MCB 2610 Exam 2

What do microorganisms generally need to grow? (nutritional requirements)

macronutrients (carbon, nitrogen, phosphorus, sulfur, oxygen, and hydrogen; potassium, sodium, magnesium, calcium and iron) and micronutrients - trace elements (Mn, Zn, Co, Mo, Ni and Cu)

What are micronutrients used for in microorganisms?

Often required for protein structure/activity, biosynthesis of ATP by electron transport-related processes

How do micronutrients get in the body? How much of them do we need? Where are they found?

Sort of get in there naturally
Required in trace amounts
Often supplied in water or in media components

all organisms require what?

carbon (backbone of all organic components present in cell), hydrogen and oxygen (found in organic molecules), and electrons (play role in energy production)

Where in nature are micronutrients found? What types of reactions do they help with? What else do they do?

Ubiquitous in nature
Serve as part of enzymes and cofactors
Aid in catalysis of reactions and maintenance of protein structure

What are diatoms?

Marine organism with silicic acid
Some organisms also require unique substances such as silicic acid used to construct the silica walls of diatoms

No matter what an organism's nutrient requirement, they require a balanced mix and if one nutrient is limited or in short supply.... what will happen?

No matter what an organism's nutrient requirement they require a balanced mix and if one nutrient is limited or in short supply the organism will have limited growth
(LIMITING FACTOR!!)

All organisms require WHAT?

Carbon, Hydrogen, Oxygen, and Electrons

What is backbone of all organic components present in cell?

Carbon

What do electrons do? (2)

Electrons play a role in energy production used for cellular work and reduction of CO2 to form organic molecules

Autotrophs

Autotrophs assimilate carbon from inorganic sources.

Heterotrophs

Heterotrophs assimilate carbon in preexisting organic form.

Organotrophs

Organotrophs acquire their electrons from organic molecules (e.g., glucose)

Lithotrophs

Lithotrophs ('rock-eaters') acquire their electrons from inorganic sources (e.g., H2 gas and elemental sulfur)

Phototrophs

Phototrophs capture light energy to produce ATP.

Chemotrophs

Chemotrophs use chemicals as energy source

If an organism used chemical energy, an organic electron source, and an organic carbon source, they would be referred to as...

Chemoorganoheterotroph

If an organism used light energy, an inorganic electron source, and an inorganic carbon source, they would be referred to as...

Photolithoautotroph

To what major nutritional group do human microbial pathogens belong?

Chemoorganoheterotroph!! Provides info of how we can grow it
Clinical lab, illness, trying to grow it - have to know needs of that organism to grow it

What is metabolic flexibility? What is one organism with this ability?

Organisms with metabolic flexibility can alter their metabolism in response to environmental changes (e.g., when oxygen is limiting they can switch their metabolism to use light as a source of energy)
E. coli - facultative anaerobe, prefers presence of oxygen, but if oxygen is limited, can switch to fermentation

Microorganisms also have an absolute requirement for 3 elements...
These elements are needed for synthesis of important molecules (e.g., amino acids, nucleic acids)

Nitrogen, Phosphorus, and Sulfur

What is nitrogen needed for?

Nitrogen is needed for amino acids, purines, pyrimidines, some carbohydrates, lipids, enzyme cofactors etc.

How is nitrogen supplied?

Supplied in numerous ways such as metabolism of amino acids, nitrates, ammonia, and some fix N2 from atmosphere

What is phosphorus needed for in microorganisms?

Phosphorus is needed for nucleic acids, phospholipids, nucleotides, some cofactors, some proteins, etc.

How is phosphorus usually supplied?

Usually supplied as inorganic phosphate, which gets directly incorporating into cells

What is sulfur needed for?

Sulfur is needed for amino acids cysteine and methionine, some carbohydrates, biotin and thiamine

How is sulfur usually supplied?

Usually supplied as sulfate via assimilatory sulfate reduction

What are growth factors?

Growth factors are organic compounds that cannot be synthesized by an organism but are essential for growth
(Some can synthesize from single carbon and organic salts... others require growth factors)

What is a growth factor requirement?

Something than an organism needs that it cannot make

E. coli has a mutation that makes it unable to make amino acids. How can we help it grow?

Add growth medium with growth factors

What are the 3 major classes of growth factors?

Amino acids, purines and pyramidines, and vitamins

What are amino acids needed for? Why do you need to include all 20 as a growth factor?

Needed for protein synthesis

Can't make 1 or the 20 amino acids, can't make proteins!!

Why are purines and pyramidines needed as growth factors?

Needed for nucleic acid synthesis
DNA and RNA

Why are vitamins needed as a growth factor?

Vitamins are small organic molecules that function as enzyme cofactors, needed in very small amounts

2 less common growth factors

Heme (cytochromes, Haemophilus influenzae) and cholesterol (required by some mycoplasmas)

Why is it so difficult to grow some organisms in a laboratory culture?

Have to meet growth/nutritional requirements but you might not know what those requirements are!!

What are 3 factors that affect microbial growth?

Nutrient concentration, effect of oxygen, and temperature

If on key nutrient, available in the lowest amount, dictates how much growth can occur over time, it is referred to as a ______________.

Limiting factor

How does a higher or lower nutrient concentration affect growth rate?

Higher nutrient concentration = faster growth and make more cells
Lower nutrient concentration = slower growth rate and make less cells

What is the relationship between obligate aerobes and oxygen?
What enzyme content do they have?
Where is it found growing in a broth culture tube?

Obligate aerobes REQUIRE oxygen.
SOD + Catalase
Top in oxic zone, Wouldn't grow in anoxic zone

When do microaerophiles grow best?
What enzyme content do they have?
Where is it found growing in a broth culture tube?

Microaerophiles grow best when there is less oxygen than normal.
SOD, +/- Catalase (low levels)
Right below top of test tube

What is the relationship between aerotolerant anaerobes and oxygen?
What enzyme content do they have?
Where is it found growing in a broth culture tube?

Aerotolerant anaerobes aren't harmed by oxygen but don't use it, either.
SOD, but NO Catalase
Grows through whole tube

What is the relationship between obligate anaerobes and oxygen?
What enzyme content do they have?
Where is it found growing in a broth culture tube?

Obligate anaerobes cannot grow when oxygen is present.
No SOD, No Catalase
In anoxic zone (bottom)

What is the relationship between facultative anaerobes and oxygen?
What enzyme content do they have?
Where is it found growing in a broth culture tube?

Facultative anaerobes CAN use oxygen but can also grow in the absence of oxygen.
SOD + Catalase
Produces more ATP in oxic zone

In a test tube of liquid medium, where is the anoxic zone?

Bottom
Oxic zone is on top since oxygen isn't very soluble in liquid

How do we determine effects of oxygen on microbial growth? (its toxicity)

Often determined by what defenses are available against oxygen's negative effects in the cell
We have mechanisms to detoxify oxygen

What is oxygen easily reduced to toxic reactive oxygen species (ROS)?

superoxide radical (O2-)
hydrogen peroxide (H2O2)
hydroxyl radical (OH)

Aerobes produce protective enzymes against toxic oxygen which are..... (3)

Superoxide dismutase (SOD)
Catalase
Peroxidase

What does superoxide dismutase (SOD) detoxify?

Superoxide anion (O2-)

What does catalase and peroxidase detoxify?

Hydrogen peroxide (H2O2)

All strict anaerobic microorganisms lack or have very low quantities of....
Why?

Superoxide dismutase (SOD) and catalase
Because the can't tolerate O2 / can't detoxify

How do you grow strict anaerobes? (3 method)
What do you need to include?

Growth in an anaerobic culture medium containing reducing agents (thioglycollate or cysteine)
Work in an anaerobic chamber
GasPak jar
Plastic bag or pouch system

What is a GasPak system used for? How does it work?

Anaerobic environment
Put petri dish in
Add water, H gas and CO2
Indicator strip changes color to indicate anaerobic environment
Convert hydrogen and oxygen to water
Used when organisms are not as sensitive

What is the optimal pH ranger for acidophiles growth? What's an example?

pH < 5.5
Fungi
On orange in fridge lol

What is the optimal pH ranger for neutrophiles growth? What's an example?

pH 5.5 to 8.5
E. coli

What is the optimal pH ranger for alkalophiles growth? What's an example?

pH > 8.5

How do hypotonic environments affect microbial cells?

Lower osmotic concentration outside
water enters the cell
cell swells may burst

How do hypertonic environments affect microbial cells?

Higher osmotic concentration outside
Water leaves the cell
Membrane shrinks from the cell wall (plasmolysis) may occur

Halophiles

Grow optimally in the presence of NaCl or other salts at a concentration above about 0.2M
Phile = low, halo = salt

Extreme halophile

Require salt concentrations of 2M and 6.2M
Require extremely high concentrations of potassium
VERY SPECIFIC
Cell wall, proteins, and plasma membrane require high salt to maintain stability and activity (e.g., Halobacterium in Dead Sea)

Halotolerant

Can tolerant higher salt but does not grow optimally

Moderate halophile

Salt between 0.4M and 3.4m

What is water activity (aw)? What is it dependent on and how is it reduced?

Amount of water available to organisms
(NOT amount in the medium)
Dependent on solute concentration
Reduced by interaction with solute molecules (osmotic effect)

If the solute concentration is higher, how is the water activity affected?

Lower aw
Reduced by adsorption to surfaces (matric effect)
Organisms can;t pull water from here

Most bacteria require an water activity (aw) of what value?

>0.9

Osmotolerant microbe

Can grow over wide ranges of water activity (e.g., Staphylococcus aureus 3M NaCl)

Xerophile

Can grow on "dry" surfaces
Might appear wet but water is stuck

How does temperature affect microbial growth?

Affects macromolecular structure, membrane fluidity, and enzyme function
(different optimal temps for growth)
Microbes cannot regulate their internal temperature and high temperatures may inhibit enzyme functioning and be lethal

Why is it difficult for organisms to grow at low water activity (aw)?

They would have to pull water out of matrix away from solute molecules, that takes energy
Lower aw foods in fridge

Psychrophiles

0C to 20C
Pink snow, need water

Mesophiles

20C to 45C
Eukarya

Thermophiles

55C to 85C
Bacteria

Hyperthermophiles

85C to 113C
Archaea

Psychrotrophs

0C to 35C
Range of fridge

Adaptations of thermophiles (3)

1. protein structure stabilized by a variety of means
e.g., more H bonds
e.g., more proline
e.g., chaperones
2. histone-like proteins stabilize DNA
3. membrane stabilized by variety of means
e.g., more saturated, more branched and higher molecular weight lipids
e.g., ether linkages (archaeal membranes)

How is the protein structure of thermophiles stabilized?

more H bonds
more proline
chaperones

How is the membrane of thermophiles stabilized?

More saturated, more branched and higher molecular weight lipids
Ether linkages (archaeal membranes)

Culture media can be classified based on what three things?

Chemical constituents from which they are made
Physical nature
Function

What is an example of solid media?

Agar
(dehydrated powder from red algae)
Above 85C to melt

What are the 2 chemical classifications of culture media?

Complex (unknown chemical composition)
Defined/synthetic (precisely defined chemical composition)

What are the 3 physical classifications of culture media?

Liquid, semisolid, solid

What are the 4 functional classifications of culture media?

Supportive (general purpose, unsure of what organism needs)
Enriched
Selective
Differential

Examples of complex media

Nutrient broth
Tryptic soy broth
MacConkey Agar
Extract/digest
LB broth
COMPLEX IF IT HAS PEPTONE, YEAST EXTRACT, fusion

Media components include

Peptones
protein hydrolysates prepared by partial digestion of various protein sources
Extracts
aqueous extracts, usually of beef or yeast
Agar
sulfated long chain polysaccharide used to solidify liquid media; most microorganisms cannot degrade it
NOT A FOOD SOURCE
usually extracted from red algae

What is agar?

Sulfated long chain polysaccharide used to solidify liquid media; most microorganisms cannot degrade it
usually extracted from red algae

Supportive or general purpose media: function and examples

Assuming will support the growth of many microorganisms
e.g., tryptic soy agar / broth
E. coli, Salmonella

Enriched media: function and examples

General purpose media supplemented by blood or other special nutrients
e.g., blood agar or chocolate agar
Human pathogens grow better in these conditions

What is blood agar used for?

Enriched media
streptococcus media
Clearing zone = hemolyzed = broke down RBCs from sheep
Whole sheeps blood, not heated in autoclave

Selective media: function and examples

favor the growth of some microorganisms and inhibit growth of others
e.g., MacConkey agar which selects for gram-negative bacteria
Test JUST for fecal matter in water or JUST for Salmonella

Differential media: function and examples

Distinguish between different groups of microorganisms based on their biological characteristics / appearance
Blood agar: hemolytic (clearing zones) vs not
MacConkey agar: lactose fermenters vs not

What type of media is MacConkey agar? What does it consist of? What does it test for?

Selective and differential
Contains lactose, dyes (crystal violet & neutral red), and bile salts - selects for gram negative bacteria and for organisms that produce acid from lactose fermentation
Red if fermenters, white/tan if not

E. coli appears red in MacConkey agar. What does this mean?

E. coli ferments lactose to acid

Three basic methods for separating cells on a plate

Streak plate method - best for isolating pure cultures
Spread plate method - best for quantifying
Pour plate method - best for quantifying

What method for separating cells on a plate is best for isolating pure cultures? What else can you see with this method?

Streak plate method
Can see different colony morphologies
Can see colonies

One of the benefits of a solid medium is...

Cells are held in place on the surface and can be isolated.

Brilliant green agar
What organism is identified?
Is it selective or differential? How?

Salmonella
Selective for Gram-negative
Differentiates Shigella (which do not ferment) [red/white] from those that do ferment [yellow/green]

Eosin methylene blue agar
What organism is identified?
Is it selective or differential? How?

Gram-negative enterics
Selective: inhibits Gram-positive
Differential: E. coli are purple with green sheen, Enterobacter aerogenes are pink (ferment)
Colorless = don't ferment

MacConkey
What organism is identified?
Is it selective or differential? How?

Gram-negative enterics
Selective: CV and bile salts inhibit Gram-positive
Differential: lactose and pH indicator - lactorse fermenters (red), nonfermenters (white/tan) [No acid fermented!!]

Why are peptones, yeast extract and beef extract added to growth media?

Do not know growth requirements, trying to get it to grow
For complex media - general all purpose medium
First try at growing, throw in all purpose things (peptone, etc.)

What technique involves spreading a mixture of cells on an agar surface so that individual cells are well separated from each other?

Streak plate / T-streak