LABORATORY Flashcards
Haemophilus influenzae: Specimens
- Cerebrospinal fluid
- Sputum, other types of respiratory specimens
- Blood
- Pus
- Material from genital ulcer (H. ducreyi)
small, palestaining, gram-negative coccobacilli. Occasionally, slender filamentous cells may be observed
Haemophilus species
pale-staining, gram-negative coccobacilli, often arranged in clustered groups (“school of fish”) or loosely coiled parallel chains (“railroad tracks”)
H. ducreyi
morphology is rarely seen in clinical specimens
H. ducreyi
Organisms may be inside and outside of polymorphonuclear leukocytes (PMNs).
H. ducreyi
Used for rapid diagnosis of H. influenzae type b infections.
Antigen detection
[?] or other commercially available methods for the detection of the type b capsular antigen in CSF,, serum, and urine.
Neufeld-Quellung reaction
prepared by adding rabbit or horse blood to an agar base when the temperature of the medium is high enough (about 80°C) to lyse the red cells to release the growth factors without inactivating V factor (NAD), which is heat-labile
Chocolate Agar
is not suitable for recovery of Haemophilus species that require V factor for growth
Conventional sheep blood agar
Conventional sheep blood agar contains X factor but not V factor because of the presence of [?] in native sheep blood.
V-factor-inactivating enzymes
It is a selective medium containing beef heart infusion, peptones, yeast extract, and defibrinated horse blood (5%) containing both X and V factors.
Haemophilus Isolation Agar
is added to inhibit the other normal respiratory tract flora.
Bacitracin (300 µg/mL)
Other isolation media that provide both X and V factors:
i. Casman’s Agar with 5% lyzed blood
ii. Fildes enrichment agar
iii. Levinthal agar
Principle:
Several bacterial species, including Staphylococcus aureus, produce V factor (NAD) as a metabolic by-product. Therefore tiny colonies of Haemophilus species. may be seen growing on sheep blood agar very close to colonies of bacteria capable of producing V factor; this is known as the satellite phenomenon (or satellitism).
Staphylococcus Streak Technique (using 5% Sheep BAM)
Procedure:
i. A colony of a suspected Haemophilus species is subcultured onto a sheep blood agar plate and streaked as a lawn.
ii. A single streak of hemolytic strain of S. aureus is made through the lawn inoculum.
iii. Incubate overnight in a CO2- enriched environment at 35°C to 37°C.
iv. Staphylococcus synthesizes V factor, and lyses the red blood cells adjacent to the streak line, releasing X factor and V factor providing the necessary components for growth of Haemophilus species.
Staphylococcus Streak Technique (using 5% Sheep BAM)
Result:
Tiny moist gray colonies of the haemophili may be observed within the hemolytic area adjacent to the staphylococcal growth. Haemophilus colonies are larger at the vicinity of the staphylococcal colonies.
Staphylococcus Streak Technique (using 5% Sheep BAM)
small, gray glistening colony is H. influenzae.
Haemophilus influenzae satellitism
Incubation
- 35-37°C
- 5% to 10% CO2 atmosphere (using candle extinction jar, or CO2 incubator)
- Colonies can be observed within 24 hours, but cultures are routinely held 72 hours before being discarded as negative.
Haemophilus influenzae
Principle: X factor (hemin, hematin) and V factor (NAD) are required either singly or in combination to support the growth of various species of Haemophilus on agar media. X and V factors, each being water soluble, readily diffuse in agar culture media.
Test for Factor Requirement
Procedure:
i. Filter paper disks or strips impregnated with these factors, which are commercially available, are placed on the surface of a medium deficient in these factors, such as trypticase-soy agar or brain–heart infusion agar, which has been inoculated as a lawn with the test organism.
ii. Incubate the plate in CO2 atmosphere at 35°C for 18–24 hours.
iii. Factor requirements of the organism are
then determined, a f t e r o v e r n i g h t i n c u b a t i o n , b y o b s e r v i n g t h e patterns of growth around the paper strips or disks.
Test for Factor Requirement
A better test for X factor requirement.
Porphyrin Test
[?] detects the presence of enzymes that convert δaminolevulinic acid (ALA) into porphobilinogen, porphyrins, protoporphyrins, and heme.
Haemophilus species that possess the enzyme can synthesize [?], therefore, do not require exogenous X factor for growth.
Porphyrin Test
protoporphyrin
Procedure:
The test may be performed in broth, in agar, or on a disk.
i. With the ALA broth or agar medium, the organism is inoculated onto the medium and incubated overnight.
ii. With the disk method, the impregnated disk is moistened with water and inoculated with colonial growth, and incubated for 4 hours. After incubation, growth is examined under an ultraviolet light (Wood’s light).
Porphyrin Test
Results:
(+) Brick-red fluorescence - indicates the presence of porphyrins, does NOT require X factor.
(-) Bluish fluorescence
Porphyrin Test
