MOD8 Enterobacterales Lab Procedures

when might biochemical tests be used for IDing Enterbacterales

• low test volumes, facility doesnt have access

• supplemental tests to support ID from MALDI

• presumptive ID for some orgs

• commercial systems fail to ID isolate

carbohydrate fermentation

sugar most commonly used, can also be sugar alcohol (mannitol, sorbitol, etc)

bacterial fermentation of carbs → production of acid

acid + indicator → colour change of medium

can also produce gas → aerogenic fermentation

no gas → anaerogenic fermentation

aerogenic/anaerogenic fermentation

fermentation resulting in gas production (or lack of)

phenol red

acid: yellow

alkaline: red

→ opposite of other “x red”

bromocresol purple

acid: yellow

alkaline: purple

bromothymol blue

acid: yellow

alkaline: blue

methyl red

acid: red

alkaline: yellow

neutral red

acid: red

alkaline: yellow

peptone

source of protein

requires O2 for metabolism

results in alkaline byproduct → darkening of medium

colour depends on indicator

glucose + reversion

all Enterobacterales ferment glucose

results in weak acid → medium quickly reverts back to alkaline when incubation is extended

ingredients in triple sugar iron TSI

sugars: 1% lactose, 1% sucrose, 0.1% glucose

indicator: phenol red

ferrous sulfate/ferrous ammonium sulfate (source of ferric ions) → H2S

beef extract, yeast extract, peptones → source of nitrogen, vitamins, minerals

parts of TSI

slant → aerobic rxns

butt → anaerobic rxns; deep butt of ≥ 5cm needed to inhibit O2 exposure

cap must be loosened for incubation to allow for aerobic rxns

TSI results

slant + butt → acid (A) vs alkaline (K) vs no change (NC)

gas production → bubbles, cracks in agar, lifting of medium from tube

H2S production → blackening of media, only occurs in acidic conditions = yellow

TSI interpretation of K/A

F: glucose → monosaccharide = weak acid, only 0.1%

not enough acid produced to keep slant yellow → undergoes reversion to K due to O2 exposure

butt remains A bcs anaerobic

NF: lactose, sucrose

TSI interpretation of A/A

if isolate is Enterobacterales, know that glucose fermented

in addition, org can also ferment either lactose or sucrose

2/3 sugars are fermented → large amount of acid produced → acid in slant and butt

TSI interpretation of K/K, K/NC, NC/NC

organism doesnt ferment any carbs

darkening of medium due to peptone metabolism

organism is not an Enterobacterales

sources of error on TSI

shallow butt <5cm deep → loss of acid in butt due to O2 diffusion + peptone metabolism // false alkaline butt result

TSI capped too tightly → lack of O2 exposure, peptone not metabolized, slant not exposed to O2 // false acid result

extended incubation beyond 24h → falsely alkaline butt/slant due to loss of acid

ortho-nitrophenyl-β-galactoside ONPG

determines presence of galactosidase enzyme and ability of organism to ferment lactose (LF, LLF, NLF)

substrate ONPG-P (colourless) enters bacterial cell without need for permease

substrate is hydrolyzed by galactosidase into galactose + orthonitrophenol (yellow)

ONPG positive → able to ferment lactose

fermenting lactose

permease enzyme → brings lactose into bacterial cell from media its growing on

galactosidase enzyme → breaks lactose down in cell to glucose + galactose sugars, which get fermented

lactose fermenters LF

intrinsically have permease and galactosidase enzymes

enzymes dont need to be induced

pink on MAC @ 24h

ONPG positive

non lactose fermenters NLF

do not have either permease or galactosidase

enzymes cannot be induced

colourless on MAC

ONPG negative

late lactose fermenters LLF

NLF at 24h, LF at 48h

intrinsically have galactosidase; no permease

has DNA to produce permease but must be induced by growing isolate on medium containing lactose

takes 24h for permease enzyme to be induced + for lactose to be brought into cell → delayed fermentation

ONPG positive → test only detects galactosidase, not permease

indole

detects presence of tryptophanase → breaks down tryptophan into indole, pyruvate, ammonia

indole tube method

org + broth w tryptophan

incubate in O2 at 35deg

kovac’s reagent → paradimethylaminobenzaldehyde

positive: red

spot indole

reagent: p-Dimethylaminocinnamaldehyde

colonies must be from non-inhibitory/non-selective agar (ex BAP)

positive: robins egg blue

voges proskauer VP

detects acetoin → intermediate product in formation of butylene glycol

some Enterobacterales produce butylene glycol as result of carb fermentation

rapid VP test

growth taken from medium where fermentation occurred and added to creatine broth

add reagents → VP1: alpha naphthol VP2: 40% KOH

development of red colour w/in 15min = positive

incubated VP test

more accurate than rapid VP

doesnt require isolate to have fermented carbs

isolated added to VP broth + incubated

creatine, VP1 and VP2 added to detect acetoin

production of red colour = positive

VP1 reagent

alpha naphthol

VP2 reagent

40% KOH

citrate test

determines if organism can use citrate in test medium as sole source of carbon

metabolism of citrate requires O2 → alkaline end product

cap needs to be loose prior to incubation

indicator: bromothymol blue

positive: deep intense blue

urease

tests organisms ability to break down urea into CO2 and ammonia

ammonia alkaline + phenol red = pink, +

what form of urease is most sensitive

christensen’s urea slant

can detect small amounts of ammonia

phenylalanine deaminase test PDA

tests organisms ability to remove an amine group NH3 from phenylalanine with a deaminase enzyme

produces phenylpyruvic acid

streak slant, incubate overnight in O2, add reagent ferric chloride

positive: dark green

negative: yellow/colour of reagent

tryptophan deaminase TDA

API uses this

positive: dark brown

negative: yellow

PDA and TDA correlation

TDA results correlate w PDA results

if TDA positive, PDA is positive

if TDA negative, PDA is negative

decarboxylase tests

tests ability of orgs to remove carboxyl group from amino acid

decarboxylation

generates alkaline end product

what medium is used in decarboxylase tests

moeller’s decarboxylation medium

slight purple colour, pH 6.0

contains glucose, beef extract, peptones → nutrients

inoculated + oil overlay → anaerobic conditions

indicators: bromocresol purple and cresol red

acid: yellow // alkaline: purple

common decarboxylase tests

lysine decarboxylase LDC

ornithine decarboxylase ODC

decarboxylase control tube

contains glucose but no source of amino acid

fermentation of glucose results in yellow colour

must be positive for test to be valid

decarboxylase positive result

org ferments glucose first producing acid

acid facilitates decarboxylation if org possesses decarboxylase

results in alkaline end product

medium turns purple

decarboxylase negative result

acid is produced from glucose fermentation

no further reaction bcs org lacks decarboxylase

medium turns and remains yellow

decarboxylase sources of false positives

exposure to O2 → reversion

not adding oil overlay

over incubation

what to do if API doesnt have anything around it

fill tube only with 0.5 mcfarland soln

what to do if API is underlined

fill tube with 0.5 mcfarland soln, add oil to cupule

what to do if API is boxed

fill tube and cupule with 0.5 mcfarland soln