Microbio lab practical

Selective Media

• Contains one or more ingredients that inhibit the growth of unwanted bacteria while encouraging the growth of desired ones

  • If a microorganism is resistant to an antibiotic like ampicillin or tetracycline, then it would be added to the medium to prevent other cells, which aren’t resistant, from growing

  • Example: 7% NaCl media is selective for gram-positive organisms

    • Media lacking histidine

    • MSA is selective for gram-positive, favoring cocci

Differential Media

• Distinguishes one microorganism type from another growing on the same medium. Uses biochemical characteristics of a microorganism growing in the presence of specific nutrients/indicators added to the medium to visibly indicate the defining characteristics

• Examples: Blood agar plates (5% sheep blood)

  • Beta-hemolytic - Organisms that produce hemolysin and can completely digest RBCs

  • Alpha-hemolytic- Produce enzymes that partially digest RBCs, greenish colored growth

Both Differential and Selective Media

• Glucose fermentation broth: Contains carbohydrates and an indicator, phenol red; if fermentation occurs, the media changes color

• MacConkey media:

  • Bile salts - Inhibit the growth of gram-positive organisms and selects for gram-negative bacteria. [selective]

  • Lactose + neutral red dye - Colorless at pH 6.8, turns red at lower pH. If lactose fermentation occurs, colonies turn pink or red. [differential]

• Mannitol Salt Agar (MSA):

  • High salt concentration (7.5%) → Selective for gram-positive cocci like Staphylococcus

  • Mannitol + phenol red → fermentation turns the plate yellow, including pathogenic Staphylococcus species

  • Differentiates pathogenic from nonpathogenic strains

• Eosin Methylene Blue:

  • Eosin Y + methylene blue dye - inhibits growth of gram-positive

  • Differentiates lactose fermenters from nonfermenters

    • Acid end products of lactose fermentation turns colonies black/dark purple (E.coli), less effective = pink/purple (Klebsiella, Enterobacter)

Biochemical Tests

Fermentation of Carbohydrates

• Anaerobic degradation of carbohydrates (glucose, lactose)

• pH indicator - phenol red, red at pH 7, yellow when acidic

  • Production of acid indicates fermentation of carbohydrates

• Deeper red = organism used protein (peptone) and made broth more alkaline

• The Durham tube is used for collecting gas if produced

Oxidation-Fermentation Test (O-F test)

• Detects acid byproducts of oxidative respiration (glucose breakdown via glycolysis and cellular respiration requiring oxygen as an ultimate electron acceptor)

• Also indicates if sugar can be fermented (doesn’t require oxygen)

• Bromothymol blue dye - green at pH 7.1, yellow at pH 6, blue at pH 7.6

IMViC Series

• A series of tests that differentiate members of the Enterobacteriaceae family

• Indole test

• MR/VP test

• Citrate utilization test

Indole test

• Determines the ability to ferment the amino acid tryptophan into indole, ammonia, and pyruvic acid

  • After incubation, add Kovac solution, let stand for 5 minutes

MR/VP Test

• Methyl Red test and the Voges-Proskauer test → tests for end products of glucose metabolism

  • + MR (Red) = Large production of acid from glucose metabolism

  • + VP (Red) = Production of neutral substances like acetoin and alcohol from glucose metabolism

• 5 drops of methyl red,15 drops of VP reagent A, 5 drops of VP reagent B

  • 1 hour wait

Citrate Utilization Test

• Determines the ability of an organism to utilize citrate as a carbon source

  • Uses sodium citrate breaks down into → pyruvate

  • Also converts ammonium phosphate into → ammonia + ammonium hydroxide = increase in pH

• Bromothymol blue dye becomes blue at higher pH (alkaline)

Oxidase Test

• Tests for the presence of the oxidase enzyme, cytochrome C, an electron acceptor

• Differentiates members of the Pseudomonadaceae and Enterobacteriaceae families

• Chromogenic reducing agent, TMPD, when oxidized to cytochrome C, it turns blue

  • No cytochrome C = stays colorless

Catalase Test

• Tests for the presence of catalase, an enzyme present in cytochrome-containing aerobic and facultative anaerobes

• Using oxygen as an ultimate electron acceptor will result in toxic oxygen reactive species that need to be neutralized

Decarboxylation test

• Tests for the ability to decarboxylate amino acids, lysine, or ornithine

• Differentiates members of Enterobacteriaceae

• Bromocresol purple → purple in alkaline conditions, becomes yellow/clear due to acid production = negative decarboxylation test

  • Mineral oil

  • Decarboxylated media becomes more alkaline = stays purple/darker purple

• Lysine decarboxylase that reduces/removes a carboxyl group from lysine

• Ornithine decarboxylase converts ornithine to putrescine

Starch Hydrolysis test

• Detects the production of the exoenzyme amylase

  • This enzyme degrades starch (a polysaccharide)

• Flood plate w/ iodine after inoculation

Urease test

• Determines the ability of an organism to use urea as a source of nitrogen using the enzyme urease

• Differentiates Proteus species from other organisms, identifies Heliobacter pylori

• Alkaline byproducts of urease breakdown and turns the media from yellow to → pink/red (phenol red indicator)

SIM Deep

• Semi-solid media, tests for 3 bacterial activities

  • 1.) Reduce sulfur → forms black precipitate

    • Breaks down cysteine (contains sulfur), into hydrogen sulfide or has an enzyme, thiosulfate reductase, which catalyzes reduction of sulfur

  • 2.) Contains the amino acid tryptophan - can be broken down w/ the presence of the enzyme tryptophanase into indole, ammonia, and pyruvic acid

    • Add kovac solution later on for indole

  • 3.) A semi-solid media - if the bacteria is motile, agar will become hazy away from point of inoculation

Enzymes (What do they do and how do we test for them?)

Amylase

• An enzyme that breaks down carbohydrates, like starch, into simple sugars

• Test: Starch hydrolysis test (starch plate)

Hemolysin

• A toxin produced by some bacteria that causes the lysis of red blood cells, hemolysis

• Test: Blood agar (Containing 5% sheep blood)

Urease

• An enzyme produced by some bacteria that hydrolyzes urea into ammonia and carbon dioxide

• Test: Urease test, a rise in pH causes a color change in the media due to ammonia production

Catalase

• An enzyme that some bacteria produce to neutralize the bactericidal effects of hydrogen peroxide

• Test: Catalase test - indicates if the bacteria produces an enzyme that can break down hydrogen peroxide into water and carbon dioxide

Oxidase

• Cytochrome C oxidase, important component of the electron transport chain in aerobic bacteria

• Test: Oxidase test (card) dark purple within 10-30 seconds indicates the presence of cytochrome C oxidase, reagent TMPD used and turns dark purple

PH indicators

• Phenol red → Acidic = yellow, Alkaline = red/pink

• Methyl red → Neutral = Orange, Acidic = red, Alkaline = yellow

• Bromocresol purple → purple in alkaline conditions, becomes yellow/clear due to acid production

• Bromothymol blue → Acidic = yellow, Alkaline = blue, neutral = green

Basic Dilution Series

• Original Concentration = CFU/volume plated times dilution factor