Biochemical Tests

Good evening everyone. This is Jeremy (0702919B) from TG01. Today, I will be elaborating on several biochemical tests performed in the lab to aid in identifying bacteria.

Kliger’s Iron Agar (KIA)

KIA is a differential medium. It is similar to the Triple Sugar Iron (TSI) agar that we used in the school's lab. It tests for organisms’ abilities to ferment glucose and lactose to acid and acidic gas products. This media is commonly used to separate lactose fermenting members of the family Enterobacteriaceae (e.g. Escherichia coli) from members that do not ferment lactose. These lactose non-fermenting enterics are generally more pathogenic in the gastrointestinal tract.

Glucose fermentation creates acidic byproducts that turn the phenol red indicator in the media yellow. Upon depletion of glucose, organisms that can ferment lactose will continue to produce acidic byproducts and the media will remain yellow. Gaseous products produce might lift the agar off the bottom of the tube.

Non lactose fermenters metabolize amino acids and proteins in the media, creating alkaline byproducts that turn the indicator red at the slant.

Non glucose and lactose fermenters will use solely amino acids and proteins, creating a red slant and unchanged color at the butt.

Organisms that sulfer reducing enteric will produce H₂S that creates a black precipitate at the butt.

	Slant	Butt	Examples
Lactose fermenters	Yellow	Yellow	Escherichia coli
Non lactose fermenters	Red	Yellow OR Black (H2S production)	Shigella dysenteriae Proteus mirabilis (produce black ppt)
Non glucose and lactose fermenters	Red	Unchanged	Pseudomonas aeruginosa

Simmons’ Citrate Agar

Simmons’ citrate agar tests the ability of an organism to use citrate as a sole carbon source and ammonium ions as a sole nitrogen source. The medium contains citrate, ammonium ions, and also a pH indicator, bromothymol blue. Organism that can grow on Simmons’ citrate agar are capable of metabolizing citrate as the sole carbon source and ammonium ions as the sole nitrogen source. This will create alkaline byproducts which will turn bromothymol blue indicator blue.

Growth on Simmon’s citrate agar or an intense blue color formation is an indication of a positive result.

Urease Test

This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus mirabilis is a rapid hydrolyzer of urea.

Well, that is all for now. Thank you for your attention.

Jeremy.