Microbial growth

Question 1

What do all living things need to grow?

Answer 1

sources of carbon and energy

• over eons bacteria has evolved
• ingenious strategies to find, acquire, and metabolize a wide assortment of food sources.
• This owes to the remarkable plasticity of microbial genomes.

Question 2

what allows us to control the growth of bacteria

Answer 2

• Understanding how bacteria use food to increase cell mass and, ultimately, cell number
• allows us to manipulate them to make useful products (find optimal conditions and nutrients
  note: growth = inc in both cell mass (biomass need biochemical techniques to measure) and cell numbers

Question 3

Answer 3

• microbes evolved to grow in diff environments so they have diff nutritional requirements dependent on ecological niche
• not every microbe sues every element

Question 4

what are the essential nutrients that must be supplied by the environemnt for microbes

Answer 4

Macronutrients need mg to g quantities

• Major elements in cell macromolecules: C, O, H, N, P, S (remember)
• Cations necessary for protein function: Mg2+, Ca2+, Fe2+, K+
  
  • protein folding or catalysis

Micronutrients (mg to microgram quantities some nanogram)

• trace elements required for enzyme function: Co, Cu, Mn, Zn

*when want to grow bacteria need to supply these, and consider in what form they use it, do they derive carbon from other organic molecules? do they fix CO2?

Question 5

How do microbes get carbon to build biomass

Answer 5

All earths life forms are based on carbon which they squire diff ways

• autotrophs (primary producers, make inorganic carbon available for other organisms)
• fix CO2 and assemble into organic molecules (mainly sugars)
• ex: assimilation/fixing CO2, and inorganic C-source
• plants, algae, autotrophic bacteria

Heterotrophs

• use organic molecules made by autotrophs, use to make their own biomolecules and generate energy

Question 6

How do mirobes obtain energy

Answer 6

• all organisms require an energy source

phototrophs obtain energy from chemical reactions triggered by light

• make ATP

Chemotrophs obtain energy from ox-red rxns

• energy generation involves transfer of electrons from e- donor to e- acceptor with release of energy (respiration involved, ETC)
• chemolithotrophs use inorganic mol as a source of electrons
  
  • only found in bacteria
• chemoorganotrophs obtain electrons from organic molecules

Question 7

what is the role of nitrogen in life

Answer 7

• N2 makes up approx 79% of eather atmosphere but unavailable for use by most organisms
• bacteria play key role in global nitrogen cycle
• nitrogen fixers (Diazotrophs) possess nitrogenase enzyme which converts N2 to ammonium ions NH₄⁺
• only a few bacteria can fix N2, ex = Rhizobium, root endosymbionts of leguminous plants

Question 8

what is the most basic consideration when trying to culture an organism

Answer 8

1. How the cell generates energy: Phototrophy or Lithotrophy
2. The form of Carbon utilized by the cell: Heterotrophy or autotrophy

(many autotrophs inhibited by the presence of organic molecules)

3. The form of nitrogen used: Diazotrophy

*figure compares heterotrophs and autotrophs

Question 9

what conditions and considerations are required for measuign growth in the lab

Answer 9

• optimal nutritional and environmental conditions

*assumes good understanding of the growth requirements (nutritional energy, physical, chemical)

Note: less than 1% of organisms can be grown in pure culture, so instead grow mixed populations (real life they exist in complex multispecies communities)

• consider best case scenario (bioremediation agents, microbial production of drugs, enzymes, recombinant proteins)
• consider the worst case scenario (pathogens, food spoiling agents, biofouling)

Question 10

what are teh different types of bacterial culture media

Answer 10

Complex / rich media are nutrient rich; the exact chemical composition is poorly defined. proves macteria with all chem that were in the previous species

• E.g. contain extracts and ‘enzymatic digests’ of yeast cell, plant and animal tissues;

Minimal defined media contain only those nutrients that are essential for growth of a given microbe.

• Exact components, their concentrations and chemical compositions are known

Enriched media are complex media to which specific growth factors or organic components are added; the bacterium is not capable of making them but needs them to grow.

• E.g. blood proteins, nucleotides, vitamins etc.,

Selective media favor the growth of one organism over another.

• E.g. high or low pH, plus antimicrobials, specific compounds or nutrients (cellulose, benzene)
• checking to see if the bacteria has capacity to grow under these condition

Differential media exploit biochemical/physiological differences between two species that grow equally well (in the medium)

• E.g. contain a pH dye that changes in colour when bacteria produce acidic or alkaline byproducts; differente bacteria based on their metabolic products/activities

Question 11

how do microbes grow

Answer 11

• e. coli requires rich media and thrive but struggle in situation where nutrients are scarce
• bacteria in most soil and water ecosystem are oligotrophs

*have optimal rate of growth at extreamly low nutrient conc, would die in rich media

• how oligotrophs thrive under extreamely low nutrient conditions remains a mystery

Question 12

what are the two main forms of culture media

Answer 12

1. Liquid or borth: cells gow in suspension

• maximize the number of cells, then centrifuge it to get biomass of cells and then cell free media with all products secreted by the cells
• allows you to study growth characteristics of pur culture

2. Solid: usually gelled with agar
   * growth in colony forming units (CFU), express number of bacteria in terms of CFU (each colony assumed to be from 1 bacteria)
   applications: trying to separate bacteria in samples, isolation of a pure culture, studying diversity in a sample, specific traits (ex: morphologies, physiology, growth, genetics, antibiotic production etc)

Question 13

what are the main techinques for isolating pure culture

Answer 13

• dilution streaking and spread plating
• allow to obtain well isolated single colonies which can be used to establish pure colonies

assume that one cell = one colony (false assumtion bc some bacteria live/occur in pairs or multiples and cannot be sep by these tehcniques)

Question 14

describe dilution streaking

Answer 14

• use a loop, pick up tiny amount of sample (loop is calibrated for this)
• drag the loop across the surface on agar plate
• flame loop to sterilize/kill bacteria
• let cool
• touch end of loop on last streak picking up some bacteria and repeat streaking
  results: filution of sample with each streaking set increases the probabiltiy of obtaining separation of single bacterium

Question 15

describe spread plates

Answer 15

• set up ten fold serial dilutions in liquid culture medium
• a small amount of each dilution is then plated onto agar
  goal: obtain a dilution that gives nicely separated colonies, select plates woth 30-300 colony forming units
• calculate numbers of bacteria in original sample

Question 16

what percent of prokaryotes are uncultured

Answer 16

>99%

• many species have adapted so well to natural habitates that still do not know how to grow in the lab
• some of these uncultured organisms depend on factors provided by other species that cohabit their niche
• others evolved to live inside other cells (intracellular parasites)
• pthers are obligately symbiotic and cannot grow/survive separated from partners

Question 17

how do most bacteria devide

Answer 17

• binart fission where a parents increases in size/volume and biomass then splits into two equal daughter cells

*pop doubles after each generation time

*chromosomes replicated as well

• note some cells have mother cell producing progeny that are smaller
• the time it takes for 1 cell to double in size and produce progeny = generation time

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Question 19

What is generation time

Answer 19

• in a favourable env with unlimited appropriate resouces and environmental conditions bacteria divide at a constant internal (ie generation time or doubling time)
• generatim time is the time it takes for the population to double

for cells undergoing binary fission: N_t = N₀ x 2ⁿ

where N_t is final cell number

N₀ is original cell number

_n is number of generations

Question 20

what is the bacteria with the fastest generation time? what is the abcteria with the slowest generation time?

Answer 20

fastest = clostridium perfringes (10 min)

slowest - mycobacterium leprae (14 days)

Question 22

what is batch culture

Answer 22

• volume of liquid emdium within a closed sytem

0 media and nutrients not refreshed

• growth in batch culture is the simplest way to model the effects of changing environemnt of growth on population culture

4 phases: lag, exponential/logarithmic, stationary, death

• changing conditions in system greatly affects bacterial physciology and growth
• nutrients gradually used up and toxic waste gradually builds
• shows ability of bacteria to adapt to chainging env

Question 23

describe the lag phase

Answer 23

• cells in inoculated sampe need time to sense and adjust to environemnt
• lag will be diff depending on the state on the inoculum
• in thi time just getting used to environment, might need time to repair

* can use spectrophotonomer to see if getting more cloudy (bateria growing)

Question 24

describe the exponential/ log growth

Answer 24

• growth rate (rate of inc in biomass) is proportional to the population size at ta given time
• such a growth rate = exponential bc generates exponential curve
• if cell divdes by binary fission the number of cells is proportional to 2ⁿ
• if gorwing quickly steeper slope

Question 25

what are the 2 phases of the exponential phase

Answer 25

- early and the late - early: cells growing at maximum rate (physiologically) possible based on conditions available \*cells ar elargest at this growth phase, have highest biomass/cell \*phase where you would want to study the cells \*may be influences by quorum sensing - late: slowing growth rate due to cell density, competition for nutrients

Question 26

describe the stationary phase

Answer 26

- cell numbers stop rising, lack of **key nutrient** (maybe phosphate), waste builds up, death = growth - to survive, cells adjust/adapt - beocme smaller- less biomass/cell (relative to the log phase) - produce **stress adaptive** response proteins - some gram-positive bacteria may sporulate (spores can survive adverse conditions), gram neg cant sporulate just small \*some bacteria produce antibiotics and noxious compounds during this phase, allows them to compete by killing others to release nutrients to keep them alive

Question 27

describe the death phase

Answer 27

- without any new nutrients cells will eventually die off - death rate is also logarithmic, but usually takes longer than the exponential phase and can be hard to predict \* as cells die others can use the nutrients they release to grow \*understanding death pahse is critical for determining food preservation and antibiotic development: food preservation- does it kill all bacteria? how fast can we will all bacteria? - if trying to maximize production how can we slow death phase to maximize production?

Question 28

compare complex and synthetic media

Answer 28

- often culture baceria in complex medium- nutrient rich with poorly defined components \*usually has yeast/beef extracts additives like amino acids, peptides, vitamins, sugars etc. - some are fastidious (particular) & require blood to be assed to basic complex medium - now called **enriched medium** - a defined selective medium where all chem components are known (like minimal media) will have slower growth bc bacteria need to synthesize more components

Question 29

why is blood added to complex media to make enriched media?

Answer 29

- require blood to reclaim the heme released from the rbc as their own - use heme as a enzyme prostheric group for enzyme function - saves a lot of energy so they can grow faster

Question 30

compare selective and differential media

Answer 30

- differenecs wrt metabolic capabilities & resistance to toxic agents is exploited in **selective media** which favourite growth of one organism over another ex: solid medium w/ bile salts and crystal violet is selective bc favours growth of gram-neg bc outer membrane is more resistant than gram pos - **differental media** expose biochemical differences between two species that grow equally well ex: E. coli and Salmonella enterica both gram neg, if grow both on solid media w/ nonfermantable carbon source, a dye and lactose only e.coli can perment lactose - produces acidic by products, lowers pH and indicated on dye (e.coli changes colour while other stays natural colour)