Kligler Iron Agar (KIA) and O/F Test Quiz

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17 Questions

Which indicator is used in LIA to detect H2S production?

Ferric ammonium citrate and sodium thiosulfate

What is the purpose of the slant and butt in LIA?

To detect aerobic and anaerobic metabolism

What is the significance of a purple slant and yellow butt on LIA?

Lysine decarboxylase positive, lysine deaminase negative

Which test is used to detect the presence of $eta$-galactosidase activity?

Ortho-nitrophenyl-$eta$-D-galactopyranoside (ONPG) test

Which agar medium is used to simultaneously detect glucose fermentation, lactose fermentation, and hydrogen sulfide production?

Triple Sugar Iron (TSI) Agar

What color change is observed on the slant after adding FeCl3 if the result is positive for TRYPTOPHAN deamination?


What is the color change observed in the UREASE test for rapid urease producers?

Bright pink

Which of the following microorganisms is a slow/weak urease producer?


Which medium is commonly used to determine motility in microorganisms?

SIM (Sulfide Indole Motility) Medium

What reaction indicates motility in the SIM (Sulfide Indole Motility) Medium?

Spread away from the stab line/hazy appearance

Which of the following is NOT a member of ENTEROBACTERIACEAE that is motile?


What is the purpose of the Oxidation-Fermentation Tests (O/F tests)?

Identifying bacteria based on their oxidation and fermentation patterns.

What happens in the Kligler iron agar tube for a non-oxidizer?

No acid is produced in both open and closed tubes.

What is the purpose of Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG)?

To determine the presence of slow lactose fermenters.

Which color indicates an alkaline environment in the O/F medium?


What does an oxidizer produce in the Kligler iron agar open tube?


What is the pH indicator used in the Oxidation-Fermentation Tests?

Bromothymol blue

Study Notes

Lysine Iron Agar (LIA)

  • LIA is used in conjunction with TSI to screen stool specimens for enteric pathogens
  • Composition: aerobic slant, anaerobic butt, lysine, peptones, and a small amount of glucose
  • Manner of Inoculation: STAB-STREAK-STAB

Reactions on LIA

  • Lysine Decarboxylation (butt/anaerobic): • K/K (Purple Slant/Purple Butt): LYSINE DECARBOXYLASE (+) LYSINE DEAMINASE (-) • K/A (Purple Slant/Yellow Butt): LYSINE DECARBOXYLASE (-) LYSINE DEAMINASE (-)
  • Note: Cadaverine neutralizes organic acids formed by glucose fermentation and reverts to alkaline state

Oxidation-Fermentation Tests

  • Purpose: Identification of bacteria based on the oxidation-fermentation pattern
  • Medium: Hugh and Leifson O/F basal medium (OFBM)
  • pH Indicator: BROMOTHYMOL BLUE
  • Uninoculated: GREEN; Acid environment: YELLOW; Alkaline environment: BLUE
  • Two tubes: one overlaid with mineral oil (closed – anaerobic) and one without mineral oil (open – aerobic)
  • FERMENTER: ACID in BOTH open & closed tube
  • OXIDIZER: ACID in OPEN tube; no acid in closed tube
  • NON-OXIDIZER: NO ACID in BOTH open & sealed tube


  • Determines the presence of Slow/Delayed/Late Lactose Fermenters (LLF)
  • ONPG is a colorless molecule structurally similar to LACTOSE
  • Can enter cell membrane without lactose permease and is converted into galactose and o-nitrophenyl (yellow chromogen) by Beta-galactosidase

IMViC Reactions


  • Used to identify organisms that produce the enzyme TRYPTOPHANASE
  • Indicator: 10% FERRIC CHLORIDE (FeCl3) – added after incubation
  • Reactions: • (+) GREEN color on slant after adding FeCl3 • (-) Slant remains ORIGINAL COLOR after adding FeCl3


  • Determines whether a microorganism can hydrolyze urea
  • Medium: Christensen’s Urea Agar or Stuart Urea Broth
  • pH Indicator: PHENOL RED
  • Reactions: • (+) BRIGHT PINK Coloration • (-) No Color Change (YELLOW)


  • Determines whether an organism is motile or non-motile
  • Medium: SIM (Sulfide Indole Motility) Medium or any semisolid motility medium
  • Streaking: STRAIGHT STAB
  • 1% triphenyltetrazolium chloride can be added to aid visualization
  • Reactions: • MOTILE (+): SPREAD AWAY from the stab line/HAZY APPEARANCE • NON-MOTILE (-): REMAIN at the stab line

Test your knowledge on Kligler Iron Agar (KIA) and the Oxidation-Fermentation (O/F) test. Learn about the purpose, medium composition, and pH indicator used in the O/F test. Identify bacteria based on their O/F pattern.

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