Lab Quiz 3 – Microbio

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selective media

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contains components that will either enhance the growth of specific organisms or inhibit growth of specific organisms

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Differential Media

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contains indicators that will show differences between organisms

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•In clinical setting - selective may screen out normal flora
•In environmental setting - may help to look for specific groups of organisms, such as fecal contamination of water

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applications of selective and differential media

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defined

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specified amounts of each ingredient

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undefined

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•("complex") :contains one or more ingredients of unknown composition and/or amount, such as yeast extract, beef extract, digest of gelatin, etc.

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nutritional components

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Selected to optimize growth of organisms being tested for (Ex/ specific vitamins)

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inhibitors

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•Makes the media selective
•Capitalizes on weakness of one group while allowing another group to grow
•Ex/ interfere with membrane permeability, enzyme function, DNA expression, osmotic pressure

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substrates

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•Generally makes media differential (Ex/Lactose fermentation capability vs. no lactose fermentation)

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indicators

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•Make a desired or expected reaction visible
•Frequently a dye that changes color as pH changes
Can also be chemicals that react with certain reaction products and produce color change

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Positive of Negative?

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This is information given so that you will know how to interpret a positive or negative identification.

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Phenylethyl Alcohol Agar (PEA)

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•Undefined, selective
•Inhibits growth of many Gram- organisms
•Selective agent: phenylethyl alcohol
--Disrupts cytoplasmic membrane of sensitive organisms, which affects its ability to act as a permeability barrier
•Used to isolate staphylococci and streptococci from mixtures of bacterial flora
--Often to screen out common contaminants E. coli and Proteus species.

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1. a rapid loss of potassium ions by the cell, which in turn affects the cell's ability to maintain pH and osmotic homeostasis
2. inhibition of DNA synthesis, likely due to loss of molecules essential for this process

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consequences of disruption of plasma membrane with PEA

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-organism is inhibited
-probable gram-

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poor growth or no growth on PEA indicates...

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-not inhibited
-probable staphylococcus, streptococcus, enterococcus, or lactococcus

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good growth on PEA indicates...

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Mannitol Salt Agar (MSA)

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•Selective and Differential
•Selective: 7.5% sodium chloride
•Differential: Fermentation of mannitol (carbohydrate)
•pH indicator: Phenol red
•Selects for staphylococci group: such as Staphylococcus aureus and Staphylococcus epidermidis
•Used for isolation and differentiation of S. aureus from other Staphylococcus species.

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growth appears but with no fermentation

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appearance of pink or red, medium remains unchanged on MSA indicates...

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staphylococcus aureus ferments mannitol, producing acids and lowering the pH of the medium

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formation of bright yellow colonies usually surrounded by a yellow halo on MSA indicates...

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-organism inhibited by NaCl
-not staphylococcus

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poor growth or no growth on MSA

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-not inhibited by NaCl
-staphylococcus

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good growth on MSA

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-produces acid from mannitol fermentation
-possible pathogenic staphylococcus aureus

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yellow growth or halo on MSA

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-does not ferment mannitol
-staphylococcus other than S. aureus

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red growth (no halo) on MSA

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MacConkey Agar

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•Selective and Differential
•Selective: bile salts and crystal violet inhibit Gram+ organisms
•Differential: Fermentation of lactose
•pH Indicator: Neutral red (colorless above 6.8 and red at less than 6.8)
•Useful for isolating and differentiating members of family Enterobacteriaceae based on ability to ferment lactose

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a pink to red color

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(MacConkey) organisms that ferment lactose to acid end-products lower the pH and their colonies turn..

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retain their normal color or the color of the medium

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(MacConkey) Lactose non-fermenters...

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ferment the lactose and appear pink on the medium

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(MacConkey) formulations without crystal violet allow growth of Enterococcus and some species of Staphylococcus, which...

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-inhibited by crystal violet and/or bile
-gram positive

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poor growth or no growth on MacConkey

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-not inhibites by crystal violet or bile
-gram negative

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good growth on MacConkey

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-produces acid from lactose fermentation
-probable coliform

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pink to red growth with or without bile precipitate on MacConkey

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-does not ferment lactose
-noncoliform

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growth is "colorless" on MacConkey

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eosin methylene blue agar

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•Selective and Differential (undefined as well)
•Contains the dyes eosin Y and methylene blue
•Inhibit most Gram+
•React with vigorous lactose fermenters whose acid end-products turn growth dark purple or black
•Differential: Lactose is fermented to acid end-products by coliforms such as E. coli and E. aerogenes, and not by pathogens such as Proteus, Shigella, and Salmonella
•Used for isolation of fecal coliforms

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-inhibited by eosin and/methylene blue
-gram+

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poor growth or no growth on EMB

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-not inhibited by eosin and methylene blue
-gram-

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good growth on EMB

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-ferments lactose with little acid production
-possible coliform

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growth is pink and mucoid on EMB

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-ferments lactose with acid production
-probable coliform

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growth is "dark" on EMB

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-does not ferment lactose
-noncoliform

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growth is "colorless" on EMB

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hektoen enteric agar

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•Selective: bile salts prevent or inhibit growth of Gram+ bacteria
•Differential:
--Fermentation of lactose, sucrose, or salicin to acid-end products
--Ability to reduce sulfur source (e.g. sodium thiosulfate) to H2S
----Indicator: ferric ammonium citrate reacts with H2S to form black precipitate (ferrous sulfide)
•pH indicators: Bromothymol blue and acid fuchsin dyes
•In medical setting, used to isolate and differentiate Salmonella and Shigella from other Gram- enteric organisms in a patient's stool sample.
•Also used in identifying fecal contamination of dairy and poultry products

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-inhibited by bile and/or one of the dyes included
-gram positive

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poor growth or no growth on hektoen

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-not inhibited by bile or any of the dyes included
-gram negative

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good growth on hektoen

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-produces acid from lactose, sucrose, and/or salicin fermentation
-not shigella or salmonella

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pink to orange growth on hektoen

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-does not ferment lactose, sucrose, or salicin, but reduces sulfur to hydrogen sulfide
-probable salmonella

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blue-green growth with black precipitate on hektoen

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-does not ferment lactose, sucrose, or salicin, or reduce sulfure
-probable shigella or rarely salmonella

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blue-green growth without black precipitate on hektoen

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decontamination

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•reduce number of pathogens to level in which items are safe to handle without protective attire.
•Physical cleaning, removal of organic material, etc.

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disinfection

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Destruction of pathogens; Liquid chemical agents, dry heat, moist heat, UV light, antiseptics (used on tissues)

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sterilization

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Complete elimination of viable microbes including spores, May include chemical, gases, incineration, dry heat, moist heat, ethylene oxide gas, ionizing radiation, etc

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•Number of organisms: Greater number will require longer kill time
• Duration of Exposure
• Concentration of disinfectant
• Temperature
• Environment - organic material such as blood, feces, vomit, etc., present?
• Endospore formation
• Other cellular characteristics such as waxy wall of tubercle bacilli

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what factors can affect microbial death?

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•Cell wall: Damage will leave cell susceptible to lysis
• Plasma membrane: surfactants disrupt membrane- leads to cell lysis
• Protein Structure/Function
Nucleic acid synthesis

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What are some of the targets for Disinfectants/Antiseptics?

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• UV-C wavelengths ranging from 100 to 280 nm are most detrimental to bacteria.
• Generally requires exposure for specific time to kill bacteria & damage DNA to extent that it is not repairable by the cell.

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lethal effects of UV radiation

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• DNA absorbs UV radiation & forms new covalent bonds between adjacent pyrimidines (pyrimidine dimers)
• Interferes with replication & transcription

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How does UV radiation damage DNA?

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1. light repair
2. excision repair (dark repair)

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DNA repair mechanisms

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light repair (photoreactivation)

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•Requires:
--Specific enzyme: DNA photolyase
--Visible light: 340-400 nm
•Effectively monomerizes the dimer
--Restore original double helix

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excision repair (dark repair)

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•Requires these enzymes:
--Endonuclease
--Helicase
--DNA polymerase I
--DNA ligase

•Both mechanisms are limited by the amount of DNA damage resulting from longer or more intense exposures

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mixed culture

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microbial culture consisting of two or more species

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pure culture

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contains only one species or strain

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streak plate

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a commonly used isolation technique

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-bacterial sample (assumed to be mixed culture) is streaked over surface of plated agar medium
-during streaking, cell density decreases, eventually leading to individual cells being deposited separately on the agar surface
-cells that have been sufficiently isolated will grow into colonies consisting of only the original cell type

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process of streak plate

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colony-forming unit (CFU)

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term used to define the cell or group of cells that produces a colony when transferred to plated media because some colonies form from individual cells and others from pairs, chains, or clusters of cells

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quadrant streak or T-streak

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used with samples suspected of high cell density (use inoculating loop)

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zigzag (continuous streak)

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used for samples containing lower cell densities (use swab)

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coliform

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normal inhabitants of the human intestinal tract; therefore, their presence in the environment may be evidence of fecal contamination

1 of 59

question

selective media

answer

contains components that will either enhance the growth of specific organisms or inhibit growth of specific organisms

question

Differential Media

answer

contains indicators that will show differences between organisms

question

•In clinical setting - selective may screen out normal flora
•In environmental setting - may help to look for specific groups of organisms, such as fecal contamination of water

answer

applications of selective and differential media

question

defined

answer

specified amounts of each ingredient

question

undefined

answer

•("complex") :contains one or more ingredients of unknown composition and/or amount, such as yeast extract, beef extract, digest of gelatin, etc.

question

nutritional components

answer

Selected to optimize growth of organisms being tested for (Ex/ specific vitamins)

question

inhibitors

answer

question

substrates

answer

•Generally makes media differential (Ex/Lactose fermentation capability vs. no lactose fermentation)

question

indicators

answer

•Make a desired or expected reaction visible
•Frequently a dye that changes color as pH changes
Can also be chemicals that react with certain reaction products and produce color change

question

Positive of Negative?

answer

This is information given so that you will know how to interpret a positive or negative identification.

question

Phenylethyl Alcohol Agar (PEA)

answer

question

answer

consequences of disruption of plasma membrane with PEA

question

-organism is inhibited
-probable gram-

answer

poor growth or no growth on PEA indicates...

question

-not inhibited
-probable staphylococcus, streptococcus, enterococcus, or lactococcus

answer

good growth on PEA indicates...

question

Mannitol Salt Agar (MSA)

answer

question

growth appears but with no fermentation

answer

appearance of pink or red, medium remains unchanged on MSA indicates...

question

staphylococcus aureus ferments mannitol, producing acids and lowering the pH of the medium

answer

formation of bright yellow colonies usually surrounded by a yellow halo on MSA indicates...

question

-organism inhibited by NaCl
-not staphylococcus

answer

poor growth or no growth on MSA

question

-not inhibited by NaCl
-staphylococcus

answer

good growth on MSA

question

-produces acid from mannitol fermentation
-possible pathogenic staphylococcus aureus

answer

yellow growth or halo on MSA

question

-does not ferment mannitol
-staphylococcus other than S. aureus

answer

red growth (no halo) on MSA

question

MacConkey Agar

answer

question

a pink to red color

answer

(MacConkey) organisms that ferment lactose to acid end-products lower the pH and their colonies turn..

question

retain their normal color or the color of the medium

answer

(MacConkey) Lactose non-fermenters...

question

ferment the lactose and appear pink on the medium

answer

(MacConkey) formulations without crystal violet allow growth of Enterococcus and some species of Staphylococcus, which...

question

-inhibited by crystal violet and/or bile
-gram positive

answer

poor growth or no growth on MacConkey

question

-not inhibites by crystal violet or bile
-gram negative

answer

good growth on MacConkey

question

-produces acid from lactose fermentation
-probable coliform

answer

pink to red growth with or without bile precipitate on MacConkey

question

-does not ferment lactose
-noncoliform

answer

growth is "colorless" on MacConkey

question

eosin methylene blue agar

answer

question

-inhibited by eosin and/methylene blue
-gram+

answer

poor growth or no growth on EMB

question

-not inhibited by eosin and methylene blue
-gram-

answer

good growth on EMB

question

-ferments lactose with little acid production
-possible coliform

answer

growth is pink and mucoid on EMB

question

-ferments lactose with acid production
-probable coliform

answer

growth is "dark" on EMB

question

-does not ferment lactose
-noncoliform

answer

growth is "colorless" on EMB

question

hektoen enteric agar

answer

question

-inhibited by bile and/or one of the dyes included
-gram positive

answer

poor growth or no growth on hektoen

question

-not inhibited by bile or any of the dyes included
-gram negative

answer

good growth on hektoen

question

-produces acid from lactose, sucrose, and/or salicin fermentation
-not shigella or salmonella

answer

pink to orange growth on hektoen

question

-does not ferment lactose, sucrose, or salicin, but reduces sulfur to hydrogen sulfide
-probable salmonella

answer

blue-green growth with black precipitate on hektoen

question

-does not ferment lactose, sucrose, or salicin, or reduce sulfure
-probable shigella or rarely salmonella

answer

blue-green growth without black precipitate on hektoen

question

decontamination

answer

•reduce number of pathogens to level in which items are safe to handle without protective attire.
•Physical cleaning, removal of organic material, etc.

question

disinfection

answer

Destruction of pathogens; Liquid chemical agents, dry heat, moist heat, UV light, antiseptics (used on tissues)

question

sterilization

answer

Complete elimination of viable microbes including spores, May include chemical, gases, incineration, dry heat, moist heat, ethylene oxide gas, ionizing radiation, etc

question

answer

what factors can affect microbial death?

question

•Cell wall: Damage will leave cell susceptible to lysis
• Plasma membrane: surfactants disrupt membrane- leads to cell lysis
• Protein Structure/Function
Nucleic acid synthesis

answer

What are some of the targets for Disinfectants/Antiseptics?

question

answer

lethal effects of UV radiation

question

• DNA absorbs UV radiation & forms new covalent bonds between adjacent pyrimidines (pyrimidine dimers)
• Interferes with replication & transcription

answer

How does UV radiation damage DNA?

question

1. light repair
2. excision repair (dark repair)

answer

DNA repair mechanisms

question

light repair (photoreactivation)

answer

•Requires:
--Specific enzyme: DNA photolyase
--Visible light: 340-400 nm
•Effectively monomerizes the dimer
--Restore original double helix

question

excision repair (dark repair)

answer

•Requires these enzymes:
--Endonuclease
--Helicase
--DNA polymerase I
--DNA ligase

•Both mechanisms are limited by the amount of DNA damage resulting from longer or more intense exposures

question

mixed culture

answer

microbial culture consisting of two or more species

question

pure culture

answer

contains only one species or strain

question

streak plate

answer

a commonly used isolation technique

question

answer

process of streak plate

question

colony-forming unit (CFU)

answer

question

quadrant streak or T-streak

answer

used with samples suspected of high cell density (use inoculating loop)

question

zigzag (continuous streak)

answer

used for samples containing lower cell densities (use swab)

question

coliform

answer

normal inhabitants of the human intestinal tract; therefore, their presence in the environment may be evidence of fecal contamination

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Lab Quiz 3 – Microbio

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