Lesson 5 - Biochemical Testing

Biochemical Testing

• Determine the microbes at species level

  • Additional test that further describe and differentiate the microbes

  • Determines the specific metabolic activities

  • Very significant when you are trying to identify a microorganism

  • Uses chemicals (reagent) that is being added to inoculum or colonies of microorganisms, in which the microorganisms will react to the reagents

    • Its reactions are phenotypical (observable, can be seen with naked eye)

    • E.g., bubbles formation, changes in color, air formation

  • Used to differentiate various microorganisms

    • There are biochemical tests that is specific for a certain type of microorganism

  • Tests are based on the production of enzymes or lack of enzymes to visualize a biochemical change

  • Results are relatively rapid

  • Only needs a small amount of bacteria (microorganism)

  • Types:

    • Indole Production, Methyl Red and Voges-Proskauer Test, Citrate Utilization, Catalase Test, Oxidase Test, Urease Test

Indole Production

• Its purpose is to identify organism with tryptophanase

  • Performed at bacterial species to determine their ability to convert tryptophan to indole

Indole Production | Positive

an indole production:

• Pink- to wine- colored ring after addition of Kovac’s Reagent

• Escherichia coli

Indole Production | Negative

an indole production:

• No color change after addition of the reagent

• Klebsiella pneumoniae

Kovac’s Reagent

Consists of Isoamyl alcohol para-dimethylenebenzaldehyde (DMAB) and concentrated HCL (hydrochloric acid)

Methyl Red and Voges-Proskauer Test

Its purpose is to differentiate members of Enterobacteriaceae family

Methyl Red and Voges-Proskauer Test | Methyl Red Test

Used to identify if a bacteria produces acid during glucose fermentation

Methyl Red and Voges-Proskauer Test | Voges-Proskauer Test

Used to identify if a bacteria produces acetoin

Methyl Red Test | Positive

methyl red test

• Bright Red Color +5-6 Drops Methyl Red Reagent

• Escherichia coli

Methyl Red Test | Negative

methyl red test

• Yellow color

• Enterobacter aerogenes

Voges-Proskauer Test | Positive

voges-proskauer

• Red color

  • + 12 drops-Reagent A*

  • + 4 drops-Reagent B*

• Escherichia coli

Voges-Proskauer Test | Negative

voges-proskauer

• Yellow color

• Enterobacter aerogenes

*A reagent

Refers to Alpha-naphthol

*B-reagent

Refers to 40% KOH with creatine

Citrate Utilization

• Its purpose is to identify organisms capable of using sodium citrate as Carbon source and using Ammonium salts as Nitrogen source

  • Used to determine bacteria’s ability to use sodium citrate as the only source of carbon and inorganic ammonium as a source of nitrogen

  • Sodium Citrate to Carbon

  • Ammonium Hydrogen Phosphate to Nitrogen

Citrate Utilization | Positive

a citrate utilization that has:

• Growth on the medium

• Bromothymol blue indicator turning from green to blue

• Enterobacter aerogenes

Citrate Utilization | Negative

a citrate utilization has:

• Absence of growth

• Escherichia coli

Catalase Test

• Its purpose is to differentiate catalase-positive micrococcal and staphylococcal species from catalase-negative streptococcal species + 30% hydrogen peroxide

  • Used to determine if a bacteria can produce a catalase (an enzyme which is produced by a microorganism that uses oxygen in their respiration; protects bacteria from toxic by-product of oxygen)

  • Results are rapid

  • You will only use an inoculated microorganisms which is put into a slide to view under the microscope

  • When you put a reagent and it bubbles, it is positive in ________

Catalase Test | Positive

catalase test

• Copious bubbles are produced

• Staphylococcus aureus

Catalase Test | Negative

catalase test

• No or few bubbles are produced

• Streptococcus pyogenes

Oxidase Test (Kovac’s Method)

• Its purpose is to identify the differences between Enterobacteriaceae and other gram-negative bacilli

  • Uses filter paper with 1% tetramethyl-p-phenylenediamine dihydrochloride

  • Results are rapid

  • Used to identify bacteria that produce cytochrome C oxidase (enzyme used in electron transport chain)

  • Every bacteria that turns positive, is an aerobic bacteria

  • They use oxygen as a terminal electron acceptor in their respiration

Oxidase Test (Kovac’s Method) | Positive

oxidase test

• Development of a dark purple color within 10 seconds

• Pseudomonas aeruginosa

Oxidase Test (Kovac’s Method) | Negative

oxidase test

• Absence of color

• Escherichia coli

Urease Test (Christensen’s Method)

• Its purpose is to know the organism’s ability to produce enzyme urease (that hydrolyzes urea)

  • Used to identify bacteria that is capable of hydrolyzing urea with the use of urease

  • Hydrolysis of urea forms a weak base ammonia

  • Urea > Hydrolysis > Ammonia

  • “urea” is a waste product especially when we pee

  • Uses butt-slut medium

Urease Test (Christensen’s Method) | Positive

urease test

• From orange to magenta

• Proteus vulgaris

Urease Test (Christensen’s Method) | Negative

urease test

• No color change

• Escherichia coli

Triple Sugar Iron Agar Test

• Its purpose is to determine whether Gram-negative rod ferments glucose and lactose or sucrose and forms hydrogen sulfide

  • Like a differential medium

  • Uses differential agar medium

  • Determines different strains or properties of a microorganism (bacteria)

  • Tests acid and gas production (positive result)

  • To see if they can ferment glucose, sucrose, or lactose

  • If an organism can ferment those three sugars, it will produce hydrogen sulfide

Triple Sugar Iron Agar Test | Alkaline Slant/no change in the butt

triple sugar iron agar test

• Triple sugar non-utilizer

• Alkaline slant and butt

• Pseudomonas aeruginosa

Triple Sugar Iron Agar Test | Alkaline Slant/Acid Butt

triple sugar iron agar test

• Glucose Fermentation Only

• Shigella flexneri

Triple Sugar Iron Agar Test | Acid Slant/Acid Butt

triple sugar iron agar test

• Glucose, Sucrose, and/or Lactose Fermenter

• Escherichia coli

Triple Sugar Iron Agar Test | Black Precipitate

triple sugar iron agar test

• Hydrogen Sulfide Production

• K/A

• Proteus mirabilis

Triple Sugar Iron Agar Test | Bubbles or crack in tubes

triple sugar iron agar test

• Carbon Dioxide Production and Hydrogen

• Escherichia coli

Identification IMVIC Test

• Its purpose is to identify/differentiate member of Family Enterobacteriaceae

  • Consists of indole test, methyl red test, Voges-Proskauer test, and citrate test

Ammonia

• A chemical made by bacteria in your intestine and body cells while processing proteins

  • Waste products of human body

  • Excreted through urine

Catalase

• Refers to an enzyme which is produced by microorganism that uses an oxygen in their respiration

  • Protects bacteria from toxic by-product of oxygen

Indole

• an important small molecule signal utilized by many bacteria to coordinate various forms of behavior

  • plays a role in biofilm formation

cytochrome C oxidase

an enzyme primarily found in aerobic organisms that use oxygen as their terminal electron acceptor in respiration