://www.emedicine.com/med/topic/936.html

Centers for Disease Control and Prevention. (2013, March 15). Chapter 9: Identification and
Characterization of Haemophilus influenzae. Retrieved February 8, 2013, from Centers for Disease
Control and Prevention: http://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-
hi.html

American Society of Microbiology. (2010). Clinical Microbiology Procedures Handbook (3rd ed.).
Washington DC, USA: American Society of Microbiology.

Puri J, T. V. (1999). Prevalence of antimicrobial resistance among respiratory isolates of
Haemophilus influenzae. Indian Pediatrics , 36 (10), 102932.

John, T., Cherian, T., Steinhoff, M., Simoes, E., & John, M. (1991). Etiology of acute respiratory
infections in children in tropical southern India. Revisiting Infectious Diseases , 13, Suppl 6:S4639.