Exercise 11

Enterobacteriaceae share these common features

• Gram-negative rods

• ALL ferment glucose

• ALL are nitrate positive

• ALL are oxidase negative

• Many (but not all) are motile

Enterobactericeae includes potential pathogens…

• Salmonella

• Shigella

• Klebsiella

• Proteus

• Serratia

Primary Pathogens

Organisms can cause disease in anyone

Opportunistic Pathogens

Organisms can only cause disease under certain conditions or in certain hosts

Family Enterobacteriaceae

Primary pathogen examples

• Shigella

• Salmonella

• Yersinia

• E. coli

• Klebsiella pneumoniae

Family Enterobacteriaceae

Opportunistic pathogen examples

• Providencia

• Morganella

• Enterobacter

• Serratia

• Proteus

• E. coli

• Klebsiella pneumoniae

A provided “Unknown will have 2 organisms…

E. coli or Enterobacter aerogenes

AND

Salmonella or Proteus mirabilis

Day 1: Isolation

Goal: Obtain isolated colonies of the 2 organisms from the mixed culture

3 zone streak plates on EMB, MAC, SS, and BSA

Eosin Methylene Blue agar (EMB)

Selective for Gram-negative and differential for lactose fermentation

MacConkey agar (MAC)

Selective for Gram-negative and differential for lactose fermentation

Salmonella-Shigella agar (SS)

Selective for Gram-negative and differential for lactose fermentation and sulfur reduction

Bismuth Sulfite agar (BSA)

Selective for Salmonella spp. and differential for sulfur reduction

Day 2

Goal: Identify 2 different colonies on plates

• Colony 1: lactose (+); Sulfur (-) - probable E. coli or E. aerogenes

• Colony 2: Lactose (-); Sulfur (+) - probable Salmonella or Proteus spp.

what is positive for Lactose fermentation

EC and EA are positive

• We used EMB, MAC, and SS plates

Sulfur reduction

Salmonella and Proteus are positive

• We used SS and BSA plates

MacConkey agar

• Crystal violet and bile salts inhibit Gram positives

• Neutral red detects lactose fermentation

• Results:

  • Growth w/ pink color → Gram (-) and lactose (+)

  • Growth w/out pink color → Gram (-) and lactose (-)

Eosin Methylene Blue agar

• Eosin and methylene blue inhibit Gram positives

• Eosin and methylene blue detect lactose fermentation

Eosin Methylene Blue agar Results

• Results:

  • Growth w/ pink to lavender color → Gram (-) and weak lactose fermentation (+)

  • Growth w/ dark purple or green sheen → Gram (-) and strong lactose fermentation (+)

  • Growth w/out color change → Gram (-) and lactose (-)

Salmonella-Shigella agar

• Brilliant green, bile salts, and sodium citrate inhibit Gram positives and many Gram negatives

• Neutral red detects lactose fermentation and iron indicates sulfur reduction

Salmonella-Shigella agar Results

• Results:

  • Growth w/ pink to red color → Gram (-), lactose (+), sulfur (-)

    Growth w/out color change → Gram (-), lactose (-), sulfur (-)

    Growth w/ black precipitate → Gram (-), lactose (-), sulfur (+)

Bismuth Sulfite agar

• Bismuth sulfite and brilliant green dye inhibit Gram positives and many Gram negatives

• Iron and bismuth sulfite detect sulfur reduction

Bismuth Sulfite agar Results

• Results:

  • Growth w/out black precipitate Gram (-) and sulfur (-)

  • Growth w/ black precipitate Gram (-) and sulfur (+)

Biochemical Tests

Used to identify unique biochemical characteristics since selective and differential plates alone aren’t enough to differentiate:

• E. coli from E. aerogenes

• Salmonella from Proteus mirabilis.

Remember: We have lactose (+)/ sulfur (-) and lactose (-)/ Sulfur (+) we must identify.

Day 3: Confirmatory

Read results

E. coli and E. aerogenes

• Indole, methyle red, Voges-Proskauer, and citrate (IMViC)

• E. Coli → Indole (+); MR (+); VP (-); Citrate (-)

• E. aerogenes → Indole (-); MR (-); VP (+); Citrate (+)

• Salmonella and Proteus mirabilis

  ◦ Urea broth and SIM (H2S and motility only)

Tryptone Broth detects…reagent…

• Detects tryptophanase: hydrolyzes tryptophan → indole, pyruvate and ammonia (NH3)

• Add 3-5 drops of Kovac’s reagent

Tryptone Broth Results…

• (+) = cerise/red

• (-) = no change in the reagent color

Methyl Red (MR) tests for… reagent…

• Tests for mixed acid fermenters

• Add 3-4 drops of methyl red reagent

Methyl Red (MR) Results…

• (+) = reagent remains red

• (-) = red color is not retained

Voges-Proskauer (VP) tests for… Reagent..

• Tests for 2,3-butanediol fermenters

• Add 10 drops of VP I (alpha naphthol) then 10 drops of VP II (KOH)

Voges-Proskauer (VP) Results

• (+) = red color

• (-) = no red color

Simmons Citrate detects… indicator…

• detects utilization of citrate as sole carbon source for growth

• Indicator: bromothymol blue

Simmons Citrate Results

• (+) = growth, possibly accompanied by blue color

• (-) = no growth

What is SIM medium used for?

Used to determine three bacterial activities: sulfur reduction, indole production and motility

SIM - Sulfide

H2S produced by bacterium reacts with Fe in medium to produce FeS

(+) = black precipitate

SIM - Motility

Motility

(+) = cloudy

Urea Broth detects…

Urea broth detects the enzyme urease which degrades urea → 2 ammonia (NH3) and carbon dioxide (CO2)

Whats the indicator for Urea Broth?

Indicator: Phenol red

• detects pH changes

pH changes for Urea broth and results

◦ As pH increases > 8.1 → cerise

◦ Neutral pH → red

◦ As pH decreases → yellow

◦ Positive = cerise color (alkaline)

◦ Negative = yellow for acidic, red for neutral

Salmonella should be _____ (positive/negative) when inoculated in urea broth.

negative

Salmonella species are urease-negative, meaning they test negative when inoculated into urea broth or media