Differential & Selective Media, Chemical Control, Antibiotics
Microbiologists often used multiple types of media to cultivate microorganisms in order to
grow a particular organism or obtain information about the biochemical properties of the organisms that grow
Growth on MacConkey agar that is pinkish-red color indicates:
he organism has the ability to ferment lactose, and is probably a coliform.
What does Columbia CNA (C-CNA) Agar differentiate?
hemolytic patterns.
Function of dyes eosin and methylene blue:
inhibit gram-positive growth and select for gram-negative organisms
Function of crystal violet and bile salts on MacConkey Agar
inhibit most gram-positive organisms select for gram-negative organisms
What is the differential ingredient in Eosin Methylene Blue Agar (EMB)?
lactose
What is the differential ingredient in MacConkey Agar?
lactose
What does MacConkey Agar differentiate?
lactose fermentation
What does Eosin Methylene Blue Agar (EMB) differentiate?
lactose fermentation - strong vs. weak
What is the differential ingredient Mannitol Salt Agar (MSA)?
mannitol
What does Mannitol Salt Agar (MSA) differentiate?
mannitol fermentation
Additive effect of Antibiotics means
means that the overall effect of the antibiotic combination is no greater than the sum of the individual antibiotics
disk diffusion assay
method to test the effectiveness of antimicrobial chemicals
What is the pH indicator in MacConkey Agar?
neutral red
What is the pH indicator in Mannitol Salt Agar (MSA) plates?
phenol red
Function of colistin & nalidixic acid in Columbia CNA (C-CNA) Agar
prohibit the growth of most gram-negative Gram-positive organisms are allowed to grow.(Staphylococcus and Streptococcus)
Epsilometer or E test
quantitatively measures the minimal inhibitory concentration (MIC) of the antibiotic for the microorganism being tested
What type of media is Mannitol Salt Agar (MSA)?
selective and differential
How are selective properties of microbiological media different from differential properties of microbiological media?
selective media composed ingredients to inhibit the growth of certain microbes, while allowing others to grow differential media contains ingredients or chemicals that help to visually distinguish which species do or don't carry out a specific biochemical process.
Three commonly used types of media are
selective media, differential media enriched media.
Antimicrobial therapy is based on
selective toxicity
What does Mannitol Salt Agar (MSA) selects for?
selects for halotolerant bacteria
why is solid media is usually employed with selective and differential media?
so that individual colonies may be isolated.
Why are the C-CNA and blood plates incubated in the Anaeropak box?
so they can be incubated in an environment enriched with carbon dioxide.
Antibiotics are
substances produced as metabolic products of one microorganism which inhibit or kill other microorganisms;
Often, most differential media contain a___,___and__ indicator
substrate chemical indicator pH indicator
Define. Selective media
suppresses the growth of some microorganisms while allowing the growth of others (i.e., it selects for certain microbes)
Growth on mannitol salt agar indicates
that the organism is Halotolerant
Growth on Eosin Methylene Blue agar which exhibits a green metallic sheen indicates Example
the organism can ferment lactose to form strong acid end-products. (Q1) is not inhibited by eosin and methylene blue Escherichia coli
Growth on Eosin Methylene Blue agar, which is a pink color indicates Example
the organism can ferment lactose to form weak acid end-products (Q2 Maybe Q3) is not inhibited by eosin and methylene blue Enterobacter aerogenes
Growth on Eosin Methylene Blue agar indicates Example:
the organism can grow in the presence of the dyes eosin and methylene blue and is likely a gram-negative (Q4) is not inhibited by eosin and methylene blue Morganella Morganii
A change in the color of the Mannitol Salt Agar (MSA) from pink to yellow indicates:
the organism has the ability to ferment mannitol (red arrow)
Absence of growth on Eosin Methylene Blue agar indicates Example
the organism is Gram-positive is inhibited by eosin and methylene blue Streptococcus mutans
Growth on MacConkey agar indicates:
the organism is gram-negative Resistant to crystal violet and bile salts
Growth on Columbia CNA agar indicates
the organism is resistant to the antibiotics colistin and naladixic acid, and is likely a gram-positive.
Antimicrobial chemotherapy is
the use of chemicals to inhibit or kill microorganisms in or on the host
What does Blood agar differentiate?
Differentiation among Gram-positive cocci Bacteria with hemolysins
Which chemical would you choose if you wanted maximum effectiveness against both E. coli and S. aureus?
Clorox Most effective
If you wanted to select for Gram-positive bacteria, what type(s) of agar could you use?
Columbia CNA Agar
What type of media is Blood Agar?
Differential
Example of Gram-negative bacteria that ferment lactose
Enterobacter aerogenes (Q1) Escherichia coli (Q2)
Example of Gram-positive cocci bacteria Alpha hemolytic
Enterococcus Faecalis
Bacteria: Growth + alpha hemolysis on C-CNA plates
Enterococcus faecalis
Compare and contrast the Disk Diffusion and Epsilon tests for determining antibiotic susceptibility. Is one better than the other? Why or why not?
Epsilon test is better because the measurement is more accurate
What does this media select for Columbia CNA (C-CNA) Agar?
Gram- positive Resistan to colistin and nalidixin acid
What does it mean when bacteria wont grow on MacConkey agar?
Gram- positive (Q3) Streptococcus mutans is inhibited by bile salts and crystal violet
Beta hemolysis on blood plate results
Growth surrounded by a complete clearing of the blood indicates beta hemolysis. complete lysis of RBC and hemoglobin.
Alpha hemolysis on blood plate on blood plates results in
Growth surrounded by a partial clearing of the blood (often a greenish-grey color) partial lysis of RBC and hemoglobin.
Gamma hemolysis on blood plates results in
Growth which produces no changes in the blood indicates the bacteria are non-hemolytic.
What does Mannitol Salt Agar select for?
Halotolerant Bacteria
What does it mean when bacteria grow on MacConkey agar?
It is Gram-negative and not inhibited by bile salts and crystal violet
An unknown bacterium produces colorless colonies when inoculated onto an EMB plate. Predict what you would see if you inoculated that same unknown onto MacConkey agar, and why:
It will grow because the organism is gram-negative
Examples of selective media:
MSA - mannitol salt agar Mac - MacConkey agar EMB -eosin-methylene blue agar C-CNA- Columbia C-CNA agar.
Examples of differential media
MSA- mannitol salt agar, C-CNA blood agar Mac- MacConkey agar EMB- Eosin-methylene blue agar.
Which Selective media, selects for Gram-negative bacteria?
Mac - MacConkey agar Halotolerant EMB -eosin-methylene blue agar
If you wanted to select for Gram-negative bacteria only, what type(s) of agar could you use?
MacConkey Agar EMB
Example of Gram-negative bacteria that does not ferment lactose
Morganella Morganii (Q4) Salmonella typhimurium (Q4)
Example of Halotolerant organisms
SES Enterococcus faecalis (yellow) Staphylococcus xylosus (yellow) Staphylococcus epidermidis (pink)
What type of media is Columbia CNA (C-CNA) Agar?
Selective and differential
What type of media is Eosin Methylene Blue Agar (EMB)?
Selective and differential
What type of media is MacConkey Agar?
Selective and differential
Bacteria: Growth + Beta hemolysis on C-CNA plates
Staphylococcus xylosus
Example of Gram-positive cocci bacteria Beta hemolytic
Staphylococcus xylosus Streptococcus zooepidemicus
Example of Gram-positive cocci bacteria Gamma hemolytic
Streptococcus mutans
selective toxicity means
That the agent used must inhibit or kill the microorganism without seriously harming the host
Chemical agents work through two main mechanisms:
(1) Alteration of cell walls or cytoplasmic membranes (2) Interference with protein and nucleic acid structure.
2 general classes of antimicrobial chemotherapeutic agents:
(1) Antibiotics (2) Antimicrobial chemotherapeutic chemicals
synergistic effect of antibiotics means
(Arrow) the overall effect of the antibiotic combination is greater than the sum of the individual antibiotics. Antibiotics enhance the effect of each other.
The goal of antibiotic combination therapy is
(a) reduce the incidence of antibiotic-resistance (b) allow lower doses of antibiotics to be used (c) enhance the agents' antibacterial activity.
Describe how the disk diffusion assay works.
1. filter disks are impregnated with the chemical to be tested, and are placed on a plate inoculated w/ bacteria 2. plates are incubated 3. As a chemical diffuses into the agar, it becomes less concentrated
What the selective component Mannitol Salt Agar (MSA)?
7.5% sodium chloride
In order to be selectively toxic
Agent must interact with microbial function/structure that is either not present in the host, or is substantially different from that of the host.
Which organisms grow on the blood plates?
All organisms
The three types of hemolysis are:
Alpha hemolysis -partial/green Beta hemolysis - complete Gamma hemolysis-no hemolysis
Hemolysis patterns
Alpha- partial hemolysis Beta- complete hemolysis Gamma- no hemolysis
Disk Diffusion Assay or Kirby-Bauer test.
Antibiotic sensitivity testing to determine the susceptibility of bacteria to various antibiotics.
Define the term "zone of inhibition".
Area around the disk where the growth has been inhibited Organism is susceptible to a chemical
What is the differential ingredient Blood agar?
Blood
What is the differential ingredient in Columbia CNA (C-CNA) Agar?
Blood
How does Blood agar allows distinction among bacteria?
By the bacteria ability to lyse red blood cells hemolysins (exotoxins that lyses of RBC)
Which Selective media, selects for Gram-positive bacteria?
C-CNA- Columbia C-CNA agar. MSA - mannitol salt agar
An unknown bacterium produces colorless colonies when inoculated onto an EMB plate. Predict what you would see if you inoculated that same unknown onto C-CNA Agar agar, and why:
The organism will not reproduce because C-CNA does not allow gram-negative organisms to grow
What property or properties of gram-negative organisms would likely make them more resistant to crystal violet and bile salts than gram-positive organisms?
The outer membrane
size of this zone of inhibition depends on
The sensitivity of the bacteria to the specific chemical and the chemical's ability to diffuse through the agar.
Example of Halotolerant organisms that ferment mannitol
Yellow Enterococcus faecalis Staphylococcus xylosus
Organisms that are able to ferment the mannitol will produce ____ _____products which __ the pH. Causing the phenol red indicator to turn ____
acid fermentation lower yellow
Antibiotics used in combination can have 2 effects
additive effect synergistic effect
Define. Differential media
allows growth of more than 1 microorganism of interest but with morphologically distinguishable colonies based on specific biochemical properties of the organisms.
Which media, is only differential?
blood agar
Examples of enriched media
blood agar chocolate agar
Antimicrobial chemotherapeutic chemicals are
chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.
What are the selective ingredients in Columbia CNA (C-CNA) Agar?
colistin & nalidixic acid
Define.Enriched media
contains specific growth factors needed by fastidious bacteria to support their growth.
What are the selective ingredients in MacConkey Agar?
crystal violet and bile salts
Mueller-Hinton agar is used to
determine additive and synergistic effects of drug combinations by placing the two antibiotic discs on a lawn of cells
What are the selective ingredients Eosin Methylene Blue Agar(EMB)?
eosin & methylene blue dyes
What does Eosin Methylene Blue Agar (EMB) select for?
gram-negative bacteria
What does MacConkey Agar select for?
gram-negative bacteria
