Dynamics of Prokaryotic Growth

Chapter 4

4.1 Principles of Prokaryotic Growth

Robert Koch (1843-1910)

Developed the strategies for cultivating bacteria

Defined growth requirements

Media formulations

First to use agar for semisolid media

Bacterial replication

Binary fission

Doubling time varies by species and conditions

Growth can be calculated

Nt = N0 x 2n

Nt = total cells in a given timeN0 = starting population of cellsn = number of cell divisions

Assume t = 20 min (3 per hour)3 x 4 hours = 12 doublings

If N0 = 10 then10 x 212 = 40,960 bacteria

4.2 Bacterial Growth in Nature

Biofilms

Bacteria in nature tend to attach to solid surfaces

These bacteria encase themselves in polysaccharide coatings to form communities

Collectively, these communities are referred to as biofilms

Biofilm communities are highly organized

Intracellular communication

Channels

Cellular movement within the biofilm (usually mediated by pili)

Biofilm Activities

Ear infections

Dental decay

Bioremediation

Sewage treatment

Toxic waste sites

Heavy metals

Intracellular “warfare” (i.e., competition)

4.3 Obtaining a Pure Culture

Isolation of pure cultures is mandatory for studying bacteria

This is usually accomplished using semi-solid media composed with agar

Agar is a polysaccharide obtained from marine algae

It melts at about 95° C and remains liquid to 45° C

Media are made with agar and usually sterilized in an autoclave

Media are cooled to 60° C, then dispensed into Petri dishes or tubes

After cooling, the agar solidifies, providing a semi-solid surface

The streak plate method for obtaining a pure culture

Agar media in Petri dish

Collect a broth culture with a sterile loop

Streak the plate to deposit individual bacteria at sites on the plate

Incubate for 24-48 hr

Single bacterium grows to millions, forming a colony on the plate

Maintaining a Stock Culture

Once a colony is obtained it is considered pure

This colony can be picked and inoculated into another tube or plate (subculturing) to provide a stock of the purified culture for short-term use (weeks)

Long-term storage (years)

This purified culture can also be grown in broth and lyophilized (freeze-dried)

It can also be diluted 1:2 in glycerol and frozen at -70° C

4.4 Bacterial Growth in Laboratory Conditions

The Growth Curve

Bacteria exhibit distinct kinetic profiles of growth in the laboratory in closed cultures (systems)

These profiles generally are the same, although time-course between species can be different

Continuous (Open) Cultures

Removal of toxic metabolites

Replenishment of medium

Conducted in culture tanks called fermenters

Internal sensors

Computer controlled

4.5 Environmental Factors that Influence Microbial Growth

4.6 Nutritional Factors that Influence Microbial Growth

Heterotrophs

Carbon source is organic carbon

Medically important bacteria

Autotrophs

Carbon source is inorganic carbon

Perform carbon fixation, the conversion of inorganic carbon into organic carbon

Photoautotrophs use photosynthesis

CO2 + H2O ➔ Glucose + O2

4.7 Cultivating Prokaryotes in the Laboratory

Complex Media

Contain a variety of biomolecule precursors

Concentrations of precursors can vary between media

Often, the source of the precursors are extracts, which are water-soluble substances

Examples

Nutrient agar

Tryptic soy agar

Blood agar

Defined Media

Known amounts of chemicals and biomolecules are formulated into the medium

More expensive than complex media

Selective Media

Generally used for the propagation of particular bacteria

MacConkey agar selects for Gram- enterics

Bismuth sulfite agar cultures Salmonella and Proteus species to the exclusion of other bacteria

Differential Media

Media that can distinguish two or more groups of bacteria in a mixed culture

Providing Appropriate Atmospheric Conditions

Increased CO2

Capnophiles (15% CO2)

Hemophilus

Neisseria

Microaerophilic - low levels of oxygen

Anaerobe - killed by prolonged exposure to oxygen