Micro Lab 3+4

BHI with 6.5% NaCl

Differentiates microorganisms based on their ability to grow in 6.5% NaCl

Positive - Growth (turbidity)

Negative - No growth (clear)

Bile Esculin Hydrolysis

Differentiates microorganisms based on their ability to hydrolyze esculin to esculetin and dextrose. The esculetin reacts with ferric citrate in the medium to form a dark brown-black complex

Positive - Blackening of the tube

Negative - No color change or less than ½ of the slant is black

Simple and Differential

Catalase

Differentiates microorganisms based on their ability to produce the catalase enzyme. Catalase breaks hydrogen peroxide down to water and oxygen.

Positive - Bubbles

Negative - No bubbles

Coagulase

Detection of coagulase that is excreted by Staphylococcus aureus that causes fibrin clot formation

Positive - Coagulation of the plasma

Negative - No coagulation/clot formation

A excellent definitive test for confirming the identification for Staphlococcous aureus because 97% of the strains are proven to be coagulase positive - innoculating blood plasma

Hemolysis on SBA (Sheep Blood Agar)

DIfferentiates microorganisms based on their hemolytic activity on sheep blood agar with 5% sheep blood cells

Gamma - No discoloration

Aplha - greening

Beta - clearing

KOH test

Confirms gram stain based on whether 3% KOH can lyse bacterial cell wall and release DNA.

Gram Positive - Strings absent

Gram negative - Strings present

MSA (Mannitol Salt Agar)

Selects for microorganisms based on their ability to grow with 7.5% NaCl present in the agar and then differentiates organisms based on their ability to ferment mannitol

Positive - Growth + / Acid + Yellow colonies

Negative - Growth ± / Acid - Colorless colonies or no growth

Nitrate Reduction

Differentiates microorganisms based on their ability to reduce nitrate to nitrite or nitrogenous gas

Incomplete positive - Red coloration after the addition Nitrate A and B

Complete - No color change after the addition of Nitrate A and B and Zinc

Negative - Red after the addition of zinc

Differential

Oxidase

Diffferentiates microorganisms based on the presence of the enzyme cytochrome C oxidase in bacteria using oxidase reagent containing NNN tetra p

Positive - Any blue or purple color change within 30 sec

Negative - No color change within 30 sec

Phenol Red Carbohydrate Tubes

Differentiates organisms based on carbohydrate fermentation. This media contains specific carbohydrates and the pH indicator phenol red. When an organism is able to ferment the carbohydrate present, an acid or an acid and gas are produced and the indicator changes from red to yellow. If the carbohydrate is not fermented the color will remain red.

Positive - Red to yellow

Negative - Red

Starch Hydrolysis

Differentiates microorganisms based on their ability to hydroylze starch.

Positive - Dark agar with tan/brown clearing around streak line

Negative - Dark agar up to the streak line

Strict Aerobes

Require oxygen to grow and cannot survive in anaerobic conditions.

Facualitve anaerobes

Organisms that can grow in both the presence and absence of oxygen, but prefer oxygen when available.

Aerotolerant Anaerobes

Organisms that do not require oxygen for growth but can tolerate its presence without being harmed.

Strict obligate anaerobes

Organisms that cannot survive in the presence of oxygen and are killed by it.

Catalase - in terms of aerobes

Used by strict aerobes and facualtive anaerobes to convert hydrogen peroxide into water and oxygen.

Non-catalase peroxidases

Enzymes used by some aerotolerant anaerobes to break down hydrogen peroxide, providing protection from oxidative stress without producing oxygen.

Superoxide Dismutase

An enzyme that catalyzes the conversion of superoxide radicals into hydrogen peroxide and molecular oxygen, providing protection against oxidative damage.

Selective Medium

Allow only certain groups of microorganisms to grow and will inhibit the growth of others

Example: The incorporation of sodium chloride into mannitol salt agar (MSA) selects for the genus Staphylococcus but inhibits the growth of bacteria that cannot tolerate salt concentration

MSA and Bile Esculin are both selective and differential

Differential Medium

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

Example: When Staphylococcus aureus grows on mannitol salt agar, it ferment mannitol producing acid and changing a pH indicator in the media from red to yellow around the colonies

Ex: Sheep Blood Agar, Phenol Red carbohydrate tubes, starch agar plate

Beta hemolysis

Complete lysis of red blood cells around a colony

partial lysis = alpha

no change - gamma

Oxidative and fermentation reactions

If an organism can break down carbohydrates by oxidation or fermentation, acid is usually produced

red at alkaine pH values to yellow acidic pH values

Amylose and amylopectin

Two parts of the starch marco-molecule

(1) a straight chain of polymers of 200 to 300 glucose molecules

(2) a larger branched polymer of glucose

Starch Hydrolysis detection

Can be detected by adding Gramms iodine to a starch agar plate, a differential medium

See if you can produce amaylase to break down strach