Chapter 6A

Direct counts

-A special slide with an etched grid can be used. -A known volume is loaded onto the grid and cells are counted under a light microscope. -Pros: Cheap, fast, easy -Cons: You can't differentiate living vs. dead cells.

Oxygen and microbial growth

-Aerobes grow in the presence of oxygen. -Obligate aerobes REQUIRE oxygen. -Microaerophiles grow best when there is less oxygen than normal. -Anaerobic growth occurs without oxygen. -Aerotolerant anaerobes aren't harmed by oxygen but don't use it, either. -Obligate anaerobes cannot grow when oxygen is present. -Facultative anaerobes CAN use oxygen but can also grow in the absence of oxygen.

What do microorganisms need to grow ? Macronutrients

-All cells need access to large amounts of carbon, nitrogen, phosphorus, sulfur, and oxygen to build macromolecules.

Solutes and water activity

-All organisms require water! (for biochemical reactions) -Water activity (aw) is the amount of water available to an organism -If you increase the solute concentration, you will lower the amount of water available (drying is a way of preserving because organisms won't be able to grow on food with my water) -fungi have the ability to grow with lower water activity (takes energy) -zerophile: organism that can grow with a little dampness (bacteria, mold) -Most organism like 0.9 water activity or better -Staph is osmotolerant which means is enjoys the low solute concentrations but can grow in high concentration as well

What else do they need to grow? Micronutrients

-Also required by microbes -Includes several metal ions (Na+, Mg2+, Mn2+, etc.) Often required for protein structure/activity, biosynthesis of ATP by electron transport-related processes

Acquisition of nutrients

-Autotrophs assimilate carbon from inorganic sources. -Heterotrophs assimilate carbon in preexisting organic form. -Ex of an inorganic course of carbon would be carbon dioxide

Media for microbial growth

-Complex media- don't know exactly what's in it: contains some sort of peptone and yeast extract (usually use if you don't know what your microbe you are trying to grow requires) -Defined media: precisely defined chemical composition (used when you know or have an idea of microbes growth requirements) -Chemical compositions of chemical broth and chemical agar are the same (only diff. is that agar was added to make it solid) -Most organisms can not digest agar so good to add to growth media

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

-Gives you a full range of nutrients -Don't know exactly what you're providing to the organism but assume it's in there

Nutrient concentration

-Growth rate will depend on the amounts of nutrients in the environment. -One key nutrient, available in the lowest amount, will dictate how much growth can occur over time (i.e., it will be a limiting factor).

All organisms require:

-Hydrogen, oxygen, and carbon -carbon is backbone of all organic components present in cell -hydrogen and oxygen are also found in organic molecules -electrons play a role in energy production used for cellular work and reduction of CO2 to form organic molecules (most energy that a cell generates if by electron transport)

Temperature and microbial growth

-Hyperthermophiles: organisms that require very hot temps. to grow (archaea) -Thermophiles: Grow anywhere above 50 C to 100 (bacteria) -Mesophiles: the middle range, about body temp. (Eukarya) -Psychrophiles: grow at very low temps.

N, P, S

-Mircroorganisms have an absolute requirement for nitrogen, phosphorus, and sulfur -They are needed for synthesis of important molecules (ex amino acids, nucleic acids) -All cells have nucleic acids (DNA, and RNA)

Micronutrients (trace elements)

-Mn, Zn, Co, Mo, Ni, and Cu; all required in trace amounts -often supplied in water or in media components -ubiquitous (everywhere) in nature -serve as part of enzymes and cofactors -Aid in catalysis of reactions and maintenance of protein structure *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 the organism will have limited growth

Growth factor: Purines/pyrimidines

-Needed for nucleic acid synthesis

Growth factor: Amino Acids

-Needed for temporary protein synthesis

Growth factors

-Organic compounds that cannot be synthesized by an organism (must be provided) but are essential for growth -Must be supplied by environment to survive/reproduce -3 major classes: Amino acids, purines and pyrimidines, and vitamins -Less common growth factors are heme and cholesterol

Electron sources

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

Phosphorus and Sulfur

-Phosphorus is needed for nucleic acids, phospholipids, nucleotides, some cofactors, some proteins, etc. -usually supplied as inorganic phosphate -most organisms use inorganic phosphorus which is directly incorporated into their cells -sulfur is needed for amino acids cysteine and methionine, some carbohydrates, biotin and thiamine -usually supplied as sulfate via assimilatory sulfate reduction -most organisms use sulfate and reduce it by assimilatory sulfate reduction

Acquisition of Energy

-Phototrophs capture light energy to produce ATP. -Chemotrophs capture energy from oxidation of reduced organic or inorganic compounds.

Spread plate to count

-Sample is serially diluted -small volume of diluted mixture containing approximately 30-300 cells is transferred -spread evenly over surface with a sterile bent rod

Growth factor: Vitamins

-Small, organic molecules that function as enzyme cofactors, needed in very small amounts

T-Streak

-Used for mixed cultures/colonies -Purpose of it it to isolate individual colonies -Becomes diluted through a series of streaking -By the 3rd streak you can see individual colonies (shapes, structures, morphology become visible)

Strict Anaerobic Microbes

-all strict anaerobic microorganisms lack or have very low quantities of superoxide dismutase catalase -these microbes cannot tolerate O2 -anaerobes must be grown without O2 -work station with incubator -gaspak anaerobic system To grow: Growth in an anaerobic culture medium containing reducing agents (thioglycollate or cysteine) -Work in an anaerobic chamber -GasPak jar -Plastic bag or pouch system

Solutes and water activity on microbial growth

-changes in osmotic concentrations in the environment may affect microbial cells -hypotonic solution (lower osmotic concentration outside); water enters the cell, cell swells may burst -hypertonic (higher osmotic concentration outside); water leaves the cell, membrane shrinks from the cell wall (plasmolysis) may occur

Measure and counting organisms methods:

-direct counts -viable (living) cell counting -turbidity (cloudiness) measurements

Adaptive microbes

-halophiles (means salt lover): grow optimally in the presence of NaCl or other salts at a concentration above about 0.2M -extreme halophiles: require salt concentrations of 2M and 6.2M; extremely high concentrations of potassium cell wall, proteins, and plasma membrane require high salt to maintain stability and activity (e.g., Halobacterium in Dead Sea)

Temperature

-microbes cannot regulate their internal temperature -enzymes have optimal temperature at which they function optimally -high temperatures may inhibit enzyme functioning and be lethal -If temp. goes to high, proteins in the cell become denatured (can't go backwards after this) -All organisms have an optimal temperature in which they grow -Going from optimum to maximum is very steep (lose structure and die very quickly) -going from optimum to minimum is more of a slope (slower) -psychrotrophs- these are organisms that do well in the refrigerator

Nitrogen

-nitrogen is needed for amino acids, purines, pyrimidines, some carbohydrates, lipids, enzyme cofactors etc. -supplied in numerous ways such as metabolism of amino acids, nitrates, ammonia, and some fix N2 from atmosphere

Diff. oxygen sensitivities

-oxygen easily reduced to toxic reactive oxygen species (ROS) -superoxide radical -hydrogen peroxide -hydroxyl radical -aerobes produce protective enzymes -superoxide dismutase (SOD) -catalase -peroxidase

pH on microbial growth

-pH affects macromolecule structures and transmembrane electrochemical gradients. -Each microbe will have an optimal pH range for growth. -Acidophiles = pH < 5.5 -Neutrophiles = pH 5.5 to 8.5 -Alkalophiles = pH > 8.5

Thermophiles

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

Why is it difficult for organisms to grow at low aw?

-requires energy to pull water away (very energy intense)

Pour plate to count

-sample is serially diluted -diluted mixture containing approximately 30-300 cells is mixed with liquid agar -mixture of cells and agar are poured into sterile culture dishes -both may be used to determine the number of viable microorganisms in an original sample

Microorganisms change nutritional type

-some organisms have great metabolic flexibility and 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) -This provides distinct advantages to organisms that experience frequent changes in environmental conditions -If organisms couldn't do this they could become extinct

Functional media

-supportive or general purpose media support the growth of many microorganisms -e.g., tryptic soy agar -enriched media general purpose media supplemented by blood or other special nutrients -e.g., blood agar -selective media- favor the growth of some microorganisms and inhibit the growth of others ex: MacConkey agar -Differential media: differentiate between different types of organism on the same plate

Is a colony formed as a result of streaking a plate always derived from a single bacterium?

No, there are other organisms that could contaminate it -Ex: staph forms clumps, so how can you be sure that there aren't other strains in that clump