Week 3: Enterobacterales Methods

bromothymol blue colors

1. acid = yellow

2. base = blue

phenol red colors

1. acid = yellow

2. base = red

neutral red colors

1. acid = red

2. base = yellow

methyl red colors

1. acid = red 

2. base = yellow

what does MacConkey agar contain?

1. bile salts 1.5 g/L

2. lactose

3. peptones

4. crystal violet

5. neutral red

what is MacConkey agar selective for?

gram negative

how does is MacConkey agar differentiate?

1. lactose fermenters = pink

2. non lactose fermenters = pale

what is in hektoen enteric HE agar?

1. bile salts 9 g/L

2. lactose, sucrose, salicin

3. bromothymol blue and acid fuchsin

4. sodium thiosulfate and ferric ammonium citrate 

how is hektoen enteric HE agar moderately selective?

high concentration of bile salts favor classic pathogens, and selective against normal flora

how is hektoen enteric HE agar differential?

1. lactose, sucrose, or salicin fermentation = orange

2. non fermenters = blue 

3. H2S production = black precipitate 

how does Salmonella look on HE agar?

blue with black precipitate

what is in eosin methylene blue EMB agar?

1. lactose and sucrose

2. eosin Y

3. methylene blue 

what does eosin methylene blue EMB agar differentiate?

1. great lactose fermenters = metallic

2. lactose fermenters = purple 

3. non lactose fermenters = colorless 

what is in xylose lysine deoxycholate XLD agar?

1. sodium deoxycholate (bile salt)

2. phenol red 

3. xylose, lactose, sucrose 

4. lysine

5. sodium thiosulfate and ferric ammonium citrate 

how is xylose lysine deoxycholate XLD agar selective?

inhibits gram positive with sodium deoxycholate (bile salts)

how is xylose lysine deoxycholate XLD differential?

1. xylose, lactose, or sucrose fermenters = yellow 

2. non fermenters = pink 

3. lysine decarboxylation = pink 

4. H2S production = black precipitate 

what is in cefsulodin-irgasan-novobiocin CIN agar?

1. mannitol

2. neutral red 

what species has a bulls-eye appearance in CIN agar?

Yersinia

how is CIN agar selective?

its combination of antibiotics only allows Yersinia to grow

what is in triple sugar iron TSI?

1. peptones

2. lactose, sucrose, glucose 

3. phenol red 

4. sodium thiosulfate and ferrous sulfate 

what concentrations of sugars are in triple sugar iron?

1. lactose = 1%

2. sucrose = 1%

3. glucose = 0.1%

TSI colors and meaning

1. acid/fermentation = yellow

2. base/catalyzed = pink

3. H2S produced/acid = black

A/A G TSI test interpretation 

1. glucose and lactose and/or sucrose fermentation 

2. gas produced 

K/A TSI result interpretation

1. only glucose fermentation 

2. peptone catabolized 

K/K TSI result interpretation

1. no fermentation

2. peptone catalyzed 

A/A H2S TSI result interpretation

1. glucose and lactose and/or sucrose fermentation

2. H2S produced 

K/A G H2S TSI result interpretation

1. only glucose fermentation 

2. gas produced 

3. H2S produced 

how is kligler iron agar KIA different than TSI?

KIA does not contain sucrose

what does the oxidase test contain as a reagent?

TMPD

what does the oxidase test detect?

cytochrome C oxidase activity

what does a positive oxidase test look like?

purple

what does simmons citrate agar contain?

1. citrate as sole carbon source 

2. ammonium salts 

3. bromothymol blue 

what does simmons citrate agar differentiate?

1. uses citrate as sole carbon source/basic= blue

2. does not/acidic = yellow 

why is a positive citrate test basic?

organisms that metabolize citrate use the ammonium salts to release ammonia, which increases pH

what type of medium does a motility test require?

semisolid

positive and negative motility test results

1. positive = growth away from stab

2. negative = growth only in stab

what does the methyl red media contain?

1. glucose 

2. peptones 

3. methyl red 

what does the methyl red test differentiate?

1. mixed acid fermentation pathway using glucose = acid = red

2. not this pathway and peptone catalyzation = basic = yellow

what is in the voges-proskauer VP test?

1. glucose

2. peptones

3. Barritt’s A (alpha-naphthol)

4. Barritt’s B (potassium hydroxide)

what does the voges-proskauer VP test differentiate?

1. methyl carbinol intermediate from glucose fermentation = acid = red

2. no methyl carbinol and peptone catalyzation = basic = yellow 

what is in a urease test?

1. urea 

2. nutrients 

3. phenol red 

what does a urease test differentiate?

1. urease converts urea into ammonia = basic = red 

2. no urease = acidic = yellow

what amino acids are tested for decarboxylation?

1. lysine

2. ornithine

3. arginine 

decarboxylation of amino acids leads to production of ________, which ___________ the environment

amines, alkalinize

what is in the ornithine decarboxylase test?

1. ornithine 

2. glucose

3. bromocresol purple 

what does the ornithine decarboxylase test differentiate?

1. ornithine decarboxylation = basic = purple and turbid

2. none = acidic = yellow 

what does the lysine iron agar LIA test contain?

1. peptones

2. lysine

3. glucose

4. bromocresol purple 

5. sodium thiosulfate and ferric ammonium citrate 

ferrous sulfate of the TSI test requires a _____ pH, while the ferric ammonium citrate of the LIA test requires a _______ pH

low, high

what does the lysine iron agar LIA test differentiate?

1. lysine deamination = no pH change = red slant

2. lysine decarboxylation = basic = purple butt 

3. no lysine decarboxylation = acidic = yellow butt 

4. H2S production = black precipitate in butt 

what does the motility indole ornithine MIO test contain?

1. ornithine 

2. kovacs reagent 

3. bromocresol purple 

what does an indole test differentiate?

1. indole production from tryptophan breakdown = positive and red ring

2. no tryptophan breakdown = negative and yellow 

what does the ONPG test contain?

o-nitrophenyl-beta-galactopyranoside

what does the ONPG test differentiate?

1. late lactose fermenters = positive and yellow 

2. non lactose fermenters = negative and no color change 

somatic O antigens

1. heat stable

2. polysaccharide part of LPS

flagellar H antigens

heat labile

capsule K antigens

1. Vi antigen in Salmonella 

2. on top of O antigens

3. boil sample to destroy K antigens to reveal O antigens 

what is added to the bacterial suspension in a latex agglutination test?

specific antiserum 

methods that utilize neutral red

1. MacConkey agar

2. CIN agar

methods that utilize methyl red

methyl red test

methods that utilize phenol red

1. XLD agar

2. TSI test

3. KIA test

4. urease test

methods that utilize bromothymol blue

1. HE agar

2. simmons citrate agar

methods that utilize bromocresol purple

1. ornithine decarboxylation test

2. LIA test

3. MIO test