Microbiology 258 Practical 2

Mannitol Salt Agar (MSA) is used to?

Used to isolate bacteria that tolerate high salt e.g., Stapylococcus spp

Why is it selective for those that can tolerate high salt concentration?

It contains 7.5% salt and inhibits all other bacteria that cannot survive in such environment

What is the differential component that distinguishes between those bacteria that can cause fermentation to produce an acid and those that cannot?

Mannitol (carbohydrates)

Why would MSA have a yellow color?

When it becomes acidic it turns from red to yellow

MSA isolates what particular strain?

Staphylococcus strains

Peptone in MSA plate is?

Source of carbon and nitrogen

MacConkey Agar (MAC) is used to?

Used to isolate Gram-negative enteric organisms and differentiate between them based on lactose utilization  

What is the differential component that distinguishes between those bacteria that can ferment lactose and those that cannot?

Lactose

MAC contains two selective agents that are?

Bile salts and Crystal violet that selects gram (-) bacteria

Why does MAC go from pink to red?

It goes pink to red when in acidic pH

Why does MAC go colorless?

When in basic pH

What does it mean if bacteria grows on MAC?

It is gram (-)

What does it mean if bacteria grows on MAC and it is pink?

It is a lactose fermenter

What does it mean if bacteria grows on MAC and it is colorless?

Non-lactose fermenter

The two inhibitors, crystal violet and bile salts, prevent what?

The growth of gram-positive bacteria

Eosin Methylene Blue Agar (EMB) is used to?

Used to isolate enteric based on their ability to ferment lactose

What are the two carbohydrate sources that EMB use?

Lactose and sucrose

What are the two selective agents for EMB and what do they select/inhibit?

Eosin Y and Methylene blue that select gram (-) and inhibits many gram (+)

Why does EMB form crystals at low pH?

Because of Eosin Y and Methylene blue

What do strong lactose fermenters appear like on EMB plate?

Appear dark purple or black colonies with green metallic sheen

On EMB plates, bacteria that can use lactose but are not strong lactose fermenters generate?

Pink colonies

Why would EMB plate colonies appear colorless?

Non-fermenters use proteins and amino acids as sources of carbon and energy which leads to an increase in pH and the colorless colonies

Hektoen Enteric Agar (HEA) is used to?

Used to isolate salmonella spp and shigella spp in stool samples

What are the two differential properties used to distinguish bacteria?

1) the presence of 3 carbohydrates (lactose, sucrose, and salicin) 2) sodium thiosulfate

When bromothymol blue gets more acidic, what color does it change?

It gets more yellow

When acid fuchsin gets more acidic, what color does it change?

It gets more red

Sodium thiosulfate can form hydrogen sulphide when?

When reduced and it will form a black precipitate

Bacteria that can use lactose but are not strong lactose fermenters generate?

Pink colonies

What are the two separate differential tests that exist within HE agar?

1) differentiate fermenting bacteria from non-fermenting bacteria. 2) Reveals an organism’s ability to produce hydrogen sulfide gas

What can reduce sulphur?

Salmonelle can reduce sulphur and it combines with ferric ammonium citrate and forms a black precipitate

Blood Agar (BA) is used to?

Used to isolate bacteria that can lyze/breakdown red blood cells

What are the 3 distinct hemolysis patterns that are formed from hemolysins?

Alpha (partial), Beta (complete), and Gamma (no hemolysis)

What is the appearances of the 3 distinct hemolysis patterns?

Alpha: produces a greenish/brownish zone around the colonies

Beta: produces a clear zone around the colonies

Gamma: produces no color change in the agar

How do hemolysins work?

They work by aggregating on the surface of cells and forming pores

Which agar is used routinely in clinical microbiology labs in hospitals and clinics?

Blood agar

Chocolate agar is used to?

Used to isolate bacteria that require additional nutrients to grow

Why is chocolate agar referred to as enriched media?

Because additional nutrients have been added to it

What are “fastidious” organisms?

Organisms that require extra vitamins and co-factors found inside human cells in order to grow in cultured media

What are the 3 principal products produced by fermentation?

• Gas (often CO2) which is detected by a small, inverted test tube called a Durham tube

• Alcohol

• Acid

What are the tests for fermentation?

• Phenol red broth

• Methyl red (MR-VP) broth

What color is phenol red at a low pH and high pH?

Yellow at low pH and red/pink at high pH

What is microbial fermentation?

Breaking down a monosaccharide sugar to pyruve, then the pyruvate is then broken down to organic acids, alcohol, CO2, and ATP

What is deamination?

A mode of protein digestion where the amine group (NH2) is removed from an amino acid resulting in the alkaline (basic) end product ammonia (NH3) and a weak organic acid

Methyl Red-Voges Proskauer test is used to?

Used to differentiate enteric bacterial species based on which fermentation pathway is used to metabolize glucose

The MR test determines?

If the organism fermented glucose via the mixed acid fermentation pathway

The VP test determines?

If the organism fermented glucose via the 2,3-butanediol pathway

Which reagent is used for MR test?

Methyl red reagent

Which reagent is used for VP test?

Reagent A and Reagent B

In MR-VP what does peptone, glucose, and potassium phosphate do?

Peptone provides the protein, glucose provides fermentable carbohydrates, and the potassium phosphate prevent pH changes

In the presence of atmospheric oxygen, acetoin is converted to?

Diacetyl and alpha naphthol

In some bacteria, single-step reduction is?

Nitrate to nitrite (NO3 —> NO2)

Dentrification is?

Where nitrate is converted to nitrogen (NO3 à NO2 à NO à N2O à N2) (Nitrate à Nitrite à Nitric oxide à Nitrous oxide à Molecular nitrogen gas)

What does it mean if no color change with addition of reagents?

Nitrate was not reduced to nitrite or reduced to something other nitrogenous compound

Why is Zinc added?

To catalyze reduction of any remaining nitrate to nitrite

What does it mean if nitrate is present when zinc is added and the media turns red?

Organism unable to reduce nitrate to nitrite

If gas production in Durham tube, that means?

Positive for dentrification

What does Nitrate reductase do?

Allows for reduction of nitrate to nitrite

Oxidase is used to?

Used to identify presence of cytochrome c oxidase which is part of complex 4 in ETC

Tetramethyl-p-phenylenediamine (TMPD) is?

A reagent used to indicate the presence of cytochrome c oxidase in a microbe

When TMPD donates an electron to cytochrome C and itself becomes oxidized, what happens?

It turns purple

When resources are limited, rather than being used as protein building blocks, amino acids can be utilized for?

Energy

Every amino acid contains?

An amino (NH2) functional group, carboxyl (COOH), and R group

What is the enzyme that removes the carboxyl group from the amino acid lysine?

Lysine decarboxylase

Bromocresol purple is purple at? Yellow at?

Purple at high pH and yellow at low pH

If the organism ferments glucose what will happen?

The pH will drop and the broth will turn yellow

If the organism ferments glucose and decarboxylates lysine, what happens?

The broth will turn yellow and then subsequently to purple as the basic amine products build up

Why is mineral oil added to the top of the broth?

To seal out external oxygen and thus promote fermentation of the organism

DNA hydrolysis tests what?

Tests the ability of an organism to produce DNase

What is DNase?

An enzyme that breaks down (depolymerizes) DNA

It is often best to view results behind what for DNase?

Behind a white background, such as a sheet of paper

Starch is what?

A complex polysaccharide composed of glucose monomers that are linked together through glycosidic bonds

Triple Sugar Iron (TSI) test is designed to?

Designed to differentiate bacteria based on glucose fermentation, lactose or sucrose fermentation, gas production, and sulfur reduction

What is the indicator for TSI?

Phenol red

In TSI how is phenol red affected with pH?

High pH is red and low pH is yellow

What turns the TSI tube yellow?

Glucose fermentation which lowers the pH

What does the tube look like when there is glucose only fermentation?

The tube has a red slant with yellow butt due to the slowed fermentation by anaerobic conditions

What does the tube look like when no fermentation and only protein breakdown?

Red butt and/or red slant

How does sulfur reduction occur?

Reduction of sodium thiosulfate produces H2S and then ferrous sulfate reacts with H2s to produce black precipitate

How is gas production (CO2) made evident in TSI test?

By cracks, fissures, lifting of the agar

In TSI, Yellow slant/yellow butt

glucose and lactose and/or sucrose fermentation with acid accumulation in slant and butt

In TSI, Red slant/yellow butt

Glucose fermentation only with acid production. Proteins catabolized aerobically in the slant with alkaline production (reversion)

In TSI, Red slant/red butt

No fermentation. Proteins catabolized aerobically and anaerobically with alkaline products

In TSI, Black participate in agar

Sulfur reduction

In TSI, Cracks or lifting of agar

Gas production

What three properties does SIM Sulfur reduction test for?

Sulfur reduction, Indole production, Motility

Indole production is tested by?

The addition of Kovac’s reagent

If Kovac’s reacts with indole, it turns?

The reagent layer red

Tryptophanase serves as?

The catalyst for the hydrolysis of tryptophan

How to know if indole positive?

The reagent layer (top layer) is red

How to know if sulfur reduction positive?

The tube is black

How can motile organism move through the SIM semisolid media?

Because of its flagella

How can you tell there is motility in SIM?

Fuzzy or radiating growth extending from the stab line

Why should you not move the needle when inoculating the SIM?

To avoid a false positive for motility

Fluid thioglycolate media is allows for?

Allows for a test of aerotolerance (ability or inability to live in presence of oxygen)

What is resazurin?

O2 indicator that turns pink when oxidized and colorless when reduced

Obligate (strict) aerobes

require oxygen for respiration – growth at the top

Facultative anaerobes

grow in presence or absence of oxygen; can do aerobic and anerobic respiration – growth throughout the media but appears denser at the top

Aerotolerant anaerobes

do not require oxygen but are not adversely affected by it – growth is uniform throughout the media

Microaerophiles

survive only in environments containing lower than atmospheric oxygen levels – growth near the middle or upper middle of the media

Obligate (strict) anaerobes

even small amounts of oxygen are lethal – growth only in the lower regions of the media