GRAM - AND GRAM + STRUCTURES Gram negative Gram positive · Has two PM layers · Outer membrane made of lipopolysaccharides (toxic to hosts) · Thin cell wall · Has one PM layer · Cell wall has teichoic acid · Thick cell wall GRAM-NEGATIVE GRAM-POSITIVE Outer membrane Peptidoglycan Cytoplasmic PROCEDURE: 1. First stain: stain both using a purple dye 2. De-stain: alcohol wash (affects gram- b/c their thin cell wall creates develops holes, causing the dye to wash out) 3. Counter stain: a pink stain is added to cells, staining the gram- which lost the primary stain INTERPRETING BACTERIAL GROWTH CURVES Growth curve BACTERIAL GROWTH CURVE Stationary Log Growth Lig # of Bacteria Death LAG · Lag: organisms reproduced in new conditions · Log: constant doubling time (nutrients/space available) · Stationary: lack of nutrients + waste build up · Death: nutrient exhaustion, waste > nutrients · Doubling time: Nt = No 2t/n · t = time of incubation, n = doubling time Time Diauxic growth Lactose used up Second lag phase Number of cells (logarithmic scale) Glucose used up Growth on lactose Growth First lag phase 1 Glucose and lactose added Growth on glucose Time Time of incubation (hr)
Growth rate: Nº = No × 2t/n NI = Final population size No = Initial population size or number of cells inoculated t = time of incubation n = doubling time MICB 211 CFU micb bacteria CFU and morph CFU morph 30 2.5 2.0 20 1.5 1.0 10 0.5 0 Thumb Index Middle Ring 0.0 Pinky Conclusions: · Index finger had the most bacteria while the pinky finger had the least Amount of bacteria mostly decreased with each finger + so did morph but less so PREDICTING GENE REGULATION MODELS · Regulatory proteins: transcription factors o High concentrations: substrates always bind Low concentrations: substrates never bind ? Activators: bind at low concentrations ? operator is BEFORE promoter o Repressors: bind at high concentrations ? operator is AFTER promoter
Substrate Concentration Substrate binds to protein? (ex: ArgR) Complex binds to the operator? Genes expressed? (ex: argC) Activator High Yes No No Low No Yes Yes Repressor High Yes Yes No Low No No Yes Bacteria's sugar preference · Glucose = one of the most preferred energy sources o Consumed first, then the secondary sugar · When glucose is present, cell stops transport of other sugars + metabolizes preferred sugar LAC OPERONS · Low glucose: CAMP made => attaches to CAP => binds to DNA => RNA Pol binds => high transcription of lac operon O (no glucose, yes CAP) o CAMP promotes lactose metabolism when there is no glucose . High glucose: no cAMP => doesn't bind to DNA => basal transcription ? (yes glucose, no CAP) o CAMP does not promote lactose metabolism when there is glucose Sugar: Bound to the promoter? LacI (repressor) CAP (activator) Expression of lactose metabolism genes Glucose only Yes No No Lactose only No Yes Yes Glucose and lactose No No Basal Neither Yes Yes No