1/9 MIcroBiologyMicroorganisms! -Microbes used directly used by humans: dietary supplements, pesticides, bioremediation, some food (yogurt, beer, etc), pharmaceuticals! -Microbes with ecological funtions: nitrogen, sulfur, carbon, phosphorous cycles. NItrogen fixation, organic decomposition, food source for higher organisms, primary producers (photosynthesis), secondary producers! -Microbes living on/in humans: bacteria of skin, portions of digestive tract, reproductive cells, more microbial cells than human cells!, herbivorous animals! -Pathogens (disease causing microbes): Plant pathogens, animal pathogens, microbial diseases (lower respiratory infections, HIV, tuberculosis, etc. Microbiology - The study of organisms that are normally too small to be seen by the naked eye. - micro-meter scaleTypes of Microbes - Prokaryotes (no true nucleus) and eukaryotes (membrane-enclosed nucleus)*Universal tree of life, domains:bacteria, archaea, eukarya. No virus=not living particlesDefinition of Life - cells + organization, biological evolution, reproduction, homeostasis, development + growth, respond to environmentFirst Evidence of Microbes - observation under microscope, bacteria culture, Where Do Microbes Come From?Spontaneous Generation - microorganisms arise from nonliving material, or from “particles” of larger organismsTyndallization - to sterilize media by boiling requires several rounds of boilingRole of Microorganism in Disease! -not immediately obvious! -infectious disease thought to be caused by supernatural forces!Louis Pasteur! -demonstrated microorganisms carried out fermentations! -developed pasteurization! -showed the pebrine disease of silkworms was caused by protozoanKoch’s Postulates! -suspect microbe present in every case! -isolated and grown in pure culture! -pure culture able to cause disease! -suspect microbes re-isolated from hostLimitations of Koch’s Postulates! -some organisms cannot be grown in provided media, not pure culture! -using humans in completing postulates in unethical! -molecular and genetic evidence may replace this methodPure Culture Method (Koch)! -sterile surfaces (boiled potato)! -solidified nutrient media: gelatin w/ meat extracts, protein digests! ! -gelatin, degradable and low melting T (RT)! ! -agar! ! -petri dishAspects of Microbiology! -basic aspects are concerned w/ individual groups of microbes, microbial ! physiology, genetics, molecular biology and taxonomy! -applied aspects are concerned with practical problems - disease, water, food, ! and industrial microbiologyviruses=smallest(nm)Light Microscope! -bright-field! -dark-field! -phase-contrast! -fluorescence! -confocalCompound Microscope! -image formed by acrion of >2 lensesMicroscope Lenses! Magnification! ! -=objective x ocular (10x times 40x = 400x)! Resolution! ! -ability to distinguish b/w objects! Numerical Aperture! ! -light-gathering cabability of the lens!Microscope Resolution! -dres minimum distance b/w 2 objects required to resolve them (smaller dres ! ->better resolution! -lower dres -> better resolution! -lower wavelength of light source -> better resolutionOil Immersion Lenses! -if air is replaced w/ immersion oil, many light rays that did not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide will now do so. This results in and increase in resolution and numerical aperture. Working Distance - distance b/w the front surface of lens and surface of cover glass or specimen when it is in sharp focusBright-Field Microscope - produces a dark image against a bright backgroundDark-Field Microscope - image is formed by light reflected or refracted by speciment! -produces a bright image of the object against a dark background! -used to observe living, unstained preparationsPhase-Contrast Microscope! -converts slight differences in refractive index and cell density into easily detected variations in light intensity! -excellent way to observe living cells (microbial motility, detecting bacterial structures)Fluorescence Microscope ! -exposes specimen to ultraviolet, violet, or blue light! -specimens usually stained with fluorochromes, unless they have figment (chl)! -shows a bright image of object resulting from light emitted by specimenConfocal Microscopy - confocal scanning laser microscopy (CLSM) creates sharp, composite 3D image of specimentsPreparation Steps! -#1: preserves specimen - maintain cell morphology and internal structures! -#2: stain - to increase visibility of specimen, many microbes are transparentPreservation/Fixation! -Why?! -preserves internal and external structures and fixes them is position! -organisms usually killed and firmly attached to microscope slide! ! -heat fixation - routine use with bacteria and archae! ! ! -preserves overall morphology, but not internal struc. ! ! -chemical fixation - used with larger, more delicate organisms! ! ! -protects fine cellular substructure and morphologyCommon Bacterial Fixative! -formaldehyde (formalin); paraformaldehyde! -ethanolDyes! -make internal and external structures of cell more visible! -stain the subject or background! -ionizable dyes are most common! ! -basic dyes have positive charges! ! ! -methylene blue, crystal violet! ! ! -bind to negative charged molecules (eg.NA)! ! -acidic dyes have negative charges! ! ! -eosin, rose bengal! ! ! -bind to positive charged moleculesDifferential Staining! -multiple dyes are used to stain different features of microbes! -divides microorganisms into groups based on their staining properties! ! -e.g. gram stain! ! -e.g. acid-fast stain! -commonly used to detect presence or absence of structures! ! -endospores, flagella, capsules!Gram Staining! -most widely used differential staining procedure! -divides bacteria into 2 groups, gram positive (G+) and gram negative (G-), based on differences in cell wall structure! -peptidoclycanAcid-Fast Staining! -particularly useful for staining members of the genus Mycobacterium! -high lipid content in cell walls (mycolic acid) is responsible for their staining characteristicsElectron Microscope! -electrons replace light as the illuminating beam! -wavelength of electron beam is much shorter than light, resulting in much higher ! resolution! ! -1000x better resolution ! -allows for study of microbial morphology in greater detail! ! -internal structuresBacterial and Archaea Structure and Function! -prokaryotes differ from eukaryotes in size and simplicity! ! -most lack internal