Nutrition and Growth

What do cells need to grow?

nutrients, macronutrients, micronutrients

Nutrients

supply of elements required by cells for growth

Macronutrients

nutrients required in large amounts (grams)

Micronutrients

nutrients required in minute amounts (mg or microg); trace metals and growth factors (vitamins)

Macromolcules make up

~96% of dry weight; mostly water and organic materials

Macromolecules

proteins, lipids, polysaccharides, lipopolysaccharides, nucleic acids; mostly proteins and DNA, not DNA

What elements make up ~96% of dry weight of bacterial cell and are required by all life

C, O, N, H, P, S

What elements make up ~3.7% of dry weight?

K, Na, Ca, Mg, Cl, Fe (coenzymes)

Heterotrophs

require organic carbon as a source of carbon and energy; obtain C, H, O from breakdown of organic polymers or uptake of monomers (amino acids, fatty acids, organic acids, sugars, nitrogen bases, other organics); anabolic; aka organotroph

Autotrophs

synthesize organics from carbon dioxide (C source); source of energy can be light or inorganic molecules; make H/organic compounds from inorganics (H₂O, H₂S, CO₂)

Nitrogen

key element in proteins, nucleic acids, and many more; sources → NH₃ (main one; salt, secretions), NO₃^- (some; soil), organics/amino acids (some but easiest bc already reduced), N₂ with nitrogen fixing (least; decreasing movement of molecule + from air)

Phosphorus

present in nucleic acids and phospholipids; microbes usually assimilate inorganic phosphate (PO₄^3-), organic sources; limiting so can damage when surplus is added; stored in granules when in excess

Sulfur

needed for sulfur-containing amino acids (cysteine and methionine) in proteins, some vitamins (thiamine, biotin, lipoic acid); microbes assimilate organic material containing sulfur, sulfate (SO₄^2-), sulfide (H₂S)

Potassium

required by several enzymes

Magnesium

stabilizes ribosomes, membranes, and other nucleic acids; also required by many enzymes

Calcium + Sodium

required by some (marine) microbes

Trace Metals and Growth Factors

many enzymes require metal ion or small organic as a cofactor for catalysis; Iron is used for cellular respiratoin, related oxidation-reduction reactions; trace metals are required in small amounts; growth factors are organic micronutrients like vitamins (most function as coenzymes & most needed factor), amino acids, purines, pyrimidines, other organics

Culture Media

nutrient solutiosn used to grow microbes in the laboratory; can be liquid or solid medium; typically sterilized in an autoclave; different microorganisms have vastly different nutritional requirements; necessary to understand physiology and nutritional requirements and supply with nutrients in proper form and amount

Classes of Culture Media

defined, complex, selective, differential

Defined Media

exact chemical composition known; know what microbes are capable of doing; can study how it grows with something specific; doesn’t grow as well bc makes things from scratch

Complex media

composed of digests of microbial, animal, or plant products (yeast and meat extracts → organic); ex: nutrient agar

Selective Medium

contains compounds that selectively inhibit growth of some microbes (not all growth usually) but not others; relative to microbes being compared

Differential medium

contains an indicator, usually a dye, that detects particular metabolic reactions during growth

Enrichment Culture

media composition and/or incubation conditions designed to favor a group of microorganisms or genus; can be selective, differential or both & complex or defined medium; usually not a pure culture (make it a majority); sometimes followed by secondary enrichment step; always followed by screening and isolation of organism of interest; used to isolate microbes from nature

Goal of Enrichment Culture

means of isolating rare organisms with specific characteristics from heterogeneous populations

Culture Media

liquid or solid

Solid Culture Media

prepared by addition fo the gelling agent agar to liquid media; cells form isolated masses (colonies) which can help identify microbes; routinely used to determine if culture is pure of contaminated

Colony Morphology

visible characteristics; helps identify microobes

Laboratory Culture

requires aseptic technique to transfer microorganisms without contamination; transfer cells (inoculate) from liquid to liquid, solid to solid, liquid to solid, or solid to liquid; pure cultures usually require streak plate technique to make sure there are no airborne contaminants

How do we measure population growth?

direct microscopic counts; viable/plate counts; turbidimetric method

Direct Microscopic counts

Total cell count by observing and numbering cells present; liquid samples/culture with Petroff Hausser counting chamber; usually overestimate of actual number; used on natural samples; stains visualize and provide phylogenetic info or metabolic properties; DAPI reacts with DNA; fluorescent stains differentiate live and dead cells; Phylogenetic stains can determine proportion of Bacteria or Archaea

Petroff Hausser counting chamber

counting chambers with squares etched on a slide for liquid samples; cell/mL of culture (or liquid sample)

Limits of Microscopic cell counts

cannot distinguish between live and dead cells; small cells can be overlooked; need phase-contrast microscope if no stain; cell suspensions of low density (< 10⁶ cells/mL) are hard to count; need to immobilize motile cells; debris in sample can be mistaken for cells

Viable/Plate counts

measurement of cells capable of making colonies (reproducing) on solid media; can get through spread-plate method or pour-plate method (don’t like oxygen much); count colonies with 30-300 colonies; need to dilute and plate sample; assume each colony represents one cell; underestimate of actual cell number; usually accurate iwth pure cultures that plate very well

Viable/Plate Count limitations

numbers depend on inoculum size, viability, culture medium, incubation conditions; mixed cultures grow at different rates; plating inconsistencies; reporting in colony-forming units instead of number of viable cells

Great Plate Count Anomaly

microscopic counts reveal far more organisms than on plates bc microscopic may count dead cells; viable cells may not grow on medium provided and only count cells that are reproducing, not alive

Turbidimetric Measurement

cells scatter light so cell suspensions are turbid; measured with a spectrophotometer in optical density (OD) at specified wavelengths; OD is proportionally to cell number with limits; need a standard curve to relate

Turbidimetric Measurement advantages

quick and easy to perform; does not require destruction or significant disturbance of sample; same sample can be checked repeatedly; good to optimize media

Turbimetric Measurement disadvantages

sometimes problematic if microbes form clumps or biofilms or pellicles in liquid medium; need to establish linear relationship between OD, cell number via microscopic counts or viable counts (usually both)

Binary Fission

cell division following enlargement of a cell to twice its minimum size to increase the number of cells (growth); creates a septum to divide the cel land pinch off between two daughter cells; its generation time depends on nutritional and genetic factors and temperatures; each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell

Septum

partition between dividing cells; pinches off between two daughter cells

Generation time

time required for microbial cells to double in number; depends on nutritional and genetic factors and temperature

Exponential Growth

growth of a microbial population in which cell numbers double within a specific time interval; relationship between initial number of cells (N₀) present in a culture and the number present after a period of growth (N) with n being number of generations that occurred → N = N₀2ⁿ

Quantitative aspects of microbial growth

initial increase is slow but increases, resulting in huge increase in cell numbers

Batch culture

a closed-system microbial culture of fixed volume

Microbial Growth Cycle

lag phase, exponential (log) phase, stationary phase, death phase; typical growth curve for population of cells grown in closed system

1

lag

2

exponential

3

stationary

4

death

Lag phase

interval between inoculation of a culture and beginning of growth (longer with defined/without organic materials media because making from scratch); time needed for biosynthesis of new enzymes and to produce required metabolites before growth can begin; little amoutn of cells to start;

Exponential phase

cells close to metabolically identical; steeper → doubling time is shorter; rates vary greatly, influenced by media, incubation conditions, and organism itself; used to calculate generation time; continues until conditions can no longer sustain growth

Stationary phase

growth rate of population is zero; either an essential nutrient is used up or waste products accumulate; metabolism continues at greatly reduced rate; some cells grow while others die, balancing each other

Death phase

if incubation continues after cells reach stationary phase, cells will eventually die; exponential decline; viable cells remain for months or years; cryptic growth (subpopulations adapt)

Continuous Culture

an open system microbial culture of fixed volume; usually chemostat; both growth rate and population density of culture can be controlled independently and simultaneously depending on dilution rate (Flow rate of adding fresh media & removing spent medium / culture volume) and concentration of limiting nutrient; easy contamination but waste products do not accumulate; “more” natural conditions; steady state (cell density and substrate concentration do not change over time)

chemostat

most common type of continuous culture device

Experimental Uses of Continuous Culture

can maintain exponential growth phase for weeks/months; used to study physiology, microbial ecology and evolution, enrichment and isolation of bacteria from nature; growth rate controlled by dilution rate

Budding

division from unequal cell growth and forms totally new daughter cell; usually in cultures; can have cytoplasmic extensions (stalks, hyphae, appendages); alternative to binary fission

Planktonic growth

growth as suspension

Sessile growth

attached to surface; can develop into biofilms; attached polysaccharide matrix containing embedded bacteria

Biofilms form

in stages; planktonic cells attach; sticky matrix forms

Microbial mats

multilayered sheets with different organisms in each layer

Biofilm

cells enmeshed polysaccharide matrix attached to surface; 1) planktonic cells attach (flagella, fimbriae, pili) 2) colonization (growth and extracellular polysaccharide, EPS, production) 3) development (metabolic changes) 3) dispersal (colonize new sites); can study in flow chamber; prevent harmful chemicals (antibiotics) from penetrating, prevent protists from grazing, and prevent washing away of cells; implicated in joint infections from implanted medical devices; responsible for cavities and cause gum disease; foul, plug, corrode pipes; form in fuel tanks and on ship hulls; can be differently expressed genes; does not follow typical growth cycle (usually slower)

For most physiological experiments, it is usually most desirable for cells to be in the

exponential phase

Most microorganisms assimilate nitrogen as

either ammonia or nitrate

Organisms that use reduced, preformed organic molecules as carbon sources are

heterotrophs

An estimate of the number of reproductively capable cells in a sample based on the formation of colonies on solid growth media after plating dilute solutions is called a _____ cell count

viable

Media containing some ingredients of unknown chemical composition are called ______ media.

complex

The membranes of psychrophilic bacteria have relatively high levels of ________ fatty acids, which allows them to remain semi-fluid at their cardinal temperatures

unsaturated

Addition of lysozyme to a gram-positive bacterial culture in lag phase will result in

death of the cells

Addition of penicillin to a bacterial culture in log phase will result in

less growth

Mannitol salt agar (MSA) only allows the growth of halophiles. Mannitol fermenters release acid that tuns the pH indicator yellow; mannitol non-fermenters leave the medium red. Onto MSA you inoculate a halophilic mannitol non-fermenter pigmented yellow and a mannitol fermenter. The medium is acting as a _____ medium.

selective and differential

Define Medium A as defined, complex, selective, and/or differential. Explain why.

defined; contains exact known composition & does not have any selectively inhibitors or indicators

Define Medium B as defined, complex, selective, and/or differential and explain why.

complex because contains tryptone and yeast extract which have unknown exact compositions; no indicator or anything that selectively inhibits

Define Medium C as defined, complex, selective, and/or differential and explain why.

complex & differential bc has peptone and proteose peptone which have unknown exact compositions; neutral red is an indicator and there is nothing that selectively inhibits

Define Medium D as defined, complex, selective, and/or differential and explain why.

defined and selective; has exact known composition & selectively inhibits heterotrophs bc no organic carbon source and non-sulfur oxidizers bc high sulfur environment; no indicator to be differential

What is the source(s) of 1) carbon, 2) sulfur, and 3) nitrogen for organisms growing in Medium A?

glucose; MgSO₄; NaNO₃

What is the source(s) of 1) carbon, 2) sulfur, and 3) nitrogen for organisms growing in Medium B?

tryptone, yeast extract, glucose; tryptone, yeast extract; tryptone, yeast extract

What is the source(s) of 1) carbon, 2) sulfur, 3) nitrogen for organisms growing in Medium C?

peptone, proteose peptone, glucose; peptone, proteose peptone; peptone, proteose peptone

What is the source(s) of 1) carbon, 2) sulfur, 3) nitrogen for organisms growing in medium D?

CO₂ (g); (NH₄)₂SO₄, MgSO₄, S⁰; (NH₄)₂SO₄

What physiology does an organism growing in medium A have? Explain.

autotroph/heterotroph → aerobe; organic compound (glucose) for carbon source; has all other major nutrients; exposed to oxygen (so not anaerobe)

What changes would you make to the medium A/incubate conditions to grow an autotroph? explain.

replace glucose with CO₂ to have no heterotrophs grow; increase time in incubation bc takes longer to grow when making molecules from inorganics

What changes would you make to the medium B/incubation conditions to grow an autotroph? explain

replace tryptone, yeast extract, and glucose with CO₂ bc autotrophs can use inorganic carbon sources; introduce H₂S and NaNO₃ to include nitrogen and sulfur sources; also increase incubation time because takes longer to grow from inorganic compounds

What changes would you make to the medium A/incubation conditions to grow a nitrogen fixer? explain

replace NaNO₃ with N₂(g) bc nitrogen fixers use N₂ as nitrogen source and other microbes can’t

Can an organism in medium D be a pathogen? explain

yes but pathogens would need to be autotrophic sulfur oxidizers bc high sulfur environment and no organic compounds in media

What physiology does an organism growing in medium D have? explain

aerobe sulfur-oxidizing chemolithotroph bc implied carbon source is CO₂, inorganic compound; lots of sulfur compounds which need to be oxidized to form needed growth factors

Can an organism growing in medium A be a pathogen? explain

yes because provides nutrient sources for carbon, nitrogen, oxygen, hydrogen, sulfur, and phosphorous for growth; not create a pathogen but help it grow