Spore-forming Gram-positive Bacilli Flashcards
(138 cards)
1
Q
Specimens: Bacillus anthracis
[?] from beneath the eschar from cutaneous lesion.
A
Vesicular fluid or biopsy
2
Q
Specimens: Bacillus anthracis
[?] from gastrointestinal anthrax.
A
Gastric aspirates or stools
3
Q
Specimens: Bacillus anthracis
[?] in sepsis
A
Blood or cerebrospinal fluid
4
Q
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.
A
Heat shock treatment
5
Q
Gram staining: Bacillus anthracis
Large, G+ bacilli, [?]
A
single and/or in chains.
6
Q
Gram staining: Bacillus anthracis
Unstained areas within the cell indicate [?]
A
sporulation
7
Q
A primary stain-malachite green is forced into the spore by steaming the bacterial emulsion
A
Spore staining (e.g.,Schaeffer-Fulton, Dorner)
8
Q
water soluble and has a low affinity for cellular material, so vegetative cells may be decolorized with water
A
Malachite green
9
Q
is then applied to counterstain any cells which have been decolorized.
A
Safranin
10
Q
At the end of the staining process
vegetative cells will be [?], and endospores will be [?].
A
pink
green
11
Q
Both [?] that do not swell the vegetative cell and cell-free spores outside of the vegetative cells may be observed.
A
intracellular, central or subterminal spores
12
Q
Performed directly on clinical material; capsules are normally lost in culture.
A
Capsular staining
13
Q
wet preparation highlights the capsule as a transparent halo around the bacillus.
A
India ink
14
Q
Uses polychrome methylene blue.
A
McFadyean staining
15
Q
[?]-stained capsule seen surrounding the deep bluish bacilli.
A
Purple/pink
16
Q
Culture media: Bacillus anthracis
A
- 5% sheep BAM
- Polymyxin-lysozyme-EDTA-thallous acetate (PLET)
- Nutrient agar supplemented with MnSO4
- Bicarbonate agar
17
Q
Best selective medium for isolation of B. anthracis.
A
Polymyxin-lysozyme-EDTA-thallous acetate (PLET)
18
Q
inhibits or retards most G- bacteria
A
Polymyxin B
19
Q
is used to destroy vegetative cells
A
Lysozyme
20
Q
a chelating agent
A
EDTA
21
Q
a selective agent against G+ and G-bacteria
A
Thallous acetate
22
Q
Used for subcultures to stimulate sporulation
A
Nutrient agar supplemented with MnSO4
23
Q
Bicarbonate agar is a Nutrient agar with
A
sodium bicarbonate and 5-10% horse serum
24
Q
Used for subcultures to induce capsule formation.
A
Bicarbonate agar
25
Specimens should be inoculated to a [?] and [?]
blood agar and a selective medium (PLET).
26
Inoculate both [?] to ensure maximal recovery.
heat-treated and untreated specimens
27
Incubate either in ambient air or in [?]. Bicarbonate agar requires incubation in CO2.
5% to 10% CO2 for 24 - 48 h
28
Medium to large ([?] in diameter)
2-5 mm
29
Non-pigmented (?)
white to gray
30
[?] convex
Flat or slightly
31
Dry, rough, [?]surface
"ground-glass"
32
Irregular edges with "comma-shaped" projections
"Medusa head" or "comet tail" colonies
33
Under dissecting microscope (or a hand lens), numerous undulating [?] outgrowths consisting of filamentous chains of bacilli project from the colony.
"curled hair"
34
Sticky or tenacious consistency such that, when the colonies are touched with inoculating loop, they form standing peaks resembling
beaten egg whites
35
gamma-hemolytic on
BAM
36
Catalase test: Bacillus anthracis
Positive
37
Motility test: Bacillus anthracis
Negative (nonmotile)
38
Determined by stab inoculation of semisolid motility medium
Motility test
39
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
40
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
41
[?] type is strongly indicative of virulent B. anthracis attributed to capsule formation.
Large and mucoid colony
42
Avirulent B. anthracis produces [?] under these conditions.
rough colonies
43
Performed by stabbing a gelatin tube with the suspect colony and incubated.
Gelatin hydrolysis test
44
B. anthracis liquefies gelatin slowly producing an [?] growth.
"inverted fir tree"
45
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
46
B. anthracis are susceptible to penicillin (10 U), so a [?] will be visible.
zone of inhibition
47
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
48
The plate is examined by placing a cover slip over the [?], and observed under the high, dry objective
inoculated area
49
B. anthracis will assume [?] characteristic of the string-of-pearls reaction.
large, round cellular forms
50
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
51
has the ability to lyse B. anthracis grown aerobically on blood or other nutrient agar.
“Gamma” phage
52
[?] 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)
53
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
54
Tissue extract, prepared by boiling tissue samples in saline and filtered, is layered over anthrax antiserum in a narrow tube.
Ascoli test
55
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
56
Similar to B. anthracis, but B. cereus are [?], and are
motile with peritrichous flagella
| non-encapsulated
57
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
58
Large, feathery, spreading colonies that are cream to white or grey and have a slight green tinge
5% Sheep BAM
59
β-hemolytic for B. cereus
5% Sheep BAM
60
Bacteria that ferment [?] produce [?] and form colonies that are [?] with as the pH indicator.
mannitol; acids; yellow
| phenol red
61
Bacteria that produce [?] hydrolyze lecithin in egg yolk and a [?] forms around the colonies.
lecithinase
| zone of white precipitate
62
inhibits the growth of most other bacteria.
Polymixin
63
B. cereus colonies are [?] (mannitol-negative) and surrounded by [?] (lecithinase-positive).
pink-red
| zone of precipitate
64
Polymyxin, Egg Yolk, Mannitol, Bromthymol Blue Agar (PEMBA)
Addition of [?] improve egg yolk precipitation and enhance sporulation
sodium pyruvate
65
is added as a pH indicator to detect mannitol utilization.
Bromthymol blue
66
is the selective agent.
Polymyxin B
67
B. cereus colonies are crenated, 5mm diameter,[?] with a zone of egg yolk precipitation after 18-24hr incubation.
turquoise to peacock blue
68
Best obtained by tissue [?] or by [?] using a needle and syringe to prevent contamination with normal flora.
biopsy or by aspiration
69
Use transport devices that contain [?] to protect them from oxygen exposure
pre-reduced anaerobically sterilized (PRAS) media
70
Deliver to the laboratory
promptly
71
Processed in the
biologic safety cabinet
72
Processing of specimen with minimal exposure to
atmospheric oxygen
73
Use of combination of
selective and nonselective media
74
In most instances, [?] is the recommended temperature for primary isolation of anaerobes.
35°C to 37°C
75
Use of
anaerobic incubation system
76
Specimens: Clostridium perfringens
Material from wounds, pus, and tissue
77
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
78
Spores are large, subterminal, oval and swell the sporangia
Clostridium perfringens
79
often described as "boxcar-shaped"
Clostridium perfringens
80
rarely produced when cultured on agar in the laboratory
Clostridium perfringens
81
Nonmotile, encapsulated
Clostridium perfringens
82
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
83
Identification tests: Clostridium perfringens
Biochemical characterisitcs:
- Gelatinase
- Lecithinase
- Lipase
- [?]
- [?]
- Gelatinase (+)
- Lecithinase (+)
- Lipase (-)
- Proteolytic
- Saccharolytic
84
Nagler test: Medium
Egg Yolk Agar (EYA) e.g., Lombard-Dowell medium, or Modified McClung medium
85
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
86
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
87
Lecithinase (+)
A zone of opacity in the antitoxin-free half only but not on other half due to neutralization of the alpha toxin.
88
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
89
Lecithinase (-)
A zone of opacity on both sides of the plate or no reaction on the agar.
90
EYA does NOT only differentiate bacterial species based on their lecithinase BUT also
lipase production
91
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
92
Reverse CAMP test
A CAMP positive Group B Streptococcus
center of [?], and C.
perfringens is streaked perpendicular
to it.
sheep blood agar
93
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
94
This is because of [?] produced by C. perfringens interacts with CAMP factor and produce synergistic hemolysis.
alpha toxin
95
Proteolysis of Milk: Medium
Litmus Milk
96
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
97
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
98
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
99
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"
100
Specimens: Clostridium tetani
Material from wound
101
[?] 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
102
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
103
are round and terminal within swollen sporangia giving a drumstick, lollipop or tennis racket appearance.
Spores
104
Motility
Motile
105
Capsule
Non-encapsulated
106
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
107
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
108
Clostridium tetani: Biochemical characterisitcs:
- Gelatinase
- Lecithinase
- Lipase
- [?]
- [?]
Biochemical characterisitcs:
- Gelatinase (+)
- Lecithinase (+)
- Lipase (-)
- Proteolytic
- Asaccharolytic
109
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
110
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
111
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
112
The mice are observed for up to 3 days for the development of tetanus in unprotected group.
Lethal effect and neutralization test in mice
113
The animal usually dies within [?]. No symptoms appear in the control animal.
48 hours
114
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
```
115
Demonstration of C.botulinum in these specimens is highly suggestive of C. botulinum because [?] is rare.
carriage
116
Large (0.5-0.8 x 3-6 um), gram-positive, straight rods; occur singly or in pairs.
C.botulinum
117
Spores forms readily and abundantly, are usually subterminal within a swollen
sporangium giving it a characteristic [?] appearance,
“snow-shoe”
118
C. botulinum: Capsule
Non-encapsulated
119
Colonies are gray-white; circular to irregular; usually β-hemolytic.
C. botulinum: On anaerobic BAM
120
C. botulinum: Biochemical characterisitcs:
- Gelatinase
- Lecithinase
- Lipase
- [?]
- [?]
- Gelatinase (+)
- Lecithinase (-)
- Lipase (-)
- Proteolytic
- Asaccharolytic
121
by lethal effect and neutralization test in mice
Serum toxin bioassay
122
Enzyme linked-immunosorbent assay (ELISA)
Serology
123
Polymerase chain reaction (PCR)
Molecular genetic assay
124
Clostridioides difficile: Specimens
Watery or semisolid, unformed fecal specimens
125
Gram-positive straight rods; may produce chains of up to six cells aligned end to end.
Clostridioides difficile
126
Spores are spores oval and subterminal within a swollen sporangium.
Clostridioides difficile
127
Clostridioides difficile: Motility
Motile
128
Clostridioides difficile: Capsule
Nonencapsulated
129
Heat shock or alcohol treatment to eliminate contaminating vegetative cells.
spore selection technique
130
Enrichment broth supplemented with sodium taurocholate[?] to enhance spore germination.
sodium taurocholate
131
Colonies are large (2-4 mm), white, circular, matte to glossy, convex, opaque; nonhemolytic.
Clostridioides difficile: On anaerobic BAM
132
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").
133
Clostridioides difficile: On Cycloserine-cefoxitin fructose agar (CCFA)
Cultures show [?] under longwave UV light.
yellow-green fluorescence
134
Produce [?] odor due to production of [?].
"horse manure" (horse barn)
| para-cresol
135
Biochemical characterisitcs:
- Gelatinase
- Lecithinase
- Lipase
- Proteolytic
- Saccharolytic
Biochemical characterisitcs:
- Gelatinase (+)
- Lecithinase (-)
- Lipase (-)
- Proteolytic
- Saccharolytic
136
a. Stool cytotoxin test
is filtered and inoculated to Hep-2 human diploid cell cultures.
Diarrheal stool
137
a. Stool cytotoxin test
The demonstration of cytopathic effect that is neutralized by [?] indicates the presence of [?] (positive test).
specific antiserum
toxin
138
Toxigenicity test: Serology
ELISA, Latex agglutination test
