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Clin Micro 1 > Enterobacteriaceae ID > Flashcards

Enterobacteriaceae ID Flashcards

1
Q

ONPG test is used…

A

To ID late lactose fermenters

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2
Q

Principles of ONPG test

A

1) transport of lactose molecule across cell membrane active transport system
- porin proteins
- beta-galactoside permease
2) needs enzyme to break apart lactose
- beta-galactosidase

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3
Q

ONPG principle: delayed lactose orgs require:

A

1) time to produce permease

2) exposure to lactose to synthesize permease

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4
Q

ONPG requires ____ but DOES NOT require ____

A

REQUIRES: beta-galactosidase

DOES NOT REQUIRE: beta-galactoside permease

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5
Q

ONPG stands for…

A

Orthonitrophenyl galactopyranoside

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6
Q

ONPG procedure

A

ONPG disk placed in 0.5 mL saline and inoculated w/ 3-4 isolated colonies

Incubated for 1-24 hrs in 37C heat block

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7
Q

ONPG result interpretation

A

POSITIVE = disk and/or saline turns YELLOW color

NEGATIVE = colorless = lactose neg

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8
Q

Delayed lactose (+) GNRs:

A

Serratia (95%)

Citrobacter (50-60%)

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9
Q

GNRs that are LACTOSE (+) and ONPG (+)

A 
E. coli 
Enterobacter
Citrobacter
Serratia 
Klebsiella

SEECK

also: pink ChEEKS

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10
Q

GNRs that are LACTOSE (-) and ONPG (-)

A

Proteus
Providencia
Morganella
Edwardsiella

MEPP

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11
Q

Indole test principle

A

If org has tryptophan deaminase, it will break down tryptophan (amino acid) to indole

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12
Q

Indole MIO tube result interpretation

A

POSITIVE for indole = any evidence of red ring at top of tube

NEGATIVE = yellow

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13
Q

Two procedures for indole test

A

1) MIO tube

2) spot indole test

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14
Q

Indole MIO tube test procedure

A

stab MIO tube and incubate overnight at 37C in air incubator; oil added to top of tube before incubation (facilitates anaerobic rxn)

Next day, add few drops of kovac’s reagent and mix gently

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15
Q

Indole spot test procedure

A

Place 2-3 drops of indole reagent (para-dimethylaminobenzaldehyde) on a piece of filter paper
Rub a colony on filter paper/reagent spot

*DO NOT use colonies from a MAC plate for spot test; can give false negative rxn

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16
Q

Indole spot test results interpretation

A

POSITIVE for indole = color change to turquoise

NEGATIVE = salmon/pink or no color change

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17
Q

Citrate utilization principle

A

To ID orgs that use citrate as SOLE carb/energy source (media contains only citrate)

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18
Q

Citrate utilization tube test procedure

A

Org is stabbed into slant and streaked up slant surface and incubated overnight @ 37C

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19
Q

Citrate utilization results interpretation

A

POSITIVE = color change from GREEN to BLUE

**ANY VISIBLE GROWTH ALONG SLANT INDICATES A POSITIVE RXN (incubate for an additional 24 hrs)

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20
Q

Citrate utilization test precautions

A

inoculate lightly

Make sure cap is loose

If neg, reincubate for total of 48 hrs

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21
Q

Urease test principle

A

If org has urease, urease will break down urea and produce CO2 + H2O + NH3

NH3 causes basic pH shift

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22
Q

Urease tube test procedure

A

inoculate a tube containing ~1mL of urea broth (contains pH indicator phenol red) and incubate overnight @ 37C in air incubator

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23
Q

Urease tube test results interpretation

A

POSITIVE for urease = hot pink color change

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24
Q

PAD test principle

A

If org has PAD it will convert phenylalanine (amino acid) to phenylpyruvic acid which will react with 10% ferric chloride

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25
PAD stands for…
Phenylalanine deaminase
26
PAD test procedure
Org streaked onto slant (containing 1% phenylalanine) surface and incubated overnight @ 37C; next day 4-5 drops of 10% ferric chloride added; invert several times to mix
27
PAD test results interpretation
POSITIVE for pad = dark green color change NEGATIVE = yellow or no color change
28
DNase test principle
If org has DNase it will break down DNA to nucleotides which creates an alkaline pH
29
DNase plate test procedure
Org streaked onto DNase plate containing toluidine blue dye (pH indicator) and incubated overnight to 48 hrs @ 37C in air incubator - don’t need to streak for isolation - use positive control org as well as test org
30
DNase plate test results interpretation
POSITIVE for DNase activity = pink halo around colony
31
Motility test principle
Testing for presence of flagella
32
Motility tube test procedure
Stab org into media and incubate overnight @ 37C * stab straight in come straight out * TTC reagent can be added to add red color to motility rxn
33
Motility tube test results interpretation
POSITIVE for motility = media surrounding the inoculation line will be cloudy NEGATIVE = very visible growth only along line of inoculation
34
Ornithine decarboxylase test principle
If org has enzyme ornithine decarboxylase it will break down ornithine (amino acid) to produce putrescene which creates a basic pH
35
Ingredients of MIO tube
Ornithine Tryptophan 0.1% glucose Bromcresol purple
36
Amino acids that can be used for decarboxylase rxn
1) ornithine 2) lysine 3) arginine
37
Ornithine decarboxylase MIO tube test procedure
Stab org into MIO tube media, add mineral oil to top, tube incubated overnight @ 37C
38
Ornithine decarboxylase results interpretation
POSITIVE for ornithine decarboxylase = starts purple —> yellow —> back to purple Negative = purple —> yellow and stays yellow
39
Orgs POSITIVE for ONPG
``` E. coli Enterobacter (aerogenes, cloacae) Citrobacter (freundii, koseri (diversus), amalonaticus) Klebsiella (pneumoniae, oxytoca) Serratia (marcescens, liquefaciens) ```
40
Orgs POSITIVE for indole
E. coli C. koseri K. oxytoca indole EcCkKo
41
Orgs POSITIVE for citrate
Enterobacter Citrobacter Klebsiella Serratia
42
Orgs POSITIVE for Urease
Proteus Morganella Yersinia
43
Orgs POSITIVE for PAD
Providencia Morganella Proteus
44
Orgs POSITIVE for MRVP
MR (+) E. coli ? P. vulgaris VP (+) Enterobacter Klebsiella ? Serratia marcescens
45
Orgs POSITIVE for ornithine decarboxylase
Enterobacter C. koseri Serratia E. coli SEEC
46
Orgs POSITIVE for motility
E. coli Enterobacter Proteus Salmonella
47
Orgs POSITIVE for DNase
Serratia
48
Biochemical tests used for differentiation of Enterobacteriaceae (9)
``` TSI ONPG Indole Decarboxylase (ornithine) Citrate Urease PAD Motility MR-VP DNase ``` CUPOID MTM
49
Orgs POSITIVE for H2S
Salmonella Proteus C. freundii Come back to this
50
function of beta galactosidase
breaks down lactose ---> glucose and galactose
51
function of beta galactoside permease
transports lactose into cell
52
MIO stands for...
motility, indole, ornithine
53
MR-VP test is used...
to determine if an unidentified GNR utilizes the mixed acid fermentation (MAF) pathway or the butanediol/butylene glycol pathway (BGP) to break down sugars
54
Name this test
Positive DNase test (pink ring around colonies)
55
MRVP principle
MR: Escherichia produces large amts of mixed acids when it ferments sugars via MAFP —> results in low pH VP: bacteria such as Enterobacter and Klebsiella break down sugars via butanediol pathway (BGP) —-> results in neutral pH —-> acetoin product
56
MRVP procedure/principle continued
Inoculate ~1 mL of glucose broth and incubate overnight @ 35C Split broth equally between two tubes Tube 1: Methyl red (pH indicator) added —-> if org uses MAFP pH of broth will go acidic and pH indicator will turn RED (yellow if neutral) Tube 2: alpha-naphthol and KOH are added —-> if org uses BGP acetoin will be produced —-> KOH breaks down the acetoin into diacetyl —-> alpha-naphthol catalyzes rxn btwn diacetyl and peptones in the media —-> results in RED color change (yellow if negative)
57
IMViC stands for….
Set of tests used to ID enteric GNRs: Indole Methyl Red Voges-Proskauer Citrate
58
Escherichia IMViC profile
+ + - - (opposite of Enterobacter and K. pneumo)
59
Enterobacter and K. pneumo IMViC profile
- - + + (opposite of E. coli)
60
Enterobacteriaceae common characteristics (4)
1. Facultative anaerobes 2. non-fastidious 3. Most are normal human fecal flora 4. Ubiquitous in nature (except for intestinal pathogens)
61
Enterobacteriaceae lab characteristics (5)
1. Reduce nitrate to nitrites 2. Oxidase (-) 3. Ferment glucose 4. Gram neg pleomorphic rods 5. Divided into lactose (+) and lactose (-)
62
Enterobacteriaceae colony morphology
BAP: large grey mucoid colonies CHOC: large grey mucoid Generally larger than staphs/streps Variable hemolysis
63
TSI ingredients
0.1% glucose 1.0% lactose 1.0% sucrose 2.0% peptones Phenol red Sodium thiosulfate Iron salts
64
TSI procedure
Inoculate by stabbing butt straight down slant, squiggle up slant as you pull needle out Incubate in air inc with loose cap Read at 18-24 hrs
65
TSI is used to…
Differentiate GROUPS of common GNRs
66
Groups differentiated by TSI (5)
1. Orgs that CAN FERMENT GLUCOSE 2. Orgs that CAN ferment GLUCOSE and LACTOSE OR SUCROSE 3. Orgs that CANNOT FERMENT GLUCOSE, LACTOSE, or SUCROSE 4. H2S production 5. SUCROSE NEG/LACTOSE NEG
67
TSI rxns/interpretation: If an org can utilize GLUCOSE ONLY
Slant: rapidly oxidize (aerobic rxn) glucose on slant —> acids —> after 8-12 hrs slant turns yellow (all enterics) —> are glucose is used up org will revert to using peptones as energy source —> peptones broken down —> ammonia produced —> pH raised (alkaline) —> slant turns RED Butt: ferment (ana rxn) glucose in butt —> acids (fermentation slower than oxidation so may take 24-48 hrs to use up all glucose) —> YELLOW RECORDED AS: K/A (alk/acid)
68
TSI rxns/interpretation: If an org can utilize glucose, AND lactose or sucrose
Abundant carbs in slant and butt, org will pump out acids for several days RECORD AS: A/A *if an org is lactose (-) on MAC AND sucrose (-) —> RECORD AS K/A
69
TSI rxns/interpretation: If an org CANNOT utilize glucose, lactose, or sucrose
Suggestive of NFB - not enterics NFBs will utilize the peptones in slant and break down the peptones in the butt (but sometimes rxn in butt is neg) RECORD AS: K/K or K/N
70
TSI rxns/interpretation: H2S production
Two step process: 1. Org will utilize the sodium thiosulfate in media —> prod H2S gas 2. Gas will react with the ferric ions in media and prod ferrus sulfide —> black ppt RECORD AS: K/H2S or A/H2S **on black butt rxn, assume that there is also an acid rxn but is covered up due to black color
71
TSI rxns/interpretation: Gas production
Bubbles and/or crack in butt RECORD AS: A/AG or K/AG *gas prod not used for ID
72
Kliger Iron Agar (KIA) ingredients
Glucose + lactose (rest is same as TSI)
73
Lysine Iron Agar (LIA) ingredients and fxn
Lysine + glucose Fxn: deamination rxn and decarboxylation rxn
74
Four major groups of GNR:
1. Enteric GNR (70-80%) 2. NFB (10-15%) 3. Fastidious GNR (10-15%) 4. Misc and infrequently encountered GNR (1%)
75
Coliforms vs. enterics
Coliforms: usually a term used in environmental testing Enterics: found in human enteric system (intestines, GI tract)
76
3 pathogenic categories of enteric pathogens
1. Opportunistic pathogens - UTIs, wounds, septicemia, LRTI, meningitis 2. Intestinal pathogens - Salmonella, Shigella, E. coli (certain strains), Yersinia enterocolitica **NOT NF** 3. Non-intestinal pathogens - Yersinia pestis (bubonic plague)
77
Manual ID of orgs:
GS selective/differential plating media Biochemical tests Usually a profile of 3-4 key tests that differentiate from other orgs
78
Automated ID of orgs:
20-30 biochemical tests and large data bases that orgs gets run through to find a statistical match based on rxns Instruments: microscan, vitek
79
Monosaccharide vs Disaccharide
Mono: glucose Di: lactose + sucrose

Clin Micro 1 (3 decks)

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