Lab 9 (TSIA)

What is a selective media biochemical test?

Selective media designed to enhance isolation by inhibiting growth of some organisms while encouraging growth of others

What is a differential media biochemical test?

Differential media is many types of media that may also contain indicators to expose differences between organisms

What are coliforms?

Subgroup of Enterobacteriaceae that produce acid and gas from lactose fermentation.

What is the most prominent member of coliforms?

E.coli

Where do coliforms usually live?

Most coliforms are normal inhabitants of the human intestinal tract

What does the presence of coliforms possibly indicate?

fecal contamination

What do the nutritional components in biochemical tests do?

Typically selected to obtain optimal growth

What do inhibitors do in biochemical tests?

Make the medium selective. Designed to prevent growth of specific growth of microorganisms while allowing other organisms to grow

What do substrates do in biochemical tests?

Almost always what makes the medium differential. Differing abilities to preform a specific chemical reaction differentiates organisms?

What do indicators do in biochemical tests?

• Make expected reaction visible

• Frequently a dye that changes color as pH changes

• Also can be chemicals that react with products of a reaction and produce a color change

What is the preferred source of metabolic activity?

Fermentation

What is the process of fermentation in biochemical test?

Process of extracting energy from the oxidation of organic compounds such as carbohydrates (glucose, sucrose, lactose)

T/F Organisms capable of fermenting glucose will exhaust glucose supply before utilizing sucrose or lactose

True

What products does fermentation produce?

• Fermentation produces acid byproducts, lowering the pH

• Many types also produce hydrogen gas

What do microbes utilize and produce when carbohydrates are depleted or when they’re incapable of carbohydrate fermentation?

• Microbes utilize peptones or amino acids

  • Produce NH3, raising the pH

What are the categories of microbe fermentation? (what they’re capable of)

• Only capable of fermenting glucose

• Capable of fermenting glucose and sucrose and/or lactose

• Incapable of carbohydrate fermentation

On what basis does TSIA differentiate bacteria?

• Sugar fermentation

  • Glucose

  • Sucrose/ Lactose

• Sulfur reduction

• Gas production

Why does a TSIA slant have a shallow agar slant with a deep butt?

Provides aerobic and anaerobic growth environments

How do you inoculate a TSIA slant?

Inoculated by a stab in the butt followed by fishtail streak of the slant

What does a TSIA slant contain?

• 3 carbohydrates (glucose, sucrose, and lactose)

• ferrous sulfate and sodium thiosulfate as sources of oxidized sulfur

What are the indicators in TSIA?

• For fermentation:

  • pH indicator: phenol red

• For sulfur reduction:

  • Ferrous sulfate

What results do you see when TSIA is inoculated with a glucose ONLY fermenter?

• Fermentation of glucose produces acid, which lowers pH and turns medium yellow throughout

• Glucose concentration is very low (0.1%) and so it is depleted very quickly

• As glucose diminishes, organisms in the aerobic region (slant) begin to break down available amino acids, producing NH3 and raising the pH turning the pH pink

  • Only occurs in the slant because of the anaerobic conditions in the butt

What results do you see when TSIA is inoculated with a organisms capable of fermenting glucose and lactose and/or sucrose?

• Fermentation of glucose also lowers pH and turns the medium yellow throughout

• Because lactose and sucrose concentration are 10x higher than glucose, greater acid production keeps both the slant and butt yellow

What results do you see in TSIA when inoculated with an organism incapable of carbohydrate fermentation?

Utilizes peptone and amino acids, alkalinizes the medium and turns it red (will look almost like an uninoculated tube)

How is gas production observed in TSIA slants? How is it produced?

• Produced by fermentation of the carbohydrates

• Appears as fissures in the medium or will lift the agar off the bottom of the tube

How is hydrogen sulfide production produced in TSIA slants?

• May be produced by the reduction of thiosulfate in the medium or by the breakdown of cysteine in the peptone

• Indicator (ferrous sulfate) reacts with the hydrogen sulfide to form a black precipitate

What conditions need to be present for thiosulfate reduction? What does this indicate?

• Acidic conditions

• Black precipitate indicates BOTH sulfur reduction and fermentation

• If the black precipitate obscured the color of the butt, it MUST be yellow

How do we record results for TSIA?

• Slant/ butt/ gas/ H2S

  • Yellow = acidic (A)

  • Pink/red = alkaline (K)

  • Using + or - to indicate result for gas and H2S production