BIO 326R 1st Edition Lecture 4Outline of Last Lecture I. Gram positive and Gram Negative OverviewII. Gram positivea. Peptidoglycani. NAG and NAMb. Techoic acidsIII. Gram Negativea. Peptidoglycani. Inner leaflet and outerleaflet1. LPSa. Lipd A, core, o-antigenOutline of Current Lecture I. Gram Negativea. Periplasmb. Braun’s lipoproteinsc. PorinsII. Growth of Bacterial Cella. Growth mediab. Inoculumc. Binary fissionIII. Measuring GrowthThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.a. Absorptionb. Viable countsc. Direct countingCurrent LectureI. Gram Negative Continued- Periplasm= between the inner and outer membrane (15-30nm thick)o Gel-likeo Diffusion is 3 times slower than cytoplasm More viscous than cytoplasm (periplasm>cytoplasm>membrane)o Thin layer of peptidoglycan in the periplasmo Oxidizing agent Outer membrane and periplasm are oxidizing and the inner membrane is reducingo Proteins Transport, peptidoglyan, biosynthetic proteinso 20% of cell volume- Why two membranes?o Decreased permeability- If the purpose of peptidoglycan is to protect the membrane, then why in gram negative is there a second membrane over peptidoglycan if membranes are unstable and unprotected?-- Lipoproteinso Lipoproteins= covalently bonded to peptidoglycan to tether the outermembrane to the cello Braun’s Lipoprotein= 600,000 copies in an average E. coli cell ~25% of all protein in E. coli cell are lipoproteins Peptidoglycan is ~5nm from inner leaflet of the outer membrane- With two membranes how does anything permeate?o Porins= channel proteins in outermembrane that have pores for transport Trimers of a single protein Channel big enough for glucose, but too small for proteins (~600da) In gram positive, channels are much larger Specific pores in outer membrane- Allows transit by B12II. Growth of Bacterial Cell- Growth media= liquid or solid with nutrients for bacterial growtho Solid= agar (gel-like)- Inoculum= starting source of a bacterial culture- Binary Fissiono Complicated but just need to think of it simply as: One cell, cell copies DNA, cell splits in two dividing the DNA, two cellsIII. Measuring Growth- Absorption= how cloudy liquid is o More cloudy, more growtho Other names for this are turbidity and optical densityo Measured by a spectrophotometer Measures absorbance—how much light “gets through” the sampleo Problems with this No differentiation between live and dead bacteria- Still done though because it’s fast and easy- Viable counts= only count the live cellso Dilute a culture, count bacteria, dilute again, count bacteria again Calculate total number of bacteria based on dilution factoro Problem with this Slow, only works for bacteria that can grow in vitro, clumping causes inaccurate count- Direct counting= bacteria is added to microscope chambers of defined size and bacteria is countedo Clumping problem is solved but…o Problem with this No differentiation between live and
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