Spore-forming Gram-positive Bacilli Flashcards
Specimens: Bacillus anthracis
[?] from beneath the eschar from cutaneous lesion.
Vesicular fluid or biopsy
Specimens: Bacillus anthracis
[?] from gastrointestinal anthrax.
Gastric aspirates or stools
Specimens: Bacillus anthracis
[?] in sepsis
Blood or cerebrospinal fluid
Specimen processing before plating on solid media:
[?] at 62°C to 65°C for 15 to 20 minutes kills contaminating vegetative cells, so only endospores survive.
Heat shock treatment
Gram staining: Bacillus anthracis
Large, G+ bacilli, [?]
single and/or in chains.
Gram staining: Bacillus anthracis
Unstained areas within the cell indicate [?]
sporulation
A primary stain-malachite green is forced into the spore by steaming the bacterial emulsion
Spore staining (e.g.,Schaeffer-Fulton, Dorner)
water soluble and has a low affinity for cellular material, so vegetative cells may be decolorized with water
Malachite green
is then applied to counterstain any cells which have been decolorized.
Safranin
At the end of the staining process
vegetative cells will be [?], and endospores will be [?].
pink
green
Both [?] that do not swell the vegetative cell and cell-free spores outside of the vegetative cells may be observed.
intracellular, central or subterminal spores
Performed directly on clinical material; capsules are normally lost in culture.
Capsular staining
wet preparation highlights the capsule as a transparent halo around the bacillus.
India ink
Uses polychrome methylene blue.
McFadyean staining
[?]-stained capsule seen surrounding the deep bluish bacilli.
Purple/pink
Culture media: Bacillus anthracis
- 5% sheep BAM
- Polymyxin-lysozyme-EDTA-thallous acetate (PLET)
- Nutrient agar supplemented with MnSO4
- Bicarbonate agar
Best selective medium for isolation of B. anthracis.
Polymyxin-lysozyme-EDTA-thallous acetate (PLET)
inhibits or retards most G- bacteria
Polymyxin B
is used to destroy vegetative cells
Lysozyme
a chelating agent
EDTA
a selective agent against G+ and G-bacteria
Thallous acetate
Used for subcultures to stimulate sporulation
Nutrient agar supplemented with MnSO4
Bicarbonate agar is a Nutrient agar with
sodium bicarbonate and 5-10% horse serum
Used for subcultures to induce capsule formation.
Bicarbonate agar
Specimens should be inoculated to a [?] and [?]
blood agar and a selective medium (PLET).
Inoculate both [?] to ensure maximal recovery.
heat-treated and untreated specimens
Incubate either in ambient air or in [?]. Bicarbonate agar requires incubation in CO2.
5% to 10% CO2 for 24 - 48 h
Medium to large ([?] in diameter)
2-5 mm
Non-pigmented (?)
white to gray
[?] convex
Flat or slightly
Dry, rough, [?]surface
“ground-glass”
Irregular edges with “comma-shaped” projections
“Medusa head” or “comet tail” colonies
Under dissecting microscope (or a hand lens), numerous undulating [?] outgrowths consisting of filamentous chains of bacilli project from the colony.
“curled hair”
Sticky or tenacious consistency such that, when the colonies are touched with inoculating loop, they form standing peaks resembling
beaten egg whites
gamma-hemolytic on
BAM
Catalase test: Bacillus anthracis
Positive
Motility test: Bacillus anthracis
Negative (nonmotile)
Determined by stab inoculation of semisolid motility medium
Motility test
is indicated by the spreading of growth beyond the line produced by the inoculating needle, or by microscopic exam of hanging drop preparation of bacterial suspension.
motility
Suspect colony is heavily inoculated by
streaking into bicarbonate agar and incubated for 24 to 48 hr at 37 °C in a 20% CO2 atmosphere.
Growth on bicarbonate agar
[?] type is strongly indicative of virulent B. anthracis attributed to capsule formation.
Large and mucoid colony
Avirulent B. anthracis produces [?] under these conditions.
rough colonies
Performed by stabbing a gelatin tube with the suspect colony and incubated.
Gelatin hydrolysis test
B. anthracis liquefies gelatin slowly producing an [?] growth.
“inverted fir tree”
Involves spreading a portion of a nutrient or blood agar plate with the culture under test, and placing a 10U penicillin disc at some point within the area of spread. and incubated overnight at 35–37 °C.
Susceptibility to penicillin
B. anthracis are susceptible to penicillin (10 U), so a [?] will be visible.
zone of inhibition
Test organism is heavily streaked on agar plates (e.g., MHA) containing [?] unit of penicillin G per ml, and incubated at
0.5
37 °C for 3 to 6 hours
The plate is examined by placing a cover slip over the [?], and observed under the high, dry objective
inoculated area
B. anthracis will assume [?] characteristic of the string-of-pearls reaction.
large, round cellular forms
Susceptibility to gamma phage
Suspect colony is streaked to a blood agar plate and a drop of gamma phage and a drop of [?] to serve as a control are added to separate areas of the streaked plate The plate is incubated overnight at 35–37 °C.
sterile water
has the ability to lyse B. anthracis grown aerobically on blood or other nutrient agar.
“Gamma” phage
[?] in the region of confluent growth where the gamma phage was applied results from the phage’s ability to lyse the bacterial cells.
A plaque (clear area)
Definitive identity of a suspect B. anthracis isolate used to be done by inoculating the organism into a white mouse or a guinea-pig and confirming the cause of death by smear or isolation.
Animal pathogenicity test
Tissue extract, prepared by boiling tissue samples in saline and filtered, is layered over anthrax antiserum in a narrow tube.
Ascoli test
The development of [?] at the junction of the two fluids within 10 minutes at room temperature denotes a positive results for Ascoli test
white ring of precipitate
Similar to B. anthracis, but B. cereus are [?], and are
motile with peritrichous flagella
non-encapsulated
Heat treatment at [?] or [?] is effective for killing vegetative cells and retaining spores for most Bacillus spp.
70°C for 30 minutes or 80°C for 10 minutes
Large, feathery, spreading colonies that are cream to white or grey and have a slight green tinge
5% Sheep BAM
β-hemolytic for B. cereus
5% Sheep BAM
Bacteria that ferment [?] produce [?] and form colonies that are [?] with as the pH indicator.
mannitol; acids; yellow
phenol red
Bacteria that produce [?] hydrolyze lecithin in egg yolk and a [?] forms around the colonies.
lecithinase
zone of white precipitate
inhibits the growth of most other bacteria.
Polymixin
B. cereus colonies are [?] (mannitol-negative) and surrounded by [?] (lecithinase-positive).
pink-red
zone of precipitate
Polymyxin, Egg Yolk, Mannitol, Bromthymol Blue Agar (PEMBA)
Addition of [?] improve egg yolk precipitation and enhance sporulation
sodium pyruvate
is added as a pH indicator to detect mannitol utilization.
Bromthymol blue
is the selective agent.
Polymyxin B
B. cereus colonies are crenated, 5mm diameter,[?] with a zone of egg yolk precipitation after 18-24hr incubation.
turquoise to peacock blue
Best obtained by tissue [?] or by [?] using a needle and syringe to prevent contamination with normal flora.
biopsy or by aspiration
Use transport devices that contain [?] to protect them from oxygen exposure
pre-reduced anaerobically sterilized (PRAS) media
Deliver to the laboratory
promptly
Processed in the
biologic safety cabinet
Processing of specimen with minimal exposure to
atmospheric oxygen
Use of combination of
selective and nonselective media
In most instances, [?] is the recommended temperature for primary isolation of anaerobes.
35°C to 37°C
Use of
anaerobic incubation system
Specimens: Clostridium perfringens
Material from wounds, pus, and tissue
Gram-positive (or gram-variable) bacilli, usually 0.8 to 1.5 µm in diameter × 2 to 4 µm long and have blunt ends; occur singly or in chains.
Microscopy: Clostridium perfringens
Spores are large, subterminal, oval and swell the sporangia
Clostridium perfringens
often described as “boxcar-shaped”
Clostridium perfringens
rarely produced when cultured on agar in the laboratory
Clostridium perfringens
Nonmotile, encapsulated
Clostridium perfringens
Colonies are large, gray to grayish yellow; circular, glossy, dome shaped, entire, translucent, typically surrounded by a double zone of hemolysis (AKA target hemolysis)
On anaerobic BAM
Identification tests: Clostridium perfringens
Biochemical characterisitcs:
- Gelatinase
- Lecithinase
- Lipase
- [?]
- [?]
- Gelatinase (+)
- Lecithinase (+)
- Lipase (-)
- Proteolytic
- Saccharolytic
Nagler test: Medium
Egg Yolk Agar (EYA) e.g., Lombard-Dowell medium, or Modified McClung medium
Bacterial lecithinase (phospholipase) breaks down lecithin (a normal component of egg yolk) to [?] resulting in an opaque halo, surrounding the colony when grown on the egg yolk agar medium.
insoluble diglycerides
Procedure: Nagler test
i. Label and dry an [?] and mark the plate into two halves.
ii. Inoculate [?] of C. perfringens type A antitoxin in half of the plate, spread over the surface of agar using a spreader and allow to absorb and dry.
iii. Mark the side of the plate in which the antitoxin is [?].
iv. Streak the test organism in a straight line from the [?] half of the plate to toxin containing side. (Repeat the same procedure with control strains on the same plate.)
v. Incubate anaerobically at
vi. Examine the plate for an around the inoculum and inhibition by antitoxin.
- egg yolk media plate
- 60 µl
- inoculated
- toxin free agar
- 35-37°C for 24-48 hrs
- opalescent halo
Lecithinase (+)
A zone of opacity in the antitoxin-free half only but not on other half due to neutralization of the alpha toxin.
C. perfringens type A antitoxin is not specific for C. perfringens; a positive Nagler reaction can also be produced by [?] if heavy inoculum are used.
C. bifermentans, C. sordellii , and C. baratii
Lecithinase (-)
A zone of opacity on both sides of the plate or no reaction on the agar.
EYA does NOT only differentiate bacterial species based on their lecithinase BUT also
lipase production
While the degradation of lecithin present in the egg yolk results in the formation of opaque precipitate around the colonies, the [?] enzyme hydrolyzes the fats within the egg yolk, which results in an [?] on the colony surface.
lipase
iridescent sheen
Reverse CAMP test
A CAMP positive Group B Streptococcus
center of [?], and C.
perfringens is streaked perpendicular
to it.
sheep blood agar
Following incubation at 37°C for 24-48
hours in anaerobic conditions, the [?] zone of enhanced hemolysis pointing towards [?] is seen.
“bow tie”
S. agalactiae
This is because of [?] produced by C. perfringens interacts with CAMP factor and produce synergistic hemolysis.
alpha toxin
Proteolysis of Milk: Medium
Litmus Milk
The major substrates in skim milk capable of transformation are the milk sugar lactose and the milk proteins [?].
Litmus incorporated in the medium is both a pH and [?] indicator.
At pH [?], the medium is blue or violet-colored. It is a differential used to determine several characteristics of bacteria.
casein, lactalbumin, and lactoglobulin
oxidation-reduction
6.5
The action of bacteria on milk can be categorized as follows:
i. Fermentation of lactose demonstrated when the litmus turns [?] as a result of acid production.
ii. Coagulation of casein by sufficient acids produced as evidenced by the formation of a [?]. If the casein is converted to paracasein by the enzyme rennin, a clear, watery liquid called
“whey” is produced at the top of a thoroughly coagulated tube.
iii. Breakage of [?] indicates gas production.
iv. Peptonization due to digestion of casein as evidenced by a
clearing of the medium and dissolution of the clot.
v. Action of proteolytic enzymes on [?] with production of ammonia or basic amines resulting in an alkaline reaction (blue color).
vi. Reduction of the litmus in the depths of the tube due to the action of reductase enzymes with the resultant removal of oxygen to form the decolorized [?] compound.
- pink
- curd or clot
- coagulum
- lactalbumin
- leucolitmus
Procedure: Proteolysis of Milk
i. Inoculate tubes of Litmus Milk with [?] pure cultures. For the study of anaerobic organisms, sterile mineral oil can be layered over the medium
following inoculation.
ii. Incubate tubes in ambient air at [?] for up to 14 days.
iii. Record reactions at [?] during the incubation process.
- 18- to 24-hour
- 35 ± 2°C
- various intervals
Action of C. perfringens on Litmus Milk.
In litmus milk, lactose is fermented with acid production which is indicated by
the change in color of litmus from [?].
The acid coagulates the [?] (acid clot). The clotted milk is broken up due to large amount of [?]. This phenomenon is known as
blue to pink-red
casein of milk
gas production
“stormy clot” or “stormy fermentation”
Specimens: Clostridium tetani
Material from wound
[?] and [?] of the wound site often fail to reveal the organism. The diagnosis rests on the clinical picture and a [?] of injury.
Direct gram-stained smears
anaerobic cultures
history
Gram-positive bacilli,[?] and [?] measuring 0.4-0.6 x 2-5 um, becoming gram negative after 24-hr incubation; occur singly or in pairs.
long and slender
are round and terminal within swollen sporangia giving a drumstick, lollipop or tennis racket appearance.
Spores
Motility
Motile
Capsule
Non-encapsulated
Colonies are small, gray; matte surface, irregular to rhizoid margin, translucent, flat; narrow zone of β-hemolysis due to hemolysin, tetanolysin.
Clostridium tetani: On anaerobic BAM
Colonies tend to swarm producing thin veil of growth over the entire agar surface. Stiff blood agar, which contains 4% to 6% agar, instead of the usual 1.5%, is sometimes used in plating media to minimize swarming.
Clostridium tetani: On anaerobic BAM
Clostridium tetani: Biochemical characterisitcs:
- Gelatinase
- Lecithinase
- Lipase
- [?]
- [?]
Biochemical characterisitcs:
- Gelatinase (+)
- Lecithinase (+)
- Lipase (-)
- Proteolytic
- Asaccharolytic
Neutralization test on stiff BAM
One-half of the medium is inoculated with [?] (1,500 units per ml), while the other half does not contain any antitoxin. Strains of C. tetani are stab inoculated on each half of the plate and incubated [?] for [?].
tetanus antitoxin
anaerobically for 48 hours
Neutralization test on stiff BAM
The colonies of C. tetani show hemolysis without any [?], but do not show any hemolysis on the [?].
This is due to the inhibition of [?] by antitoxin.
antitoxin
part of the agar with antitoxin
hemolytic activity of toxin
Lethal effect and neutralization test in mice
[?] in saline or culture suspension is injected into the hind leg/ tail of two mice, one of which is protected by giving [?] units of tetanus antitoxin 1 hour before the test (control animal), the other one is not (test animal).
Tissue homogenate
1000 units
The mice are observed for up to 3 days for the development of tetanus in unprotected group.
Lethal effect and neutralization test in mice
The animal usually dies within [?]. No symptoms appear in the control animal.
48 hours
Used for demonstration of botulinum toxin and/or C. botulinum
Serum feces gastric contents or vomitus exudate or tissue sample from the wound (for wound botulism) suspect food
Demonstration of C.botulinum in these specimens is highly suggestive of C. botulinum because [?] is rare.
carriage
Large (0.5-0.8 x 3-6 um), gram-positive, straight rods; occur singly or in pairs.
C.botulinum
Spores forms readily and abundantly, are usually subterminal within a swollen
sporangium giving it a characteristic [?] appearance,
“snow-shoe”
C. botulinum: Capsule
Non-encapsulated
Colonies are gray-white; circular to irregular; usually β-hemolytic.
C. botulinum: On anaerobic BAM
C. botulinum: Biochemical characterisitcs:
- Gelatinase
- Lecithinase
- Lipase
- [?]
- [?]
- Gelatinase (+)
- Lecithinase (-)
- Lipase (-)
- Proteolytic
- Asaccharolytic
by lethal effect and neutralization test in mice
Serum toxin bioassay
Enzyme linked-immunosorbent assay (ELISA)
Serology
Polymerase chain reaction (PCR)
Molecular genetic assay
Clostridioides difficile: Specimens
Watery or semisolid, unformed fecal specimens
Gram-positive straight rods; may produce chains of up to six cells aligned end to end.
Clostridioides difficile
Spores are spores oval and subterminal within a swollen sporangium.
Clostridioides difficile
Clostridioides difficile: Motility
Motile
Clostridioides difficile: Capsule
Nonencapsulated
Heat shock or alcohol treatment to eliminate contaminating vegetative cells.
spore selection technique
Enrichment broth supplemented with sodium taurocholate[?] to enhance spore germination.
sodium taurocholate
Colonies are large (2-4 mm), white, circular, matte to glossy, convex, opaque; nonhemolytic.
Clostridioides difficile: On anaerobic BAM
Clostridioides difficile: On Cycloserine-cefoxitin fructose agar (CCFA)
Colonies are 4 mm in diameter or larger, yellowish, rhizoid that have birefringent crystalline internal structures
(“speckled opalescence”, or “ground glass”).
Clostridioides difficile: On Cycloserine-cefoxitin fructose agar (CCFA)
Cultures show [?] under longwave UV light.
yellow-green fluorescence
Produce [?] odor due to production of [?].
“horse manure” (horse barn)
para-cresol
Biochemical characterisitcs:
- Gelatinase
- Lecithinase
- Lipase
- Proteolytic
- Saccharolytic
Biochemical characterisitcs:
- Gelatinase (+)
- Lecithinase (-)
- Lipase (-)
- Proteolytic
- Saccharolytic
a. Stool cytotoxin test
is filtered and inoculated to Hep-2 human diploid cell cultures.
Diarrheal stool
a. Stool cytotoxin test
The demonstration of cytopathic effect that is neutralized by [?] indicates the presence of [?] (positive test).
specific antiserum
toxin
Toxigenicity test: Serology
ELISA, Latex agglutination test
