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[8] LABORATORY DIAGNOSIS > Spore-forming Gram-positive Bacilli > Flashcards

Spore-forming Gram-positive Bacilli Flashcards

1
Q

Specimens: Bacillus anthracis

[?] from beneath the eschar from cutaneous lesion.

A

Vesicular fluid or biopsy

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2
Q

Specimens: Bacillus anthracis

[?] from gastrointestinal anthrax.

A

Gastric aspirates or stools

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3
Q

Specimens: Bacillus anthracis

[?] in sepsis

A

Blood or cerebrospinal fluid

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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

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5
Q

Gram staining: Bacillus anthracis

Large, G+ bacilli, [?]

A

single and/or in chains.

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6
Q

Gram staining: Bacillus anthracis

Unstained areas within the cell indicate [?]

A

sporulation

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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)

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8
Q

water soluble and has a low affinity for cellular material, so vegetative cells may be decolorized with water

A

Malachite green

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9
Q

is then applied to counterstain any cells which have been decolorized.

A

Safranin

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10
Q

At the end of the staining process

vegetative cells will be [?], and endospores will be [?].

A

pink

green

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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

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12
Q

Performed directly on clinical material; capsules are normally lost in culture.

A

Capsular staining

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13
Q

wet preparation highlights the capsule as a transparent halo around the bacillus.

A

India ink

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14
Q

Uses polychrome methylene blue.

A

McFadyean staining

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15
Q

[?]-stained capsule seen surrounding the deep bluish bacilli.

A

Purple/pink

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16
Q

Culture media: Bacillus anthracis

A
  1. 5% sheep BAM
  2. Polymyxin-lysozyme-EDTA-thallous acetate (PLET)
  3. Nutrient agar supplemented with MnSO4
  4. Bicarbonate agar
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17
Q

Best selective medium for isolation of B. anthracis.

A

Polymyxin-lysozyme-EDTA-thallous acetate (PLET)

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18
Q

inhibits or retards most G- bacteria

A

Polymyxin B

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19
Q

is used to destroy vegetative cells

A

Lysozyme

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20
Q

a chelating agent

A

EDTA

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21
Q

a selective agent against G+ and G-bacteria

A

Thallous acetate

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22
Q

Used for subcultures to stimulate sporulation

A

Nutrient agar supplemented with MnSO4

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23
Q

Bicarbonate agar is a Nutrient agar with

A

sodium bicarbonate and 5-10% horse serum

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24
Q

Used for subcultures to induce capsule formation.

A

Bicarbonate agar

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25
Q

Specimens should be inoculated to a [?] and [?]

A

blood agar and a selective medium (PLET).

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26
Q

Inoculate both [?] to ensure maximal recovery.

A

heat-treated and untreated specimens

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27
Q

Incubate either in ambient air or in [?]. Bicarbonate agar requires incubation in CO2.

A

5% to 10% CO2 for 24 - 48 h

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28
Q

Medium to large ([?] in diameter)

A

2-5 mm

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29
Q

Non-pigmented (?)

A

white to gray

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30
Q

[?] convex

A

Flat or slightly

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31
Q

Dry, rough, [?]surface

A

“ground-glass”

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32
Q

Irregular edges with “comma-shaped” projections

A

“Medusa head” or “comet tail” colonies

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33
Q

Under dissecting microscope (or a hand lens), numerous undulating [?] outgrowths consisting of filamentous chains of bacilli project from the colony.

A

“curled hair”

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34
Q

Sticky or tenacious consistency such that, when the colonies are touched with inoculating loop, they form standing peaks resembling

A

beaten egg whites

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35
Q

gamma-hemolytic on

A

BAM

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36
Q

Catalase test: Bacillus anthracis

A

Positive

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37
Q

Motility test: Bacillus anthracis

A

Negative (nonmotile)

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38
Q

Determined by stab inoculation of semisolid motility medium

A

Motility test

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39
Q

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.

A

motility

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40
Q

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.

A

Growth on bicarbonate agar

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41
Q

[?] type is strongly indicative of virulent B. anthracis attributed to capsule formation.

A

Large and mucoid colony

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42
Q

Avirulent B. anthracis produces [?] under these conditions.

A

rough colonies

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43
Q

Performed by stabbing a gelatin tube with the suspect colony and incubated.

A

Gelatin hydrolysis test

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44
Q

B. anthracis liquefies gelatin slowly producing an [?] growth.

A

“inverted fir tree”

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45
Q

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.

A

Susceptibility to penicillin

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46
Q

B. anthracis are susceptible to penicillin (10 U), so a [?] will be visible.

A

zone of inhibition

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47
Q

Test organism is heavily streaked on agar plates (e.g., MHA) containing [?] unit of penicillin G per ml, and incubated at

A

0.5

37 °C for 3 to 6 hours

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48
Q

The plate is examined by placing a cover slip over the [?], and observed under the high, dry objective

A

inoculated area

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49
Q

B. anthracis will assume [?] characteristic of the string-of-pearls reaction.

A

large, round cellular forms

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50
Q

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.

A

sterile water

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51
Q

has the ability to lyse B. anthracis grown aerobically on blood or other nutrient agar.

A

“Gamma” phage

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52
Q

[?] in the region of confluent growth where the gamma phage was applied results from the phage’s ability to lyse the bacterial cells.

A

A plaque (clear area)

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53
Q

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.

A

Animal pathogenicity test

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54
Q

Tissue extract, prepared by boiling tissue samples in saline and filtered, is layered over anthrax antiserum in a narrow tube.

A

Ascoli test

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55
Q

The development of [?] at the junction of the two fluids within 10 minutes at room temperature denotes a positive results for Ascoli test

A

white ring of precipitate

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56
Q

Similar to B. anthracis, but B. cereus are [?], and are

A

motile with peritrichous flagella

non-encapsulated

57
Q

Heat treatment at [?] or [?] is effective for killing vegetative cells and retaining spores for most Bacillus spp.

A

70°C for 30 minutes or 80°C for 10 minutes

58
Q

Large, feathery, spreading colonies that are cream to white or grey and have a slight green tinge

A

5% Sheep BAM

59
Q

β-hemolytic for B. cereus

A

5% Sheep BAM

60
Q

Bacteria that ferment [?] produce [?] and form colonies that are [?] with as the pH indicator.

A

mannitol; acids; yellow

phenol red

61
Q

Bacteria that produce [?] hydrolyze lecithin in egg yolk and a [?] forms around the colonies.

A

lecithinase

zone of white precipitate

62
Q

inhibits the growth of most other bacteria.

A

Polymixin

63
Q

B. cereus colonies are [?] (mannitol-negative) and surrounded by [?] (lecithinase-positive).

A

pink-red

zone of precipitate

64
Q

Polymyxin, Egg Yolk, Mannitol, Bromthymol Blue Agar (PEMBA)

Addition of [?] improve egg yolk precipitation and enhance sporulation

A

sodium pyruvate

65
Q

is added as a pH indicator to detect mannitol utilization.

A

Bromthymol blue

66
Q

is the selective agent.

A

Polymyxin B

67
Q

B. cereus colonies are crenated, 5mm diameter,[?] with a zone of egg yolk precipitation after 18-24hr incubation.

A

turquoise to peacock blue

68
Q

Best obtained by tissue [?] or by [?] using a needle and syringe to prevent contamination with normal flora.

A

biopsy or by aspiration

69
Q

Use transport devices that contain [?] to protect them from oxygen exposure

A

pre-reduced anaerobically sterilized (PRAS) media

70
Q

Deliver to the laboratory

A

promptly

71
Q

Processed in the

A

biologic safety cabinet

72
Q

Processing of specimen with minimal exposure to

A

atmospheric oxygen

73
Q

Use of combination of

A

selective and nonselective media

74
Q

In most instances, [?] is the recommended temperature for primary isolation of anaerobes.

A

35°C to 37°C

75
Q

Use of

A

anaerobic incubation system

76
Q

Specimens: Clostridium perfringens

A

Material from wounds, pus, and tissue

77
Q

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.

A

Microscopy: Clostridium perfringens

78
Q

Spores are large, subterminal, oval and swell the sporangia

A

Clostridium perfringens

79
Q

often described as “boxcar-shaped”

A

Clostridium perfringens

80
Q

rarely produced when cultured on agar in the laboratory

A

Clostridium perfringens

81
Q

Nonmotile, encapsulated

A

Clostridium perfringens

82
Q

Colonies are large, gray to grayish yellow; circular, glossy, dome shaped, entire, translucent, typically surrounded by a double zone of hemolysis (AKA target hemolysis)

A

On anaerobic BAM

83
Q

Identification tests: Clostridium perfringens

Biochemical characterisitcs:

  • Gelatinase
  • Lecithinase
  • Lipase
  • [?]
  • [?]
A
  • Gelatinase (+)
  • Lecithinase (+)
  • Lipase (-)
  • Proteolytic
  • Saccharolytic
84
Q

Nagler test: Medium

A

Egg Yolk Agar (EYA) e.g., Lombard-Dowell medium, or Modified McClung medium

85
Q

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.

A

insoluble diglycerides

86
Q

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.

A
  • egg yolk media plate
  • 60 µl
  • inoculated
  • toxin free agar
  • 35-37°C for 24-48 hrs
  • opalescent halo
87
Q

Lecithinase (+)

A

A zone of opacity in the antitoxin-free half only but not on other half due to neutralization of the alpha toxin.

88
Q

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.

A

C. bifermentans, C. sordellii , and C. baratii

89
Q

Lecithinase (-)

A

A zone of opacity on both sides of the plate or no reaction on the agar.

90
Q

EYA does NOT only differentiate bacterial species based on their lecithinase BUT also

A

lipase production

91
Q

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.

A

lipase

iridescent sheen

92
Q

Reverse CAMP test

A CAMP positive Group B Streptococcus
center of [?], and C.
perfringens is streaked perpendicular
to it.

A

sheep blood agar

93
Q

Following incubation at 37°C for 24-48

hours in anaerobic conditions, the [?] zone of enhanced hemolysis pointing towards [?] is seen.

A

“bow tie”

S. agalactiae

94
Q

This is because of [?] produced by C. perfringens interacts with CAMP factor and produce synergistic hemolysis.

A

alpha toxin

95
Q

Proteolysis of Milk: Medium

A

Litmus Milk

96
Q

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.

A

casein, lactalbumin, and lactoglobulin
oxidation-reduction
6.5

97
Q

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.

A
  • pink
  • curd or clot
  • coagulum
  • lactalbumin
  • leucolitmus
98
Q

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.

A
  • 18- to 24-hour
  • 35 ± 2°C
  • various intervals
99
Q

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

A

blue to pink-red
casein of milk
gas production
“stormy clot” or “stormy fermentation”

100
Q

Specimens: Clostridium tetani

A

Material from wound

101
Q

[?] and [?] of the wound site often fail to reveal the organism. The diagnosis rests on the clinical picture and a [?] of injury.

A

Direct gram-stained smears
anaerobic cultures
history

102
Q

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.

A

long and slender

103
Q

are round and terminal within swollen sporangia giving a drumstick, lollipop or tennis racket appearance.

A

Spores

104
Q

Motility

A

Motile

105
Q

Capsule

A

Non-encapsulated

106
Q

Colonies are small, gray; matte surface, irregular to rhizoid margin, translucent, flat; narrow zone of β-hemolysis due to hemolysin, tetanolysin.

A

Clostridium tetani: On anaerobic BAM

107
Q

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.

A

Clostridium tetani: On anaerobic BAM

108
Q

Clostridium tetani: Biochemical characterisitcs:

  • Gelatinase
  • Lecithinase
  • Lipase
  • [?]
  • [?]
A

Biochemical characterisitcs:

  • Gelatinase (+)
  • Lecithinase (+)
  • Lipase (-)
  • Proteolytic
  • Asaccharolytic
109
Q

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 [?].

A

tetanus antitoxin

anaerobically for 48 hours

110
Q

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.

A

antitoxin
part of the agar with antitoxin
hemolytic activity of toxin

111
Q

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).

A

Tissue homogenate

1000 units

112
Q

The mice are observed for up to 3 days for the development of tetanus in unprotected group.

A

Lethal effect and neutralization test in mice

113
Q

The animal usually dies within [?]. No symptoms appear in the control animal.

A

48 hours

114
Q

Used for demonstration of botulinum toxin and/or C. botulinum

A 
Serum
feces
gastric contents or vomitus
exudate or tissue sample
from the wound (for wound botulism)
suspect food
115
Q

Demonstration of C.botulinum in these specimens is highly suggestive of C. botulinum because [?] is rare.

A

carriage

116
Q

Large (0.5-0.8 x 3-6 um), gram-positive, straight rods; occur singly or in pairs.

A

C.botulinum

117
Q

Spores forms readily and abundantly, are usually subterminal within a swollen
sporangium giving it a characteristic [?] appearance,

A

“snow-shoe”

118
Q

C. botulinum: Capsule

A

Non-encapsulated

119
Q

Colonies are gray-white; circular to irregular; usually β-hemolytic.

A

C. botulinum: On anaerobic BAM

120
Q

C. botulinum: Biochemical characterisitcs:

  • Gelatinase
  • Lecithinase
  • Lipase
  • [?]
  • [?]
A
  • Gelatinase (+)
  • Lecithinase (-)
  • Lipase (-)
  • Proteolytic
  • Asaccharolytic
121
Q

by lethal effect and neutralization test in mice

A

Serum toxin bioassay

122
Q

Enzyme linked-immunosorbent assay (ELISA)

A

Serology

123
Q

Polymerase chain reaction (PCR)

A

Molecular genetic assay

124
Q

Clostridioides difficile: Specimens

A

Watery or semisolid, unformed fecal specimens

125
Q

Gram-positive straight rods; may produce chains of up to six cells aligned end to end.

A

Clostridioides difficile

126
Q

Spores are spores oval and subterminal within a swollen sporangium.

A

Clostridioides difficile

127
Q

Clostridioides difficile: Motility

A

Motile

128
Q

Clostridioides difficile: Capsule

A

Nonencapsulated

129
Q

Heat shock or alcohol treatment to eliminate contaminating vegetative cells.

A

spore selection technique

130
Q

Enrichment broth supplemented with sodium taurocholate[?] to enhance spore germination.

A

sodium taurocholate

131
Q

Colonies are large (2-4 mm), white, circular, matte to glossy, convex, opaque; nonhemolytic.

A

Clostridioides difficile: On anaerobic BAM

132
Q

Clostridioides difficile: On Cycloserine-cefoxitin fructose agar (CCFA)

Colonies are 4 mm in diameter or larger, yellowish, rhizoid that have birefringent crystalline internal structures

A

(“speckled opalescence”, or “ground glass”).

133
Q

Clostridioides difficile: On Cycloserine-cefoxitin fructose agar (CCFA)

Cultures show [?] under longwave UV light.

A

yellow-green fluorescence

134
Q

Produce [?] odor due to production of [?].

A

“horse manure” (horse barn)

para-cresol

135
Q

Biochemical characterisitcs:

  • Gelatinase
  • Lecithinase
  • Lipase
  • Proteolytic
  • Saccharolytic
A

Biochemical characterisitcs:

  • Gelatinase (+)
  • Lecithinase (-)
  • Lipase (-)
  • Proteolytic
  • Saccharolytic
136
Q

a. Stool cytotoxin test

is filtered and inoculated to Hep-2 human diploid cell cultures.

A

Diarrheal stool

137
Q

a. Stool cytotoxin test

The demonstration of cytopathic effect that is neutralized by [?] indicates the presence of [?] (positive test).

A

specific antiserum

toxin

138
Q

Toxigenicity test: Serology

A

ELISA, Latex agglutination test

[8] LABORATORY DIAGNOSIS (5 decks)

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