Identification of Gram Negative Bacilli Flashcards Preview

Microbiology > Identification of Gram Negative Bacilli > Flashcards

Flashcards in Identification of Gram Negative Bacilli Deck (59):
1

- based on phenotypic characteristics

Biochemical testing

2

use of antibodies to detect expressed proteins on a bacterium

Serotyping

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Carbohydrate utilization

- lactose or glucose degradation
- traditional test for Gram-negative bacteria

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lactose degradation

- consists of glucose and galactose
- requires two enzymes: beta-galactoside permease and beta-galactosidase
- slow lactose fermenters lack permease

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Oxidation

utilize carbohydrates aerobically; glycolysis pathway (glucose to pyruvate to CO2)

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Fermentation

utilize carbohydrates anaerobically; glycolysis pathway (glucose to pyruvate to acid)

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asaccharolytic

do not utilize carbohydrates

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O/F Basal media

lower peptones; aerobic and anaerobic tubes

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O/F Media (positive for fermentation)

yellow throughout in both tubes due to acid production from fermentation under anaerobic conditions

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O/F Media (positive for oxidation)

aerobic tube (O2) is yellow at top indicating oxidative glucose utilization; anaerobic tube remains green

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O/F Media (negative for glucose utilization)

no color change in either tube

12

- contains glucose, lactose and sucrose
- much more lactose and sucrose than glucose (10:1)
- similar to KIA (no sucrose)
- ferrous sulfate and sodium thiosulfate (detects H2S)
- phenol red indicator
- slant is aerobic, butt is anaerobic

Triple Sugar Iron Agar (TSI)

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Interpreting TSI

read as slant/butt

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No fermentation (TSI)

K/K

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Glucose fermentation (TSI)

K/A

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Glucose then sucrose or lactose fermentation (TSI)

A/A

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Hydrogen sulfide (TSI)

black precipitate from acid and thiosulfate

18

Gas production (TSI)

causes bubbles or splitting in media

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Ortho-Nitrophenyl-beta-D-Galactopyranoside (ONPG) test

- tests for slow lactose fermenters
- ONPG is transported easily into bacterial cells
- hydrolyzes ONPG to galactose and O-nitrophenol (yellow)
- non lactose fermenters are clear

20

Methyl Red and Voges-Proskaur (MRVP) Test

tests glucose metabolic method

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Glucose metabolized by mixed acid pathway (MRVP)

methyl red positive

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Glucose metabolized by butylene glycol pathway (MRVP)

Voges-Proskaur positive; produces butylene glycol and acetoin

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Decarboxylase test

- tests for presence of enzymes that can remove COOH
- specific for lysine, ornithine and arginine

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Lysine decarboxylase

Lysine --> cadaverine + CO2

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Ornithine decarboxylase

Ornithine --> putrescine

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Dihydrolase test

degrades arginine to ornithine
arginine --> citrulline --> ornithine --> putrescine

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Lysine Iron Agar (LIA) Slant

contains lysine, glucose, ferric ammonium citrate, sodium thiosulfate
pH indicator is bromcresol purple
primary function of test is deamination or decarboxylation of lysine; can also detect H2S

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Deamination of lysine (LIA)

reddish color

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Decarboxylation of lysine (LIA)

remains purple

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amino acids are metabolized by deaminases that remove amine groups
includes PAD test
for differentiation of Proteus, Morganella, and Providencia

Deaminase test

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deamination produces phenylpyruvic acid
add 10% ferric chloride (green is positive)

Phenylalanine deaminase (PAD) test

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Citrate utilization

tests for organisms that use citrate as a sole carbon source
results in alkaline pH (color changes to blue +)
inoculum must be light

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DNase

endonucleases that can break down phosphodiester bonds
streak to plate and add 1N HCl; clear halo is positive

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Gelatin liquefication

Gelatinase production causes a loss of gelling (media is no longer solid in the tube)

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Indole test

Tryptophanase produced by organisms breaks down tryptophan to indole
includes Ehrlich's indole test and Kovac's test

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Ehrlich's indole test

add Ehrlich's reagent (PDAB) - positive is red color
indole is extracted by xylene

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Kovac's test

add Kovac's reagent; red color is positive

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Malonate utilization test

tests whether malonate is the sole carbon source for an organism
turns from green to blue when positive (bromothymol blud)

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Motility test

observes growth in semisolid media
stab a single line and observe growth away from line
Kleb, Yersinia and Shigella are non-motile at 37C

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Reduction of Nitrate (NO3) to Nitrite (NO2)

add NNDN or N,N-dimethyl-1 napthylamine, look for red or gas production
nitrate --> nitrite + sulfanilic acid + NNDN --> diazo red dye
if red, add Zinc, if red color, nitrate was not reduced (test verification)

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Oxidase test

determines presence of cytochrome oxidase (dimethyl-paraphenyl-dihydrochloride)
positive if purple

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Urease test

determines organisms ability to hydrolyze urea --> ammonia, water and CO2
bright pink is positive

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Combo tests

LIA (lysine differentiates Shigella and Salmonella)
MIO agar (Motility-indole-ornithine agar - differentiates Serratia and Enterobacter)
SIM media (Sulfide-indole-motility medium)

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Manual Multitest Systems (all tests based on these principles)

pH-based reactions
enzyme-based reactions
utilization of carbon sources
detection of bacterial growth
detection of volatile fatty acids

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Rapid Identification Systems

faster than conventional method
based on carbohydrate utilization or chromogenic substrates
change color quickly due to amplification of tests

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TSI Biochemical Reaction for:
Escherichia

A/A
(indole +)

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TSI Biochemical Reaction for: Proteus

A/A H2S+
(urea +, deam +)

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TSI Biochemical Reaction for:
Salmonella

K/A H2S+
(mal-, ONPG-)

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TSI Biochemical Reaction for:
Shigella

K/A
(citrate -, non-motile)

50

TSI Biochemical Reaction for:
Pseudomonas

K/K
(ox +, blue-green pigment, growth at 42 degrees C, growth in cetrimide)

51

TSI Biochemical Reaction for:
Enterobacter

A/A
(ODC +, sugars)

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TSI Biochemical Reaction for:
Arizona

A/A, H2S+
(LDC +)

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TSI Biochemical Reaction for:
Citrobacter

K/A or A/A, H2S+
(LDC-)

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TSI Biochemical Reaction for:
Providencia

K/A
(deam +)

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TSI Biochemical Reaction for:
Klebsiella

A/A
(ODC-)

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TSI Biochemical Reaction for:
Edwardsiella

K/A, H2S+
(indole +, LDC +)

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TSI Biochemical Reaction for:
Serratia

A/A
(sugars)

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TSI Biochemical Reaction for:
Proteus

K/A
(urea +, deam +)

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TSI Biochemical Reaction for:
Yersinia

K/A
(small colonies, urea +, deam -)