Flagella stains for the light microscope:Basically staining flagella is a two step process:1) Coat the flagella with something that sticksto proteins (e.g. tannic acid)2) Stain the whole organisms with a generalstain (e.g. pararosaniline or basic fuchsin)While we’re on staining, someone asked about therole of iodine in the Gram stain. I said it is amordant (=a chemical that helps fix dye on orin a cell), which is true: the iodine reacts withthe crystal violet making a larger compoundthat cannot escape through the pores in thepeptidoglycan layer of Gram+ Bacteria …Also, the decolorization step is thought to shrinkthe pores in the peptidoglycan layer thus furtherpreventing the escape of the dye in Gram+Bacteria.LECTURE 4 (cont.)GROWTHOne microbial cell can't make a difference - takesmillions of cells per ml or cc to cause any sort ofeffect. One cell of the most virulent microbe onthe planet cannot hurt you…….Growth of single-celled prokaryotes.growth and reproduction - Bacteria basicallyclone themselves when they reproduce.Binary fission (Fig. 4.3, Fig. 7.4).Budding off of smaller cells (yeast or Hyphomicrobium )(Fig. 11.24, 12.14).Fig. 12.14.Budding yeast1. Cell wall bulges out2 & 3. Nucleus divides by mitosis4. One nucleus migrates to bud5. Bud cell wall forms and bud breaks offFilamentous bacteria and fungi grow via extension of the hyphal tip (Fig. 12.15)In fungi growth is supplied by vesiclesFungi and actinomycetes also reproduce viaspores= hyphal growth followed by fragmentation inActinomycetesMICROBIAL Population GROWTHMicrobial populations grow exponentially when supplied with enough nutrients…….The time it takes to divide is called the generation time (g)Exponential growth of bacteria….EQUATION FOR EXPONENTIAL GROWTH....Bt = Bo x 2n where Bo is the initial cell number, Bt is the final cell number and n is the number of generations. Taking the log of both sides and solving for n we get....n = (ln Bt - ln Bo) / ln 2n = (ln Bt - ln Bo) / ln 2 Bo = 2 x 107 cells per ml. after 2 hours, Bt = 3.2 x 108 cells per ml. n = (19.6 – 16.8) / 0.693 = 4 generationsWhat is the generation time? (n= t/g)By rearrangement:g = ln2t / (ln Bt - ln Bo)Example Calculation:g = 0.693*2 / (19.6-16.8)= 0.5 hoursConverting g to a growth rate:We can relate G to growth rate (µ) via µ = ln2 / gwhere µ is the slope of a semi-log growth curve.One E. coli cell (g = 0.35 hr. or 20 minutes) would grow to be a mass 2000 x that of earth if it could grow unchecked for 48 hours!So why don't bacteria take over the world???Factors affecting Microbial Growth• temperature• pH• oxygen concentration• pressure• moreIn nature competition for nutrients (and space), predation and parasitism limit the exponential growth of microbes.pHFig. 4.4Fig. 4.17End of Log phase is brought about by:Exhaustion of limiting nutrientorBuild up of toxins (e.g. alcohol in yeast cultures)Primary Metabolism = relates to the growth phase Fig. 4.18Secondary Metabolism = metabolic pathways that are not involved in growth of cells take place when cells have stopped growing. e.g. production of antibiotics by bacteria and fungi. Figure 4.18 primary and secondary metabolic products (metabolites).e.g. Production of Bacitracin by Bacillus licheniformis prior to endospore formation.How do you keep a culture going?Continuous culture
View Full Document
Unlocking...