LABORATORY Flashcards
Is the preferred specimen, collected and transported in Cary-Blair medium.
Feces
is not acceptable, because glycerol is toxic to vibrios.
Buffered glycerol saline
Are also acceptable
Rectal swabs
Gram-negative curved or comma-shaped bacilli, 2–4 µm long.
V. cholerae
On prolonged cultivation, vibrios may become [?] that resemble the [?].
straight rods
Gram-negative enteric bacteria
[?]
The [?] incorporated in this medium promotes the growth of Vibrio species, while the [?] of this medium inhibits most of the commensal intestinal bacteria.
Alkaline Peptone Water (APW)
- 1% (w/v) sodium chloride
- alkalinity (pH 8.6)
APW is incubated at [?] prior to inoculation of plated media.
35°C for 12-18 hours
is done when few organisms are present, as in specimens from convalescent patients and asymptomatic carriers.
Enrichment in APW
When the patient is in very early stages of illness and is passing liquid stool, it may not be necessary to [?] stool specimens.
enrich
Colonies are typically medium to large, smooth, convex, creamy in consistency, gray-white, and have entire margins, smooth, opaque; may be β-hemolytic
5% Sheep BAM
Colonies appear transparent and usually have a characteristic cloudy or opaque zone (due to [?] activity) around colony, which becomes more definite after few minutes of [?].
Gelatin Agar (GA)
- gelatinase
- refrigeration
[?]
When these colonies are viewed in oblique light
they appear [?] to [?] colored and [?]
Gelatin Agar (GA)
- iridescent green
- bronze
- finely granular
Selective medium of choice for the isolation of V. cholerae and V. parahaemolyticus as well as other vibrios.
Thiosulfate Citrate Bile Sucrose (TCBS) Agar
TCBS Agar
stimulates growth of Vibrio species.
inhibit gram-positive bacteria
1% Sodium chloride Bile salts (oxgall)
TCBS Agar
serves as a sulfur source, in combination with [?], detects hydrogen sulfide production
Sodium thiosulfate
ferric citrate
is included as a fermentable carbohydrate for the metabolism of vibrios.
Saccharose (sucrose)
The [?] of the TCBS agar inhibits most of the commensal intestinal bacteria.
alkaline pH (8.6)
are included as indicators of pH changes in Thiosulfate Citrate Bile Sucrose (TCBS) Agar
Thymol blue and bromthymol blue
Suspicious colonies for further testing in Thiosulfate Citrate Bile Sucrose (TCBS) Agar should be subcultured to a
noninhibitory medium
examples of noninhibitory medium
gelatin agar, heart infusion agar (HIA), Kligler’s iron agar (KIA), or triple sugar iron agar (TSI).
inhibits the contaminating gram-positive bacteria in TTGA or Monsur’s agar
Sodium taurocholate
is a selective and differential agent in Taurocholate tellurite gelatin agar (TTGA or Monsur’s agar)
inhibits many gram-positive bacteria and due to the reduction reaction the colonies form a grey to black color
Potassium tellurite
helps in maintaining the elevated pH of the medium which is also inhibitory to most Enterobacteriaceae and gram-positive bacteria.
Sodium carbonate
acts as an additional carbon and energy source and is the substrate of bacterial gelatinase causing halo effect around the colony.
Gelatin
support the growth of some but not all strains of V. cholerae.
MacConkey (MAC) Agar
Overnight colonies of V. cholerae on MAC tend to be small to moderately sized (1 to 3 mm) and usually appear as lactose-negative (colorless) or [?], often resembling colonies of [?] lactose-fermenting organisms.
slightly pink
“late” or “slow”
A characteristic that separates Vbrios from the Enterobacteriaceae.
Oxidase (+)
Conduct the oxidase test with fresh growth from an [?] or any non-carbohydrate-containing medium. Do not use growth from TCBS agar.
HIA slant
TSI: A/A, no gas, no H2S;
KIA:K/A, no gas, no H2S
Gluc+, Lac-, Suc+
- Modified by the addition of 1% NaCl = [?]
- Lysine decarboxylation =
- LIA:
- Ornithine decarboxylation =
- Arginine dihydrolase =
Decarboxylase/Dihydrolase Test
- (+)
- K/K
- (+)
- (-)
[?] Principle
Vibrio species suspended in [?] will be lysed. The suspension will lose [?], and [?] will be released from the lysed cells causing the mixture to become viscous.
String Test
- 0.5% sodium desoxycholate
- turbidity
- DNA
[?] Procedure
[?]-hour colonies from HIA or other noninhibitory medium are mixed with a few drops of [?] on a glass slide. An [?] is immersed into the mixture and pulled away from the drop.
String Test
- 18- to 24
- 0.5% sodium desoxycholate
- inoculating loop
[?] Result’
Vibrio cholerae produces a [?] that becomes more [?] after [?] seconds or more (other vibrios may give an initial string reaction that diminishes or disappears [?] seconds later.
String Test
- long string
- tenacious
- 60
- 45 to 60
[?] Principle
Vibrios either require [?] or have their growth stimulated by its addition.
Growth in Salt Broths
- NaCl
[?] Procedure
The [?] are inoculated very lightly from fresh growth.
The inoculum should be light enough to prevent visible [?] before incubation of the broths.
The broths are incubated at [?] and read at [?] hours. In the absence of [?], they may be incubated for up to 7 days
Growth in Salt Broths
- 0% and 1% salt broths
- turbidity
- 35˚ to 37˚C
- 18 to 24
- overnight growth
[?]
V. cholerae grows in [?] without NaCl, and stimulated by addition of [?]
Growth in Salt Broths
- nutrient broth base
- 1.0% NaCl
[?]
Uses [?] (referred to as O/129)-impregnated disks.
Vibriostatic Test
- 2,4-diamino-6,7-diisopropyl-pteridine phosphate
[?]
V. cholerae: [?]
Other Vibrio species: [?]
However, recent strains of [?] and [?] have demonstrated resistance; therefore the dependability of this test is questionable.
Vibriostatic Test
- O129-susceptible
- O129-resistant
- V. cholerae O1
- V. cholerae O139
[?]
Addition of [?] to 24-h culture in APW with [?] and [?].
(+) result: [?] (due to nitroso indole)
Cholera red test (Nitroso-indole test)
- H2SO4
- tryptophan and nitrate
- Red color
Lysis of V. cholerae organisms when inoculated into immuned guinea pig
Pfeiffer’s phenomenon
[?]
Principle:
Used to identify V. cholerae [?] , and further subdivide into serotypes, Ogawa, Inaba, and Hikojima by [?] with specific antisera: [?], [?], and [?]
Serological Tests
- O1 serogroup
- agglutination
- anti-O1, Ogawa antiserum, and Inaba antiserum
[?]
Procedure:
i. Agglutination tests may be carried out in a [?] or on a clean glass slide.
ii. An inoculating needle or loop, or sterile applicator stick, or tooth pick is used to [?] from the surface of HIA, KIA, TSI, or other nonselective agar medium.
iii. Emulsify the growth in a small drop of physiological saline and mix thoroughly by tilting back and forth for about 30 seconds. Examine the suspension carefully to ensure that it is even and does not show clumping due to [?]. If clumping occurs, the culture is termed [?] and cannot be serotyped.
iv. Add a small drop of [?] to the suspension. Usually approximately equal volumes of antiserum and growth suspension are mixed, but the volume of [?] may be as much as double the volume of the antiserum.
v. Mix the suspension and antiserum well and then [?] back and forth to observe for agglutination.
Serological Tests
- petri dish
- remove a portion of the growth
- autoagglutination
- “rough”
- antiserum
- suspension
- tilt slide
[?]
Result:
(+) reaction: Very strong clumping will appear within [?]
Serological Tests
- 30 seconds to 1 minute.
Tests for Determining Biotypes of V. cholerae O1
B-Hemolytic on sheep blood agar CAMP TEST Voges-Proskauer (VP) Test Chicken red blood cell agglutination Susceptibility to 50 U polymyxin B Phage IV susceptibility
[?]
- Fresh [?] red blood cells can be used for this assay.
- A [?] (vol/vol) suspension of washed (3 times) and packed (by [?]) cells is made in normal saline after the final wash.
- A large loopful of the [?] is placed on a glass slide.
- A small portion of the growth from a [?] is added to the red cells with a needle or loop, and is mixed well.
- In a positive test, agglutination of the red cells occurs within [?]
Chicken red blood cell agglutination
- chicken or sheep
- 2.5%
- centrifugation
- red cell suspension
- nonselective agar slant
- 30 to 60 seconds.
[?]
- A 50-unit polymyxin B disk is used for this test, and known strains of [?] are used as controls.
Susceptibility to 50 U polymyxin B
- classical and El Tor biotypes
[?]
- The isolate to be tested is grown overnight in pure culture on a [?].
- From the overnight growth, [?] (or Trypticase soy broth) is inoculated and incubated for [?] at 35˚ to 37˚C.
- A lawn of bacteria in [?] is then seeded onto the surface of a BHI agar plate by dipping a cotton swab into the 6-hour broth and lightly inoculating (swabbing) the entire surface of the plate.
- Positive and negative control strains should also be included.
- A drop of the [?] diluted to routine test dilution is applied to the bacterial lawn.
- The plate is incubated overnight and read the next day.
- If the bacteria are susceptible to the bacteriophage they will be [?], and there will be a [?] in the bacterial lawn.
Phage IV susceptibility
- noninhibitory medium
- brain heart infusion (BHI) broth
- 6 hours
- log-phase growth (OD=0.1)
- bacteriophage
- lysed
- zone of lysis
Campylobacter jejuni: specimen
- Feces
- Rectal swabs
Gram-negative, curved, S-shaped or gull-winged, or long spiral forms.
Campylobacter jejuni
Contain antibiotics that suppress the growth of normal fecal flora.
Selective media
Fluid thioglycollate
Agar (3%) Sheep blood (10%)
Butzler’s selective medium
Bacitracin Novobiocin Colistin Cephalothin Actidione
Butzler’s selective medium
Blood agar base
Lyzed horse blood (7%)
Skirrow’s blood agar
Vancomycin
Polymixin B
Trimethoprim
Skirrow’s blood agar
Brucella agar base
Sheep blood (10%)
Blaser’s medium (Campy-BAP)
Campy CVA Agar
Vancomycin Polymixin B Trimethoprim Cephalothin Amphotericin
Blaser’s medium (Campy-BAP)
Cefoperazone
Vancomycin
Amphotericin B
Campy CVA Agar
Columbia Agar Base
Activated charcoal
Hematin
Sodium pyruvate
Campy CSM Agar
Cefoperazone
Cycloheximide
Vancomycin
Campy CSM Agar
Nutrient broth No. 2
1.2% New Zealand agar
Bacteriologic charcoal Sodiumdeoxycholate Ferrous sulfate Sodiumpyruvate Casein hydrolysate
Preston Campylobacter bloodfree medium
Cefoperazone
Preston Campylobacter bloodfree medium
Stool or rectal swab specimen is directly inoculated onto the surface of one of the recommended
selective agar media
Formed stool specimens may also be processed by emulsifying a small portion (peanut sized) in [?] before inoculating one or two drops to the surface of the agar with a Pasteur pipette; similarly, one or two drops of liquid stool specimens can be
inoculated directly
phosphate-buffered saline or broth
At 42°C for [?] hours
24, 48, and 72
Microaerophilic atmosphere of 5% O2, 10% CO2, and 85% N2, which can be achieved by:
i. Disposable gas generators (e.g., BBL CampyPak Plus disposable gas generator envelope in GasPak jar)
ii. Evacuation-replacement procedures
The use of a [?] is not recommended for cultivating campylobacters because only strains that are very aerotolerant grow in the atmosphere provided.
CO2 incubator
A [?] is not recommended because the oxygen level (12% to 17%) is too high for optimal growth of campylobacters.
candle extinction jar
C. jejuni produces two types of colonies.
i. Small, raised, grayish-brown, smooth and glistening with an entire translucent edge.
ii. Flat, mucoid, translucent, grayish and has an irregular edge.
A small percentage of strains may appear tan or [?].
Colonies tend to spread, especially when initially isolated from [?] clinical specimens.
slightly pinkish
fresh
illustrating raised, gray-white, and somewhat mucoid
colonies
Campylobacter jejuni on blood agar
a zone of inhibition forms around the nalidixic acid (NA) disk , indicating that this species is susceptible to nalidixic acid. But, it is resistant to cephalothin (CF)
Brucella agar plate showing the growth of C. jejuni
around cephalothin and nalidixic acid disks
- Staining with Warthin-Starry
- Gram’s, Giemsa’s, or H&E.
Gastric/duodenal biopsy by endoscopy
- A mucosal biopsy sample is placed into urea medium, incubated at 37 oC for 2 hours; observe for a color change.
Biopsy Urease Test (BUT), CLO-Test (Campylobacter-like Organism)
tissue section of superficial gastric mucosa demonstrating clusters of blue-black staining bacilli along the epithelial lining, consistent with the bacillary forms of H. pylori. When observed in gram-stained preparation, the individual cells are long, thick, and curved.
Silver-stained (Warthin-Starry)
A positive urease test (pink to purple color) is
considered indicative of [?] presence.
H. pylori’s
Gastric/duodenal biopsy by endoscopy: media
Brucella agar with 5% sheep blood
Trypticase soy broth with 5% sheep blood
Gastric/duodenal biopsy by endoscopy: Incubation
35-37 oC Microaerophilic atmosphere (5% O2, 10% CO2, 85% N2) Prolonged incubation (3-5 days) required.
Gastric/duodenal biopsy by endoscopy: Colonies
Small (1-2 mm), circular, convex, translucent, gray.
[?] Principle:
Based on the urease activity of H. pylori to break down urea, a chemical made up of [?] and [?], into [?[ and [?] . [?] is absorbed from the stomach and eliminated in the breath.
Urea Breath Test (UBT)
- nitrogen and carbon
- carbon dioxide and ammonia
- Carbon dioxide
Procedure:
The patient ingests radioactive [?], followed by collection of breath samples that are analyzed for the presence of labeled [?] at 60 minutes by scintillation counter.
Urea Breath Test (UBT)
- 14-C labeled urea
- 14-CO2
Detects antibodies (IgG) to H. pylori in blood samples.
Antibody Detection
Detects H. pylori antigens in stool samples
Antigen Detection
Serodiagnosis
Antibody Detection
Antigen Detection
Helicobacter pylori
Growth at
25 oC
37 oC
42 oC
-
+
+ (some strains)
Helicobacter pylori
Susceptibility to Nalidixic acid (30 µg)
R
Helicobacter pylori
Susceptibility to Cephalothin (30 µg)
S
After 24-hour incubation at 35°C, aeromonads appear as round, raised, opaque lactose-fermenting colonies with an entire edge and a smooth, often mucoid,
surface.
Growth on McConkey Agar
