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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
Q

PAD stands for…

A

Phenylalanine deaminase

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26
Q

PAD test procedure

A

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

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27
Q

PAD test results interpretation

A

POSITIVE for pad = dark green color change

NEGATIVE = yellow or no color change

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28
Q

DNase test principle

A

If org has DNase it will break down DNA to nucleotides which creates an alkaline pH

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29
Q

DNase plate test procedure

A

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
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30
Q

DNase plate test results interpretation

A

POSITIVE for DNase activity = pink halo around colony

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31
Q

Motility test principle

A

Testing for presence of flagella

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32
Q

Motility tube test procedure

A

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
Q

Motility tube test results interpretation

A

POSITIVE for motility = media surrounding the inoculation line will be cloudy

NEGATIVE = very visible growth only along line of inoculation

34
Q

Ornithine decarboxylase test principle

A

If org has enzyme ornithine decarboxylase it will break down ornithine (amino acid) to produce putrescene which creates a basic pH

35
Q

Ingredients of MIO tube

A

Ornithine
Tryptophan
0.1% glucose
Bromcresol purple

36
Q

Amino acids that can be used for decarboxylase rxn

A

1) ornithine
2) lysine
3) arginine

37
Q

Ornithine decarboxylase MIO tube test procedure

A

Stab org into MIO tube media, add mineral oil to top, tube incubated overnight @ 37C

38
Q

Ornithine decarboxylase results interpretation

A

POSITIVE for ornithine decarboxylase = starts purple —> yellow —> back to purple
Negative = purple —> yellow and stays yellow

39
Q

Orgs POSITIVE for ONPG

A 
E. coli 
Enterobacter (aerogenes, cloacae)
Citrobacter (freundii, koseri (diversus), amalonaticus)
Klebsiella (pneumoniae, oxytoca)
Serratia (marcescens, liquefaciens)
40
Q

Orgs POSITIVE for indole

A

E. coli
C. koseri
K. oxytoca

indole EcCkKo

41
Q

Orgs POSITIVE for citrate

A

Enterobacter
Citrobacter
Klebsiella
Serratia

42
Q

Orgs POSITIVE for Urease

A

Proteus
Morganella
Yersinia

43
Q

Orgs POSITIVE for PAD

A

Providencia
Morganella
Proteus

44
Q

Orgs POSITIVE for MRVP

A

MR (+)
E. coli
? P. vulgaris

VP (+)
Enterobacter
Klebsiella
? Serratia marcescens

45
Q

Orgs POSITIVE for ornithine decarboxylase

A

Enterobacter
C. koseri
Serratia
E. coli

SEEC

46
Q

Orgs POSITIVE for motility

A

E. coli
Enterobacter
Proteus
Salmonella

47
Q

Orgs POSITIVE for DNase

A

Serratia

48
Q

Biochemical tests used for differentiation of Enterobacteriaceae (9)

A 
TSI 
ONPG
Indole
Decarboxylase (ornithine)
Citrate
Urease
PAD
Motility 
MR-VP
DNase

CUPOID MTM

49
Q

Orgs POSITIVE for H2S

A

Salmonella
Proteus
C. freundii

Come back to this

50
Q

function of beta galactosidase

A

breaks down lactose —> glucose and galactose

51
Q

function of beta galactoside permease

A

transports lactose into cell

52
Q

MIO stands for…

A

motility, indole, ornithine

53
Q

MR-VP test is used…

A

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
Q

Name this test

A

Positive DNase test (pink ring around colonies)

55
Q

MRVP principle

A

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
Q

MRVP procedure/principle continued

A

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
Q

IMViC stands for….

A

Set of tests used to ID enteric GNRs:

Indole
Methyl Red
Voges-Proskauer
Citrate

58
Q

Escherichia IMViC profile

A

+ + - - (opposite of Enterobacter and K. pneumo)

59
Q

Enterobacter and K. pneumo IMViC profile

A
        • (opposite of E. coli)
60
Q

Enterobacteriaceae common characteristics (4)

A
  1. Facultative anaerobes
  2. non-fastidious
  3. Most are normal human fecal flora
  4. Ubiquitous in nature (except for intestinal pathogens)
61
Q

Enterobacteriaceae lab characteristics (5)

A
  1. Reduce nitrate to nitrites
  2. Oxidase (-)
  3. Ferment glucose
  4. Gram neg pleomorphic rods
  5. Divided into lactose (+) and lactose (-)
62
Q

Enterobacteriaceae colony morphology

A

BAP: large grey mucoid colonies

CHOC: large grey mucoid

Generally larger than staphs/streps

Variable hemolysis

63
Q

TSI ingredients

A

0.1% glucose
1.0% lactose
1.0% sucrose
2.0% peptones
Phenol red
Sodium thiosulfate
Iron salts

64
Q

TSI procedure

A

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
Q

TSI is used to…

A

Differentiate GROUPS of common GNRs

66
Q

Groups differentiated by TSI (5)

A
  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
Q

TSI rxns/interpretation:

If an org can utilize GLUCOSE ONLY

A

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
Q

TSI rxns/interpretation:

If an org can utilize glucose, AND lactose or sucrose

A

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
Q

TSI rxns/interpretation:

If an org CANNOT utilize glucose, lactose, or sucrose

A

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
Q

TSI rxns/interpretation:

H2S production

A

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
Q

TSI rxns/interpretation:

Gas production

A

Bubbles and/or crack in butt

RECORD AS: A/AG or K/AG

*gas prod not used for ID

72
Q

Kliger Iron Agar (KIA) ingredients

A

Glucose + lactose (rest is same as TSI)

73
Q

Lysine Iron Agar (LIA) ingredients and fxn

A

Lysine + glucose

Fxn: deamination rxn and decarboxylation rxn

74
Q

Four major groups of GNR:

A
  1. Enteric GNR (70-80%)
  2. NFB (10-15%)
  3. Fastidious GNR (10-15%)
  4. Misc and infrequently encountered GNR (1%)
75
Q

Coliforms vs. enterics

A

Coliforms: usually a term used in environmental testing

Enterics: found in human enteric system (intestines, GI tract)

76
Q

3 pathogenic categories of enteric pathogens

A
  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
Q

Manual ID of orgs:

A

GS
selective/differential plating media
Biochemical tests

Usually a profile of 3-4 key tests that differentiate from other orgs

78
Q

Automated ID of orgs:

A

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
Q

Monosaccharide vs Disaccharide

A

Mono: glucose

Di: lactose + sucrose

Clin Micro 1 (3 decks)

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