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
Nasophayngeal swab or aspirate is the preferred specimen
Bordetella pertussis
Very small, gram-negative coccobacilli.
Bordetella pertussis
Fluorescent antibody (FA) reagent (stain using polyclonal antibodies against B. pertussis or B. parapertussis) can be used to examine nasopharyngeal swab specimens.
Direct fluorescent antibody (DFA)
potato-blood-glycerol agar
Bordet-Gengou Agar
Potato infusion provides
nitrogen and vitamins
[?], present from the potato infusion, absorbs fatty acids which inhibits growth of B. pertussis.
Starch
is a carbon source.
Glycerol
provides essential complex growth factors.
15% sheep or horse blood
charcoal, cephalexin blood agar (CCBA)
Regan-Lowe Agar
supplies nutrients required for the cultivation of Bordetella species.
Defibrinated horse blood
promotes growth of B. pertussis.
Nicotinic acid (niacin, vitamin B3)
neutralize substances toxic to Bordetella species, such as fatty acids and peroxides.
Charcoal and starch
is a cephalosporin antibiotic that inhibits most normal flora of the nasopharynx.
Cephalexin
- 35-37°C
- Aerobic, humidified atmosphere
- generally requires 2-6 days to grow; but, must be held for up to 12 days before reporting as negative.
B. pertussis
produces small, domed, glistening colonies that resemble “mercury droplets” , “bisected pearls”, or “aluminum paint”
B. pertussis
[?] is not distinct for the identification of other Bordetella species
colony morphology
Blood or bone marrow are the specimens of choice
Brucella species
• Other specimen:
- Cerebrospinal fluid (CSF)
- Pleural fluid
- Synovial fluids
- Urine
- Abscesses
- Other tissues
Brucella species
Faintly staining, minute gram-negative coccobacilli
Brucella species
Blood and bone marrow specimens are inoculated into aerobic and anaerobic bottles, and because of their slow growth rates, conventional blood cultures should be incubated at 35°C for 3 weeks with blind subcultures into plated media.
Brucella species
Culture media for Brucella species
- Blood Agar (or Chocolate Agar) with 5% horse blood
2. Brucella Agar with 5% horse blood
- 35-37°C
- 5% to 10% CO2 in a humidified atmosphere.
- Most isolates can be detected within 5 to 7 days. Cultures should be retained for 3 weeks before they are considered negative and discarded.
Brucella species
Colonies appear small, convex, smooth, translucent, gamma-hemolytic, and slightly yellow and opalescent after at least 48 hours of incubation.
Brucella species
It uses a biphasic medium consisting of tryptose agar and tryptose broth in one bottle.
Castañeda’s Method of Blood Culture
The inoculum is added to the broth medium and when subcultures are to be made, the bottle is simply tilted to allow the broth to flow over the agar slant.
Castañeda’s Method of Blood Culture
There is NO need for frequent opening of the culture bottle to subculture; thus, reduces chances of contamination and increases the chances of isolation.
Castañeda’s Method of Blood Culture
small, Gram-negative coccobacilli, which is strictly aerobic, nonmotile, non-spore-forming and weakly catalase positive.
Francisella tularensis
fastidious and require supplementation with cysteine, cystine, or thiosulfate for growth.
Francisella tularensis
Many species of animals, esp. rabbits, deer, and
rodents are the reservoir
Francisella tularensis
Humans are infected by the bite of an arthropod vector (deerfly, ticks, mites, or lice) deerfly or tick;
by traumatic implantation on contact with infected
animal tissues; by ingestion of undercooked,
infected meat. or contaminated water; and also,
inhalation of the organism.
Francisella tularensis
It is a highly infectious agent that as few as 10 - 50 organisms can cause an infection
Francisella tularensis
facultative intracellular pathogen that localizes in reticuloendothelial cells.
Francisella tularensis
a disease of many names, including rabbit fever, deerfly fever, lemming fever, and water rat trapper’s disease
tularemia
A sudden onset of flu like symptoms (malaise, headache, fever, chills) and painful swollen lymph nodes are observed in infected individuals.
tularemia
s the most common type, which typically presents with a skin ulcer, usually as the result of a tick bite.
Ulceroglandular tularemia
The [?] at the site of infection is followed by septicemia with rapid spread to the liver and spleen; 30 percent of untreated patients die.
local abscess
[?] types are the other major syndromes that are
associated with tularemia which is influenced by the mode of infection.
Pulmonary (pneumonia) and gastrointestinal (typhoid-like)
Lab identification can be made using a medium called [?]. Most hospital laboratories don’t process this organism, however, because it is
highly contagious.
cysteine-blood agar
Protection from [?] is afforded by live attenuated vaccines and protective gloves, masks and eyewear for laboratory workers and other occupationally exposed personnel.
F. tularensis infection
Gram-negative coccobacilli with a bipolar appearance or may appear ovoid, filamentous, or as bacilli on stained smears
Pasteurella species
They are aerobes or facultative anaerobes that grow readily on ordinary bacteriologic media.
Pasteurella species
nonmotile, catalase positive, oxidase positive, and ferment glucose with weak to moderate acid production without gas
Pasteurella species
is the species which most commonly infects humans causing pasteurellosis
P. multocida
Humans can acquire the organism from dog or cat bites.
P. multocida
can quickly progress resulting in inflammation and exudate production.
Wound infections
On occasion, [?] (septicemia, arthritis, endocarditis, osteomyelitis, meningitis) and [?] forms are also possible.
systemic
pneumonic
It grows well on medium with [?] and [?], developing in 48 hours at 35-37 oC, small (1-2 mm), smooth, moist to mucoid colonies with a greenish or brownish discoloration, and musty odor resembling smell of mushroom.
blood and hemin
thin, pleomorphic, weakly-staining, gram-negative
bacilli; weakly oxidase and catalase positive, and often are motile
Legionella pneumophila
can only be grown on complex media due to their fastidious nutritional requirements
Legionella pneumophila
The primary growth factor required is , a nutrient that is also essential for F. tularensis.
L-cysteine
is also essential, and other compounds are necessary for optimal growth.
Ferric iron
Energy is derived from [?] rather than carbohydrates.
amino acids
is ubiquitous and has the ability to survive and persist in natural habitats and humanmade water supplies and distribution systems. It is widely distributed in aqueous habitats such as tap water, cooling towers, spas, ponds, and other fresh waters.
Legionella pneumophila
Transmission to humans occurs through inhalation
of aerosols from contaminated water, esp. from air
conditioning system. Human-to-human infection
and laboratory-acquired infections are not known
to occur.
Legionella pneumophila
facultative intracellular pathogen in alveolar macrophages
Legionella pneumophila
The most common presentation is acute pneumonia, which varies in severity from mild illness that does not require hospitalization (“walking pneumonia”) to fatal multilobar pneumonia. Typically, patients have high fever and cough but do not produce much sputum, a clinical picture that does not resemble typical pneumonia, thus is also referred to as “atypical pneumonia”.
Legionnaire’s disease
Extrapulmonary infections occur with or without pneumonia and has been associated with bacteremia, renal failure, liver function abnormalities, watery diarrhea, nausea, vomiting, headache, confusion, lethargy, and other CNS abnormalities.
Legionnaire’s disease
A milder, non-pneumonic form of legionellosis, resembling influenza.
Pontiac fever
Patients are previously healthy individuals who complain of flulike symptoms of fever, headache, and myalgia that last 2 to 5 days and then subside without medical intervention.
Pontiac fever
The medium of choice is [?], containing yeast extract, iron, Lcysteine, and a-ketoglutarate for bacterial growth; activated charcoal to inactivate toxic peroxides that develop in the media; and buffer with pH 6.9, the
optimum for growth of the organisms.
buffered charcoal-yeast extract (BCYE)
For contaminated specimens such as sputum, [?] should be added. Bacterial colonies that are round or flat with entire edges can usually be detected within 3 to 5 days and identified presumptively as [?].
antibiotics
Legionella
pleomorphic gram-negative bacilli frequently occurring in chains or long filaments, and often with a series of oval to elongated bulbous swellings, giving a string-of-beads appearance. It is nonencapsulated, facultative anaerobic, nonmotile, and fermentative.
Streptobacillus moniliformis
It occurs as indigenous flora in the upper respiratory
tract of rodents and is usually transmitted to humans by bites or scratches from rodents or carnivores that ingest rodents; or less commonly, after ingestion of food contaminated by rodent excrement or by traumatic injury.
Streptobacillus moniliformis
It causes streptobacillosis (“rat bite fever” or Haverhill fever). Local lymphangitis and lymphadenitis may develop up to 3 weeks later, followed by fever, chills, malaise, and, later, a general morbilliform maculopapular or petechial rash. A migratory polyarthritis develop in some patients.
Streptobacillus moniliformis
Bacteria grow slowly up to 6 days in thioglycollate broth as colonies with a puffball, cotton ball), or bread crumbs appearance.
Streptobacillus moniliformis
