- colour from oxidation of colourless TMPD (interacts with ETC) at the level of cytochrome c = dark purple indophenol - pos depends on the presence of cytochrome c (bacteria whose ETC has cytochrome c oxidase)

- certain organisms can use citrate as their sole source of carbon - pos = growth -> blue due to alkaline end products produced (bromothymol blue) - useful for differentiation of lactose fermenters - Kleb and Enterobacter utilize citrate but E. coli does not

- bacteria capable of hydrolyzing tryptophan (in peptone water) to form indole - detected by colour rxn with dimethylamino benzaldehyde (Kovac's) - tube test could check for motility before adding Kovac's by performing a wet mount - differentiation of lactose fermenters: E. coli pos and Enterobacter + Klebsiella neg

- spot test = more sensitive than tube - can't check for motility first - useful for organisms that won't grow in peptone water (anaerobes) - colony transferred ti filter paper; saturated ith spot indole reagent (p-dimethylaminocinnanaldehyde); blue/green is pos

- directly test by microscopic examination of "hanging drop" or wet prep of organisms (peptone water before Kovac's) - indirectly by observing growth in a semi-solid motility medium - motile organisms (facultative anaerobes) spread from line of inoculum = diffuse pattern - non-motile organism growth is confined to inoculum line - Tetrazolium salts = aid in reading; changes to red (red on stab line only if non-motile)

LAB - Enterobacteriaceae Flashcards by Richen Basig

Common genera in family of Enterobacteriaceae that are associated with human pathogenicity

Klebsiella
Shigella
Salmonella
Escherichia
Proteus
Citrobacter
Serratia
Hafnia
Yersinia
Enterobacter
Providencia
Morganella

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Outside their normal habitat, opportunistic pathogens can cause some serious ____________ infections

extraintestinal

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Primary intestinal pathogens

Salmonella, Shigella, Yersinia enterocolitica, E. coli O157:H7 = true pathogens
NOT commensal flora of GI

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All Enterobacteriaceae species: (4)

ferment glucose (OF=F)
reduce nitrate to nitrites
oxidase neg
nonsporulating

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Intestinal specimens (stool) for colifs

only potential pathogens are fully identified and reported

= Salmonella, Sigella, Yersinia, O157:H7)

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Extraintestinal specimens (urine, wound swabs, etc.)

any Entero- may be clinically significant

isolated on MAC
lactose or non-lactose fermenter?
further biochemical tests

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cytochromes

iron-containing hemeproteins = components f ETC; involved in aerobic respiration

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Oxidase test

colour from oxidation of colourless TMPD (interacts with ETC) at the level of cytochrome c = dark purple indophenol
pos depends on the presence of cytochrome c (bacteria whose ETC has cytochrome c oxidase)

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This may inhibit oxidase activity and thus should not be used when doing an oxidase test

MAC (selective media/carb fermentation)

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Filter paper method (Oxidase)

colony transferred onto a small piece of filter paper saturated with TMPD reagent
dark purple colour = positive (rapid)

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A differential medium, used as a screen test for enteric pathogens on the basis of carbohydrate fermentation, HS production, and gas production

Triple Sugar Iron (TSI)

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Citrate utilization

certain organisms can use citrate as their sole source of carbon
pos = growth -> blue due to alkaline end products produced (bromothymol blue)
useful for differentiation of lactose fermenters
Kleb and Enterobacter utilize citrate but E. coli does not

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Indole production

bacteria capable of hydrolyzing tryptophan (in peptone water) to form indole
detected by colour rxn with dimethylamino benzaldehyde (Kovac’s)
tube test could check for motility before adding Kovac’s by performing a wet mount
differentiation of lactose fermenters: E. coli pos and Enterobacter + Klebsiella neg

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Spot indole

spot test = more sensitive than tube
can’t check for motility first
useful for organisms that won’t grow in peptone water (anaerobes)
colony transferred ti filter paper; saturated ith spot indole reagent (p-dimethylaminocinnanaldehyde); blue/green is pos

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Motility test

directly test by microscopic examination of “hanging drop” or wet prep of organisms (peptone water before Kovac’s)
indirectly by observing growth in a semi-solid motility medium
motile organisms (facultative anaerobes) spread from line of inoculum = diffuse pattern
non-motile organism growth is confined to inoculum line
Tetrazolium salts = aid in reading; changes to red (red on stab line only if non-motile)

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Medium method for motility is not useful for these organisms

strict aerobes like Pseudomonas bc will not grow in the depth of medium (use microscopic examination)

Yersinia enterocolitica motility

motile at 22C butt not at 37C

Malonate

utilize malonate as a source of carbon?
end products = acetate and CO2
alkaline pH so bromthymol blue changes from green to blue (alkaline) in a positive test

Phenylalanine deaminase test (PAD)

test for ability to deaminate phenylalanune to phenyl pyruvic acid by adding ferric chloride as an indicator
Proteus-Providence (+); rest of colifs (-)

Urease activity

splits urea into CO2 and ammonia
phenol red turns deep cerise due to alkalinity
conventional = Christensen’s urea agar (sterile buffered nutrient agar base containing phenol red indicator)
Stuart urea broth is more buffered

Methyl Red-Voges Proskauer (MR-VP)

two tests in one
both detect acid production from fermentation of glucose
positive MR = more complete catabolism of glucose to highly acidic end products (lactic, acetic, succinic, formic acids) than VP pos (2,3-butanediol and ethanol following mixed-acid fermentation)

MR pos organisms

turning red

E. coli, Edwarsiella, Shigella, Salmonella, Citrobacter, Proteus, Klebsiella, Yersinia

MR neg organisms

turning yellow

Enterobacter, Hafnia, Serratia

VP pos organisms

pink-red

Enterobacter, Klebsiella pneumoniae, Seratia

VP neg organisms

no colour change | Scherichia, Edwarsiella, Citrobacter, Salmonella, Shigella, Yersinia, Klebsiella

MR-VP procedure

- inoculate broth and incubate - remove 1 mL of broth and place in sterile tube; 3-4 drops of methyl red; pos is distinct red at top, neg is yellow layer - add VP reagent (Barritts alpha-naphthol + 40% KOH) to OG broth; pos will be pink to red

ONPG

- orthonitrophenyl-beta-galactopyranoside - detects beta-galactosidase (cleaves lactose into glucose & galactose) - also depends on PERMEASE ( transports lactose into cells)

ONPG Positive

- lactose fermenter = E. coli, Kleb spp., Enterobacter spp. = produce B-galactosidase and permease - late lactose fermenter = Citrobacter spp, Arizona spp = only B-galactosidase so slow ** yellow in few minutes vs, 24 hours**

ONPG Negative

non-lactose fermenter = Salmonella spp, Shigella spp, Proteus spp, Providencia, Morganella = NO beta galactosidase

ONPG procedure

- broth = organism taken from medium with high lactose conctn; inoculated in ONPG broth; split beta-galactoside bond, releasing o-nitrophenol = yellow-coloured compound - disk-method = medium with high lactose -> dense suspension -> ONPG disk added = yellow if pos

Sulphide-Indole Motility (SIM)

- example of a multi-test medium - tests more than one aspect of their metabolism at a time - H2S production? indole? motility?

SIM procedure

- stab SIM medium (2/3), then incubate) - H2S if black - indole if red ring on surface of deep after adding Kovac's - motility if fan away from streak

Most prompt lactose fermenters

Escherichia, Klebsiella, and Enterobacter

Analytical Profile Index (API) strips

- ID limited number of gram neg colifs - test strips have wells containing dehydrated substrates to detect enzymatic activity - bacterial suspension used to rehydrate wells; strips incubated - colour change (may have to add reagent, etc.) - negs and pos = profile number and compare to online codebook

Serological methods for ID of Shigella, Salmonella, and E. coli O157:H7

- O (somatic antigen) - heat-stabile antigen located in cell wall, resistant to alcohol - H (flagellar) - heat-labile; resists formalin - K (capsular) - heat-labile polysaccharide; K1 of E. coli, Vi of S. Typhi; may mask O antigen

Kauffmann-White scheme

serotyping scheme for Salmonella sp. - developed upon examining rxns of different suspensions of this organism with crude antisera raised against the other antigenic group strains

Serologic grouping of Shigella is based on this antigen

O - is agglutination fails with O antisera, the suspension is heated to remove capsular antigen and is retested with O antisera - may belong to serogroups A, B, C, or D which may have multiple serotypes

Shigella serogroups

A - S. dysenteriae B - S. flexneri C - S. boydii D - S. sonnei ** A-C physiologically similar; A,B,D = major bacterial cause of GI disease

delayed D-sorbitol fermentation

E. coli O157:H7 > 24 hrs SMAC agar can be used for detection