Microlab 6 and 7

Selective Medium

Medium that allows only certain microorganisms to grow and inhibits the growth of other microorganisms

Example of Selective Medium

Ex: Dyes eosin and methylene blue when incorporated into EMB (Eosin Methylene Blue) media do not affect the growth of Gram negative bacteria but do inhibit the growth of Gram positive bacteria

Differential Media

Medium that contains substances that cause some bacteria to take on an appearance that distinguishes them from other bacteria

Differential example

EMB is also a differential media. Gram negative bacteria that vigorously ferment lactose in this medium form colonies with a metallic-green sheen. Gram negative bacteria that moderately ferment lactose in this medium form purple colonies. Non-lactose fermenting bacteria form pink or colorless colonies.

Note: Students often confuse colorless colonies with purple colonies because the media itself is purple.

You can see through the colorless colonies, but you cannot see through the purple colonies.

Carbohydrate metabolism

If an organism can breakdown carbohydrates by oxidation or fermentation, acid is usually produced.

Phenol red: red at alkaline pH and yellow at acidic pH

F(Fermentation) tube

After stabbing the F tube with an inoculating needle containing your culture, sterile mineral oil is added to the top of the tube to ensure anaerobic environment.

Contain semisold agar rather than only liquid broth

O(oxidative) tube

No oil is added to the tube after the stab inoculation

Color changes in O and F tubes

If an acid is produced in either F or O the medium changes from green to yellow

Organisms that turn both the O and F tubes yellow carry out fermentation of carbohydrates

Organisms that only turn the O tube yellow cannot carry out fermentation and produce acid only through the oxidative pathway (respiration).

Both O and F tube must be with each culture for proper results

Methyl red test/ MR Test (Mixed acid fermentation)

Escherichia, Salmonella, and Proteus ferment glucose and produce a number of organic acids such as lactic, acetic, succinic, and formic acids.

CO2 and H2 are also produced.

Positive - Red color (immediate)

Negative - Yellow/no color

Methyl red

To test for the presence of acids, the pH indicator, methyl red, is added to the medium, which turns red if acid is present. A positive test indicated that the organism has carried out mixed acid fermentation.

Vogues-proskauer test/ VP test

Some Gram Negative bacteria do not carry out mixed-acid fermentation, but rather they ferment glucose to produce limited amounts of some organic acids and primarily produce 2,3-butanediol. All species of Klebsiella and Serratia carry out the butanediol fermentation.

Positive for vogues usually negative for methyl red

Positive: Red color development within 30 minutes

Negative: No color change or a yellow to brown color should be considered negative

Acetoin

The neutral end product, 2,3-butanediol, is not detected directly but must be converted to acetoin by oxidation of the 2,3-butanediol.

Citrate test

Some bacteria can use citrate as a sole carbon source. Normally citrate is oxidatively metabolized by the krebs cycle. However, some bacteria such as Klebsiella aerogenes (previously Enterbacter aerogenes) and Salmonella typhurium can cleave citrate to produce oxaloacetate and pyruvate.

Positive - Growth with an intense blue color

Negative - No growth/no color change (remains green)

SIM: Hydrogen Sulfide Production (sulfur reduction)

Bacteria that reduce elemental sulfur to hydrogen sulfide (H2S) get their energy by coupling this reaction to the oxidation of Kreb Cycle intermediates as acetate and succinate.

• Many Gram negative rods use this process

SIM

To determine if a microorganism can reduce elemental sulfur, a SIM (Sulfur, Indole, Motility) tube is inoculated

The SIM medium contains ferrous ammonium sulfate and sodium thiosulfate which serve as indicators for the production of hydrogen sulfide (H2S).

Positive: Blackening along the stab line or throughout the tube

Negative: No color change

Indole

Some bacteria have the ability to degrade the amino acid tryptophan to produce indole, ammonia, and pyruvic acid.

Flagella

The major organelles of motility in bacteria are flagella

Chemotaxis

Flagella allow cells to move toward nutrients in the environment or move away from harmful substances, such as acids, in a process called

-10 microns out from the cell

Motility

Motility can be determined by several methods. It can be determined microscopically by observing cells in a wet mount

• drop of viable cells is placed on a slide and then covered with glass - phase-contrast microscope

Brownian motion

For the beginner, true swimming motility under the microscope must be differentiated from the Brownian motion of the cells or by the movement caused by currents under the cover glass

This motion is due to molecular bombardment of cells cause to shake or “wiggle” about but not move in any vectorial way

Semisoft agar

Another mode for determining motility involves inoculating semisoft agar medium (SIM aka Sulfur-Indole-Motility) medium. This medium has an agar concentration of 0.4% which does not inhibit bacteria from “swimming” through the medium.

Swarming Motility

Proteus mirabilis - bacteria move in every direction

After a certain distance, they divide and the progeny continue to move pit. This causes the formation of concentric rings (called swarms) on the agar plate.

When you streak for isolation but you get growth over the entire plate, you microorganism is capable of swarming motility.

Phenyalanine test

Gram-Negative bacteria such as Proteus can oxidatively deaminate the amino acid phenylalanine to produce phenylpyruvic acid and ammonia. The reaction is catalyzed by the enzyme phenlalanine deaminase

IMViC Tests

Sometimes grouping of tests can be used to differentiate organisms. For example E.coli can be differentiated from Klebsiella aerogenes by comparing four tests collectively called the IMViC test

(I-Indole M-Methyl red V-Vogues-Proskauer C- Citrate)

EMB

Positive: Growth (+)/ Acid (++) (primarily but not always E.coli)

Negative: Growth (+)/ Acid (-)

Nitrate Reduction

Positive: Red coloration after addition of Nitrate A and B reagents OR no color change after addition of zinc

Negative: Red colorarion after addition of zinc

O/F Glucose (oxidation/fermentation reaction)

Positive: (O) Oxidative Reaction: Yellow color in the open tube only (no oil)

Positive (F): Fermentative Reaction: Yellow color in both open tube and closed tube (oil added)

Negative: Neither oxidative nor fermentative reaction: Green color remains in either the O or the F tube

Phenylalanine

Positive: Green coloration

Negative: yellow coloration

Indole Production (±)

Positive: Red color at the top of agar

Negative: No color change

Motility (±)

Positive: Diffuse growth outward from the stab line (appears turbid)

Negative: Growth only along the stab line

Urea

Positive: Intense pink-red color

Negative: No color change