BI 203 Lab Quiz 8 (9/23)

Lab 8: Differential/Selective Staining

What is an enriched media?

A media that is formulated with all the necessary ingredients for a wide variety of organisms and grows a multitude of bacterial species.

What is a selective media?

A media that allows for growth of target species while inhibiting the growth of others.

What are two possible selective media ingredients?

antibiotics and salt

What is a differential media?

A media that allows for a microbiologist to distinguish between species.

What is another name for nutrient agar (NA)?

Luria Broth (LB)

What is NA/LB?

An enriched media commonly used for maintaining bacteria becuase It meets the diverse nutrient requirements of most bacteria.

What is mannitol salt agar (MSA) selective for?

Halophiles, a bacteria that thrives in high salt concentrations, such as staphylococcus species.

What is MSA differential for?

Mannitol fermentation (sugar)

What happens to the MSA plate in mannitol fermentation?

An acidic byproduct is made which lowers the pH and turns the plate a yellow color. No fermentation causes no change.

What gram type of bacteria are usually grown on MSA?

gram positive

What is MacConkey’s Agar (MCA) selective for, why?

Gram negative bacilli because bile salt and crystal violet inhibit the growth of gram positive bacteria.

What is MAC differential for and what changes does it cause?

Lactose fermentation, turns bacterial colonies pink. No fermentation causes no change.

What is eosin methylene blue agar (EMB) selective for?

Gram negative bacilli

What is EMB differential for, and what changes does it cause?

Lactose fermentation, turns bacterial colonies red. No fermentation causes no change.

What bacteria is an exception for EMB, what color does it change?

E. Coli, metallic green

What bacteria is Hektoen Enteric Agar (HK) selective and differential for?

Salmonella species

What organisms does HK inhibit growth of?

Gram positive bacteria

What two things allow for the visualization of hydrogen sulfide production in HK, and what does it form?

Ferric ammonium citrate reacts with hydrogen sulfide gas to form a black precipitate.

What is it in an HK that provides fermentable carbohydrates to encourage growth of bacteria?

Lactose, sucrose, and salicilin

What species of bacteria grows on HK?

Enterobacteriaceae species

What color colonies are produced by enteric that ferments one or more of the carbohydrates?

yellow or pink

What color colonies do non fermenters produce?

blue or green

What color do organisms that only reduce sulfur to hydrogen sulfide change the colonies to?

Black colonies or blue/green colonies with a black center

What is salmonella shigella agar (SS) selective and differential for?

gram negative bacteria

What color does lactose fermentation turn SS?

Pink, no fermentation = no change

Why can bacteria produce hydrogen sulfide in SS?

Because they contain iron, when there is an interaction iron sulfide is produced

What color does iron sulfide change the agar to?

The black precipitate creates a black center in clear colonies or entirely black colonies.

What is sheep blood agar (blood) differential for?

Hemolytic activity

What is hemolytic activity?

The ability to break down red blood cells

What gram type can grow on blood agar?

positive and negative

What are the types of hemolysis?

Alpha, Beta, Gamma

What is alpha hemolysis?

Patrial break down of RBC, creates a greenish/brown pigment

What is beta hemolysis?

complete breakdown of RBC’s that causes total clearing

What is gamma hemolysis?

No breakdown, no change

What is a catalase test used to identify?

The enzyme catalase

What does catalase do?

It facilitate the breakdown of hydrogen peroxide into oxygen and water

• 2 H2O2 → 2 H2O + O2

What does catalase protect cells from?

Oxidative damage caused by reactive oxygen species (ROS)

Where does ROS come from?

It is a byproduct of made when oxygen is metabolized

What does ROS help with?

Cell signaling and homeostasis

What does ROS do in high stress environments?

It can overproduce and damage cells

What do we used catalase test in lab for?

To differentiate gram positive cocci into staphylococcal sp. and streptococcal sp.

Catalase + = staphylococci

Catalase - = steptococci

What kind of species does catalase testing work on?

Only gram positive

What would a catalase positive bacteria in a catalase test?

Bubble

What kind of plate should the bacteria be obtained from, why?

A nutrient rich agar plate, other plates with selective or differential media can skew results because of other chemicals.

What does an oxidase identify?

Organisms that produce the enzyme cytochrome oxidase

How does an oxidase test work?

The reagent contains a chromogenic reducing agent which is a compound that changes color when it becomes oxidized

What kind of bacteria can an oxidase test be perfomred on?

Gram negative bacteria only

What comes first in Bergey’s flow chart?

Gram stain

What media should be used for gram negative bacteria?

EMB, MAC, or HK

What test should be used for gram positive bacteria?

Catalase test

What test can be done to confirm results of gram negative bacteria?

Oxidase testing

What media should be used for catalase positive bacteria?

MSA and blood agar

What media should be used for catalase negative bacteria?

blood agar