Transcript chapter04
Dynamics of Prokaryotic Growth Chapter 4 4.1 Principles of Prokaryotic Growth Robert Koch (18431910) 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 time N0 = starting population of cells n = number of cell divisions Assume t = 20 min (3 per hour) 3 x 4 hours = 12 doublings If N0 = 10 then 10 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 semisolid 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 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