1 9 MIcroBiology Microorganisms Microbes with ecological funtions nitrogen sulfur carbon phosphorous cycles Microbes used directly used by humans dietary supplements pesticides bioremediation some food yogurt beer etc pharmaceuticals NItrogen xation organic decomposition food source for higher organisms primary producers photosynthesis secondary producers reproductive cells more microbial cells than human cells herbivorous animals microbial diseases lower respiratory infections HIV tuberculosis etc Microbes living on in humans bacteria of skin portions of digestive tract Pathogens disease causing microbes Plant pathogens animal pathogens Microbiology The study of organisms that are normally too small to be seen by the naked eye micro meter scale Types of Microbes Prokaryotes no true nucleus and eukaryotes membrane enclosed nucleus Universal tree of life domains bacteria archaea eukarya No virus not living particles De nition of Life cells organization biological evolution reproduction homeostasis development growth respond to environment First 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 organisms Tyndallization to sterilize media by boiling requires several rounds of boiling not immediately obvious infectious disease thought to be caused by supernatural forces demonstrated microorganisms carried out fermentations developed pasteurization showed the pebrine disease of silkworms was caused by protozoan Role of Microorganism in Disease Louis Pasteur Koch 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 host Limitations 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 method Pure Culture Method Koch sterile surfaces boiled potato solidi ed nutrient media gelatin w meat extracts protein digests gelatin degradable and low melting T RT agar petri dish Aspects 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 microbiology viruses smallest nm Light Microscope bright eld dark eld phase contrast uorescence confocal Compound Microscope Microscope Lenses Magni cation Resolution Numerical Aperture Microscope Resolution image formed by acrion of 2 lenses objective x ocular 10x times 40x 400x ability to distinguish b w objects light gathering cabability of the lens 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 resolution Oil Immersion Lenses if air is replaced w immersion oil many light rays that did not enter the objective due to re ection 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 focus Bright Field Microscope produces a dark image against a bright background Dark Field Microscope image is formed by light re ected or refracted by speciment produces a bright image of the object against a dark background used to observe living unstained preparations Phase Contrast Microscope detected variations in light intensity structures converts slight differences in refractive index and cell density into easily excellent way to observe living cells microbial motility detecting bacterial Fluorescence Microscope exposes specimen to ultraviolet violet or blue light specimens usually stained with uorochromes unless they have gment chl shows a bright image of object resulting from light emitted by specimen Confocal Microscopy confocal scanning laser microscopy CLSM creates sharp composite 3D image of speciments Preparation Steps 1 preserves specimen maintain cell morphology and internal structures 2 stain to increase visibility of specimen many microbes are transparent Preservation Fixation Why preserves internal and external structures and xes them is position organisms usually killed and rmly attached to microscope slide heat xation routine use with bacteria and archae chemical xation used with larger more delicate organisms preserves overall morphology but not internal struc protects ne cellular substructure and morphology Common Bacterial Fixative formaldehyde formalin paraformaldehyde ethanol Dyes make internal and external structures of cell more visible stain the subject or background ionizable dyes are most common basic dyes have positive charges acidic dyes have negative charges methylene blue crystal violet bind to negative charged molecules eg NA eosin rose bengal bind to positive charged molecules multiple dyes are used to stain different features of microbes divides microorganisms into groups based on their staining properties commonly used to detect presence or absence of structures e g gram stain e g acid fast stain endospores agella capsules Differential Staining Gram Staining based on differences in cell wall structure peptidoclycan most widely used differential staining procedure divides bacteria into 2 groups gram positive G and gram negative G Acid Fast Staining characteristics particularly useful for staining members of the genus Mycobacterium high lipid content in cell walls mycolic acid is responsible for their staining Electron Microscope electrons replace light as the illuminating beam wavelength of electron beam is much shorter than light resulting in much higher resolution allows for study of microbial morphology in greater detail 1000x better resolution internal structures Bacterial and Archaea Structure and Function most lack internal membrane systems term prokaryotes is becoming blurred prokaryotes differ from eukaryotes in size and simplicity prokaryotes are divided into 2 taxa bacteria and archaea similar shape and size different cell organization Shape cocci and rods are most common vibrios resemble rods comma shaped helices spirilla rigid helices spirochetes exible helices Arrangement determined by plane of division determined by separation or not Other shapes and Arrangement mycelium network of