Selective and Differential Media (micro lab)
What is the basis of differentiation for Columbia CNA agar w/ 5% sheep blood?
level of hemolysis (the ability to lyse RBC)
What is the indicator in MacConkey agar?
neutral red dye (pH indicator)
What is the indicator in eosin methylene blue agar?
neutral red dye (pH indicator)
What is the indicator in manntiol salt agar?
phenol red (pH indicator)
What is the indicator in Columbia CNA agar w/ 5% sheep blood?
red blood cells
What is the basis of differentiation in hectoen enteric agar?
two chemical reactions - fermentation of lactose, sucrose, and salicin - reduction of sulfur (S2) to hydrogen sulfide gas (H2S)
What are the results of Columbia CNA agar w/ 5% sheep blood and how do you interpret them?
type of hemolysis, color of media bordering colonies, state of RBC - gamma, red, maintained integrity - beta, yellow, RBC are gone - alpha, slight greenish-yellow, partial damage
How can media be differential?
Every media/test is designed in such a way that it allows bacteria to be differentiated based on their ability to perform certain chemical reactions
How do we know that a chemical reaction is taking place in a media?
Products from a chemical reaction interact with an added indicator ; the indicator enables a color change
What is the selective agent in mannitol salt agar and what organism is especially selective for?
- 7.5% NaCl - Staphylococcus aureus
Types of selective and differential media for gram positive bacteria
- Mannitol salt agar - Columbia CNA agar w/ 5% sheep blood agar - Bile esculin azide agar
Types of selective and differential media for gram negative bacteria
- McConkey agar - Eosin methylene blue agar - Hektoen enteric agar
What are the selective agents in MacConkey agar?
- bile - crystal violet
What are the selective agents in bile esuclin azide agar and which does some gram negative organisms demonstrate a tolerance?
- bile: some gram negative demonstrate tolerance - sodium azide
What are the indicators in hectoen enteric agar?
- bromthymol blue and acid fushin dyes (pH indicators) - ferric ammonium citrate (ligand indicator)
What are the selective agents in eosin methylene blue agar?
- eosin Y - methylene blue
What is the indicator in bile esculin azide agar and what reaction produces color change?
- ferric citrate (ligand indicator) - esculetin + Fe3+
What are the results of bile esculin azide agar and how do you interpret them?
- ferric citrate present, darkened medium surrounding colonies, Bile Esculin - positive - absence of ferric citrate, no darkened medium, Bile Esculin - negative
What are the results of a mannitol salt agar and how do you interpret them?
- pH < 6.8, colonies are yellow, organism fermented mannitol - pH 7.4-8.4, colonies are red, organisms cannot ferment mannitol - pH > 8.4, colonies are pink, organism cannot ferment mannitol
What are the results of MacConkey agar and how do you interpret them?
- pH > 6.8, turn yellow, lactose nonfermenters - pH < 6.8, turn red, lactose was fermentated (acid accumulated during the process causes color change)
Types of indicators present in media
- pH indicators - a specific molecule that binds to the product of the chemical reaction
Why do we need selective and differential media?
- speeds up process of identifying bacteria from a clinical sample - allows for identification (gram negative vs. gram positive)
What makes media selective?
By adding chemicals that suppress the growth of one group of organisms and promote the growth of another
What is the selective agent in Columbia CNA agar w/ 5% sheep blood and how does it work?
CNA, which contains two antibiotics - colistin: affects membrane integrity - nalidixic acid: interferes with DNA replication
What is the basis of differentiation in eosin methylene blue agar?
ability to ferment lactose and sucrose
What is the basis of differentiation in bile esculin azide agar?
ability to hydrolyze esculin to esculetin and glucose
What are the results of eosin methylene blue agar and how do you interpret them?
amount of acid produced, color of colonies, level of fermentation - large amounts, green, high levels of fermentation - medium amounts, purple, average levels of fermentation - low amounts, pink, low levels of fermentation - none, no color change, no fermentation
What is the selective agent in hectoen enteric agar?
bile salts
What are the results of hectoen enteric agar and how do you interpret them?
fermentation - positive: colonies and surrounding media turn orange, yellow, or salmon - negative: colonies and surrounding media are blue-green reduction of sulfur - positive: colonies turn black - negative: colonies are blue-green
What is the basis of differentiation in MacConkey agar?
fermentation of lactose
What is the basis for differentiation in mannitol salt agar?
fermentation of mannitol