Week 7 Class 17: bacterial growth, nutrition, and differeantion

For detailed laboratory studies microbes must be grown in

pure culture

pure culture

single species

bacterial culture media can be

liquid or solid

liquid bacteril cultural media allow

•  bacteria to move freely.

  • Broth 

solid bacterial culture media is useful

to seperate mixtures of diff organisms

agar

Solid media

• Bacterial cells form colonies on solid media with agar added to make a firm surface.

colony growth

Growth of bacteria on an agar plate

bacterial cell growth

Growth of bacteria in liquid media

Isolation streaking

• allows for separation of colonies into pure cultures.

  • Goal of isolation streaking is to have isolated colonies of the organism on the plate

  • Isolated colonies can be used for obtaining pure culture and colony morphology

  • why do we want pure cultures?

Pure colonies can also be isolated using the 

spread plate technique

 spread plate technique. 

• Tenfold dilutions of the original culture are spread on agar plates. 

  • Early dilutions will show confluent growth. 

• Each colony on the agar plate represents one viable organism.

• Spread plate technique allows you to roughly calculate the number of bacteria in the original, undiluted sample

• Use this technique to determine levels of bacterial contamination in food and water

• confluent growth. 

• Growth that is continuous – essentially covers entire agar

• colony forming unit (cfu)

The bacterial cells that form a colony

• viable organism.

• is one that successfully replicates to form a colony

synthetic medium

• contains only known components. 

• Starts with water, various salts, carbon, nitrogen, and energy sources that are added in precise amounts

• Can exactly determine the factors in this media type

Bacteria can be grown in a precisely defined

synthetic medium

minimal medium

•  is a synthetic medium in which the components are limited to only those nutrients the organism needs in order to grow.

Bacteria can also be grown in a nutrient-rich but less defined

• complex medium, also called rich medium.

complex medium,

• Usually contain one or more poorly defined ingredients like yeast extract of beef extract whose exact composition is unknown

  • These additives include a rich variety of amino acids, peptides, nucleosides, vitamins, and some sugars

  • provides many of the chemical building blocks that a cell would otherwise have to synthesize on its own

  • Bacteria grows fastest in complex media

Enriched media

can be used to grow fastidious, or fussy, organisms that have a very specific set of nutritional requirements beyond basic media.

Selective media

Compounds in the media prevent some types of bacteria from growing, favoring the growth of one specific type.

Differential media

Species grow equally well but compounds in the media are metabolized differently, often distinguished by a color indicator

• Some media types are both selective and differential!

• Example: MacConkey medium

  • Selective agents are bile salts and crystal violet, which prevent growth of bacteria other than Gram-negative enteric bacteria.

  • Differential agent is lactose, which some bacteria ferment (pink) and others do not.

  • This agar is helpful for diagnosing the cause of diarrheal disease

Plated (growth) methods count only live cells in the form of

colonies

Determining the total number of cells (dead or alive)

direct cell counts

inderect cell counts

direct cell counts

• Hemocytometer is a microscope slide with shallow wells and a grid sketched into the glass surface

• By adding bacteria to the well you can count the bacterial cells per grid and estimate how many bacterial cells are in the whole sample

• Indirect cell counts

• By using fluorescence-activated cell sorter (FACS) or by measuring turbidity with a spectrophotometer

Bacterial growth is measured at the

pop. level

Most bacteria reproduce by

binary fission

binary fission

•  (one parent cell divides and forms two offspring cells).

  • Binary fission may be symmetrical or asymmetrical.

• Eukaryotic microbes divide by mitosis.

 exponential growth.

Growth in which population size doubles at a fixed rate (say, every 20 minutes)

• For example two cells divide to become four cells, then four cells divide to become eight. 

generation time

In an environment with few bacteria but plenty of resources, bacteria will divide at a constant interval

• Generation time for any organism will depend on the bacterial species, type of medium, temperature, and pH

• Population of cells doubles over one generation 

• Starting with any number of organisms at time zero (N₀), the number of organisms after n generations will be N₀ × 2ⁿ.

  • One E. coli cell that divides every 20 minutes will result in 8 cells total

Phases of Growth

Bacterial growth curve, showing the change in growth rate over time

1. lag phase

2. log phase

3. stationary phase

4. death phase

lag phase

bacteeria are preparing their cell machinary for growth

log phase

growth approximates an expontetial curve (striaght line, on a logarithmic scale)

stationary phase

cells stop growing and shut down their growth machinery while turning on stress response to help retain viability

death phase

cells begin to die at an expoentaial rate

Essential nutrients

• are those compounds a microbe cannot make itself but must gather from its immediate environment if the cell is to grow and divide. 

Elements

• Carbon (C)

• Nitrogen (N)

• Phosphorus (P)

• Hydrogen (H)

• Oxygen (O)

• Sulfur (S)

• Magnesium (Mg²⁺)

• Iron (Fe²⁺)

• Potassium (K⁺)

• Trace elements such as cobalt, copper, and zinc

Some microbes require specific compounds called

• growth factors that must be added to culture media before they will grow.

Culture media provides essential nutrients for bacteria.

• Chemically defined media

• Complex media

Some microbes, in addition to the essential nutrients mentioned in the previous slides, only need a

• carbon source (like glucose) and an energy source (also glucose) to make all their proteins, nucleic acids, and cell wall and membrane components

can grow in chemically defined minimal medium

chemically defined minimal medium

chemically defined minimal medium

• Nutritional needs of some bacteria, such as Rickettsia, are unknown or are very complex, and they cannot be grown in culture!

Carbon (organic) compounds are

an essential need for all forms of life.

• (simple and complex) are used as food, which stores energy and is a source of cellular building material for making biomass.

Not all life forms use carbon in the same way

• as carbon would be trapped in an unusable form if that was the case

Autotrophs

make their own carbon compounds starting with CO₂.

Heterotrophs

• btain carbon compounds from other organisms.

  • Each group converts carbon to a form the other group can use (carbon cycle)

All bacterial pathogens

• are heterotrophs

  • During heterotrophic metabolism organic carbon sources are disassembled to generate energy and then reassembled to make cell components like proteins and carbohydrates

  • CO₂ is produced as a result of this metabolism type and released into the atmosphere

  • Autotrophs recycle this CO₂ which prevents depletion of the worlds organic carbon sources

Organisms capture energy in two basic ways

1. Extraction of energy from chemical reactions triggered by the absorption of light

2. Oxidation-reduction reactions that remove electrons from high-energy compounds (like glucose) to produce lower-energy compounds

• Light or chemical compounds may be used as a source of energy by living things.

phototrophs

chemotrophs

lithotrophs

organotrophs

Phototrophs

use light as an energy source.

Chemotrophs

• use potential energy stored in chemical compounds as an energy source.

Lithotrophs

• use inorganic chemical compounds.

  • Hydrogen, hydrogen sulfide, ammonium, etc

Organotrophs

• use organic chemical compounds.

  • Sugars/carbohydrates

Nitrogen is needed by cells to

make proteins and nucleic acids.

The nitrogen cycle converts nitrogen to various forms.

• N₂ gas (inorganic nitrogen) makes up nearly 79% of Earth’s atmosphere, but cannot be used for biosynthesis.

• Some bacteria perform nitrogen fixation to convert N₂ to ammonium ions (NH₄⁺), which is a form that can be used for biosynthesis.

• Other bacteria transform ammonia to nitrate (nitrification) and then convert nitrate to N₂ (denitrification).

Nitrogen fixing bacteria may be living in a variety of environments

• Free-living in the soil or water

• In symbiotic associations with plants or other organisms (symbiont)

Bacteria are the most

metabolically and physiologically diverse organisms on Earth!

In addition to food, bacterial growth considerations include:

• Temperature and pressure

• Osmotic balance

• pH level

Normal” physiologic conditions vary, but are generally considered a temperature between

• 20°C (68°F) and 40°C (104°F), a near-neutral pH, a salt concentration of 0.9%, and ample nutrients.

Extremophiles

•  are bacteria, archaea, and some eukaryotic microbes that can grow in extreme environments. 

  • Many extremophile evolve to survive multiple extreme environments as longs as they aren’t conflicting environment types

• Microbes can be classified by their environmental niche

Bacterial temp will match

that of their enviorment

• Temperature directly impacts every aspect of microbial physiology

• Membrane fluidity

• Nutrient transport

• DNA and RNA stability

• Enzyme structure and function

min and max temps

• that defines their limits of growth

Optimum temperature

• s the temperature that allows all of the cell’s proteins to work most efficiently as a group

Growth stops when

• rising temperatures causes critical enzymes or cell structures like the cell membrane to fail

 too cold for the organism because enzymes become too sluggish to do the processes they need to keep the cell alive

Great biological diversity exists amongst microbes as different groups have evolved to grow within

• different thermal ranges

  • Proteins have evolved to tolerate their specific temperature range

Thermophiles

• have adapted to growth at high temperature, typically 55°C (131°F) and higher.

  • Thermophiles have membranes and proteins adapted to high temperatures.  

Hyperthermophiles

• grow at temperatures as high as 121°C (250°F), which occur under extreme pressure (for example, at the ocean floor).

Psychrophiles

• re microbes that grow at temperatures as low as –10°C (14°F). 

  • Psychrophilic microbes have proteins that are more flexible than those of mesophiles.

    • Require less heat to function

Mesophiles

include the typical “lab rat” microbes, such as E. coli and Bacillus subtilis. Their growth optima range between 20°C (68°F) and 40°C (104°F)

High pressure can

• can kill many types of bacteria

  • Autoclaves use this to their advantage!

barophiles or piezophiles.

Microorganisms at locations deep within the ocean that have adapted to grow at oppressively high pressures

Barophiles require elevated pressure to grow

Microbes can only use water that

• is not bound to ions or other solutes in solution

Water availability is measured as

• water activity (a_w), a quantity approximated by concentration. 

  • Interactions with solutes (like NaCl) lower water activity

  • The more solutes there are in a water solution (i.e. the higher the salt concentration) the less water becomes available for microbes to use for growth

Osmolarity

• is a measure of the number of solute molecules in a solution and is inversely related to a_w. 

  • The more particles that are in a solution, the greater the osmolarity and the lower the water activity

  • Limiting water availability in some foods is a way to prevent bacterial growth and limit food spoilage

• halophiles.

Some species of archaea have evolved to require high concentrations of salt (NaCl);

• Some bacteria are also tolerant of different salt concentrations

  • Some of these bacteria are also pathogenic

The concentration of H+ ions has a direct effect on

The cell’s macromolecular structures.

Extreme concentrations of either acids or bases can

• limit bacterial growth unless the organism is adapted for that environment

• The intramolecular bonds that shape proteins into active forms are sensitive to proton concentrations

  • Change in proton concentration can directly change protein shape

  • Misshapen proteins exhibit lower or no activity

• All microbes have a range of pH they can survive in, as well as an ideal pH

Neutralophiles

• are bacteria that generally grow between pH 5 and pH 8; this group includes most human pathogens.

Acidophiles

• are bacteria and archaea that live in acidic environments.

Alkaliphiles

• occupy the opposite end of the pH spectrum, growing best at values ranging from pH 9 to pH 11.

Molecular oxygen (O₂) directly impacts

•  how certain microbes grow, and if they can grow in certain environments

• Every microbial species has its own molecular oxygen requirements for growth

Whether or not a microbe can use or even tolerate oxygen depends on whether oxygen is the terminal electron acceptor following the

electron trasnsport chain

• Strict aerobes

• Require oxygen for energy metabolism

• Successfully detoxify reactive oxygen species (ROS)

  • ROS are oxygen molecules or ions with one too many or too few electrons

• Survive only in environments with oxygen

• Oxygen is a terminal electron acceptor

• Strict anaerobes

• Do not require oxygen for energy metabolism

• Generally unable to detoxify ROS, making oxygen toxic

• Survive only in environments without oxygen

• Do not use oxygen as its electron acceptor