Microbiology, principles and explorations 7th ed j black (wiley sons, 2008) 1

78793 fendpaper.3d GGS 11/6/07 18:51 Diseases and the Organisms that Cause Them BACTERIAL DISEASES—ALSO SEE APPENDIX B Disease Organism acne actinomycosis anthrax bacterial meningitis Propionibacterium acnes Actinomyces israelii Bacillus anthracis Haemophilus influenzae Neisseria meningitidis Streptococcus pneumoniae Listeria monocytogenes bacterial vaginitis Gardnerella vaginalis botulism Clostridium botulinum brucellosis (undulant Brucella sp.{ fever, Malta fever) cat scratch fever Afipia felis, Bartonella henselae chancroid Haemophilus ducreyi cholera (Asiatic Vibrio cholerae cholera) conjunctivitis Haemophilus aegyptius dental caries Streptococcus mutans diptheria Corynebacterium diptheriae ehrlichiosis Ehrlichia sp endocarditis Enterococcus faecalis food poisoning Staphylococcus aureus Streptococcus pyogenes Clostridium perfringens Clostridium botulinum Bacillus cereus Listeria monocytogenes Campylobacter sp Shigella sp Salmonella sp Vibrio parahaemolyticus gas gangrene Clostridium perfringens and others gonorrhea Neisseria gonorrhoeae granuloma inguinale Calymmatobacterium (donovanosis) granulomatis Hansen’s disease Mycobacterium leprae (leprosy) Legionnaires’ disease Legionella (legionellosis) pneumophilia leptospirosis Leptospira interrogans listeriosis Listeria monocytogenes Lyme disease Borrelia burgdorferi lymphogranuloma Chlamydia venereum trachomatis Madura foot Actinomadura, (maduromycosis) Streptomyces, Nocardia nongonococcal Chlamydia urethritis (NGU) trachomatis Ureaplasma urealyticum Type* Page Disease R, I, R, R, 580 591 97, 724–727 755 coccoid, NA 659 C, C, 444, 756 757 R, 757 R, R, CB, 613 413, 684, 767–768 730–731 R, CB, NA R, vibrio, 597 ornithosis Chlamydia psittaci (psittacosis) Oroyo fever (Carrion’s Bartonella disease, bacilliformis bartonellosis) peptic ulcer Helicobacter pylori periodontal disease Porphyromonas gingivalis and others pharyngitis Streptococcus (strep throat) pyogenes plague (black death) Yersinia pestis bubonic plague pneumonic plague pneumonia Streptococcus pneumoniae Klebsiella pneumoniae CB, 593 C, R, 679–681 645–646 Mycoplasma pneumoniae Clostridium difficile R, NA C, C, 738 720–721 413, 684 pneumonia, atypical (walking pneumonia) pseudomembranous colitis puerperal fever (childbed fever) Q fever rat bite fever C, 720 R, 413, 684 R, R, 685 685 R, 757 R, R, R, R, R, 373, 684–685, 692 413, 687–688 334, 685–686 688 595–596 C, R, 616–620 627 R, A-F 407, 763–765 R, 653–654 S, R, 612–613 757 623 413, 688–690 S, 334, 733–734 coccoid, 626–627 NA I, 1, some 591 A-F R, VAR 625–626 I, NA 626 relapsing fever rheumatic fever rickettsialpox Rocky Mountain spotted fever salmonellosis shigellosis (bacillary dysentery) skin and wound infections (scalded skin syndrome, scarlet fever, erysipelas, impetigo, etc.) syphillis tetanus toxic shock syndrome trachoma trench fever tuberculosis tuberculosis, avian tularemia typhoid fever typhus, endemic (murine typhus) typhus, epidemic typhus, recrudescent (Brill-Zinsser disease) typhus, scrub (tsutsugamushi disease) Organism Type* Page coccoid, 737–738 R, 692–694 R, 681–682 C, 643–644 R, 334, 727–729 C, 652–653 R, I, NA 128, 172, 652, 670 653 R, 694–695 Streptococcus pyogenes Coxiella burnetti Spirillum minor Streptobacillus moniliformis Borrelia sp Streptococcus pyogenes Rickettsia akari Rickettsia rickettsii C, 719 CB, NA S, R, 334, 659–660 597 597 S, C, CB, NA CB, NA 731–733 720 737 736–737 Salmonella sp Shigella sp R, R, 685–686 687–688 Staphylococcus aureus Staphylococcus epidermidis Streptococcus sp Providencia stuartii Pseudomonas aeruginosa Serratia marcescens Treponema pallidum Clostridium tetani Staphylococcus aureus Chlamydia trachomatis Rochalimaea quintana Mycobacterium tuberculosis Mycobacterium avium Francisella tularensis Salmonella typhi Rickettsia typhi C, 578 C, C, R, R, R, S, R, C, coccoid, NA CB, NA R, A-F R, A-F R, R, CB, NA 579 579 580 580–581 197, 580 620–624 765–767 614–615 593 334, 737 654–658 655 334, 729–730 686–687 736 Rickettsia prowazekii Rickettsia prowazekii CB, NA CB, NA 735 735 Rickettsia tsutsugamushi CB, NA 736 78793 fendpaper.3d GGS 11/6/07 18:51 Diseases and the Organisms that Cause Them (Countinued) BACTERIAL DISEASES—ALSO SEE APPENDIX B Disease Organism Type* Page verruga peruana (bartonellosis) vibriosis Bartonella bacilliformis coccoid, 737 Vibrio parahaemolyticus Bordetella pertussis R, 690 CB, 649–651 Yersinia enterocolitica R, 692 whooping cough (pertussis) yersiniosis *Key to types: C coccus CB coccobacillus R rod S spiral { Species I irregular Gram-negative Gram-positive VAR Gram-variable A-F acid-fast NA not applicable VIRAL DISEASES Disease Virus Reservoir Page Disease Virus aplastic crisis in sickle cell anemia avian (bird) flu bronchitis, rhinitis erythrovirus (B19) humans 743 herpes, oral usually herpes humans simplex type 1, sometimes type human humans immunodeficiency virus (HIV) Epstein-Barr humans influenza parainfluenza Burkitt’s lymphoma cervical cancer birds humans, some other mammals Epstein-Barr humans human papillomavirus humans chickenpox varicella-zoster humans coryza (common cold) cytomegalic inclusion disease Dengue fever encephalitis rhinovirus coronavirus cytomegalovirus humans humans humans Dengue Colorado tick fever Eastern equine encephalitis St Louis encephalitis Venezuelan equine encephalitis Western equine encephalitis adenovirus humans mammals birds epidemic keratoconjunctivitis fifth disease (erythema infectiosum) hantavirus pulmonary syndrome hemorrhagic fever hemorrhagic fever, Bolivian hemorrhagic fever, Korean hepatitis A (infectious hepatitis) hepatitis B (serum hepatitis) hepatitis C (non-A, non-B) hepatitis D (delta hepatitis) hepatitis E (enterically transmitted non-A, non-B, non-C) herpes, genital 660–663 648–649 HIV disease, AIDS infectious 740–741 mononucleosis 271, 587 influenza 631 277–282 583–584 277, 647–648 648 632 birds 334, 739 334, 743 277, 428, 761 761 rodents 280, 761 birds humans 280, 335, 428, 761 593–594 erythrovirus (B19) humans 277, 743 bunyavirus rodents 277, 666 Lassa fever measles (rubeola) meningoencephalitis molluscum contagiosum monkeypox arenavirus measles herpes poxvirus group mumps pneumonia paramyxovirus adenoviruses, respiratory syncytial virus poliovirus rabies poliomyelitis rabies respiratory infections Ebola virus (filovirus) Marburg virus (filovirus) arenavirus bunyavirus (Hantaan) hepatitis A orthopoxvirus adenovirus polyomavirus bunyavirus (phlebovirus) human herpes virus-6 rubella Page 277, 628 277, 555–560 740 swine, humans (type A) humans (type B) humans (type C) rodents humans humans humans 277, 280, 513 660–664 277, 280, 515, 660– 664, 757 660–664 743 277, 581–582 630, 762 586 humans, monkeys humans humans 586 humans all warmblooded animals humans none humans sheep, cattle humans 277, 768–771 758–761 682–683 652–653 667 762 742 humans (?) 277, 742 Rift Valley fever humans (?) 277, 742 roseola rodents and humans rodents 743 humans 277, 581–582 animal 665–666 humans rubella (German measles) SARS (sudden acute coronavirus respiratory syndrome) shingles varicella-zoster smallpox variola (major and minor) humans humans 277, 583–585 277, 585–586 viral enteritis warts, common (papillomas) warts, genital (condylomas) West Nile yellow fever humans humans 696 277, 586–588 humans 277, 586–588, 631–633 761 277, 280, 334, 739 277, 742 hepatitis B humans 277, 696–698 277, 699 hepatitis C humans 699 hepatitis D humans 700 hepatitis E humans 700 usually herpes simplex type 2, sometimes type influenza Reservoir humans 277, 629–631 rotavirus human papillomavirus human papillomavirus West Nile yellow fever birds monkeys, humans, mosquitoes 583 The tables of fungal and parasitic diseases appear on the following page 78793 fendpaper.3d GGS 11/6/07 18:51 Diseases and the Organisms that Cause Them (Concluded) UNCONVENTIONAL AGENTS Disease Agent Resevior Page chronic wasting disease Creutzfeldt-Jacob disease kuru prion prion prion elk, deer humans humans 773 769–770 770 FUNGAL DISEASES Disease Disease Organism Page aspergillosis blastomycosis Aspergillus sp Blastomyces dermatitidis Candida albicans Coccidioides immitis 590, 669 589–590 Filobasidiella neoformans Claviceps purpurea 668–669 candidiasis coccidioidomycosis (San Joaquin valley fever) cryptococcosis ergot poisoning 590 667–668 Organism Type Acanthamoeba keratitis African sleeping sickness (trypanosomiasis) amoebic dysentery ascariasis babesiosis balantidiasis Chagas’ disease chigger dermatitis chigger infestation Chinese liver fluke crab louse cryptosporidiosis dracunculiasis (Guinea worm) elephantiasis (filariasis) fasciiolopsiasis giardiasis heartworm disease hookworm Acanthamoeba culbertsoni Trypanosoma brucei gambiense and T brucei rhodesiense Entamoeba histolytica Ascaris lumbricoides Babesia microti Balantidium coli Trypanosoma cruzi Trombicula sp Tunga penetrans Clonorchis sinensis Phthirus pubis Cryptosporidium sp Dracunculus medinensis protozoan 439 protozoan 334, 773–775 Wuchereria bancrofti roundworm 331–332, 723 flatworm 705 protozoan 700 roundworm 312, 719 roundworm 707 leishmaniasis kala azar oriental sore liver/lung fluke (paragonimiasis) loaiasis Pneumocystis pneumonia ringworm (tinea) PARASITIC DISEASES Page Disease Disease Fasciolopsis buski Giardia intestinalis Dirofilaria immitis Ancylostoma duodenale (Old World hookworm) Necator americanus (New World hookworm) Leishmania braziliensis L donovani L tropica Paragonimus westermani Loa loa histoplasmosis sporotrichosis zygomycosis 816 protozoan roundworm protozoan protozoan protozoan mite sandflea flatworm louse protozoan roundworm Disease mad cow disease (bovine spongiform encephalopathy) scrapie tomato stunt 701 708 749 701–702 334, 775–776 598 598 704 599 702 330, 591 334, 744 flatworm 327, 669–670 roundworm 330, 595 Resevior cattle 772–773 prion viroid sheep plants 771 Organism Page Histoplasma capsulatum Pneumocystis carinii 668 Page 669 various species of 588–589 Epidermophyton, Trichophyton, Microsporum Sporothrix schenckii 589 Rhizopus sp., Mucor sp 590–591 Organism Type Page malaria Plasmodium sp protozoan 317–318, 443, 745–747 pediculosis (lice infestation) pinworm river blindness (onchocerciasis) scabies (sarcoptic mange) schistosomiasis sheep liver fluke (fascioliasis) strongyloidiasis Pediculus humanus louse 599 Enterobius vermicularis Onchocerca volvulus roundworm roundworm 711 594–595 Sarcoptes scabiei mite 598 Schistosoma sp Fasciola hepatica flatworm flatworm 328, 721–723 704 Strongyloides stercoralis Schistosoma sp Hymenolepsis nana (dwarf tapeworm) Taenia saginata (beef tapeworm) Taenia solium (pork tapeworm) Diphyllobothrium latum (fish tapeworm) Echinococcus granulosus (dog tapeworm) Toxoplasma gondii Trichinella spiralis Trichomonas vaginalis Trichuris trichiura roundworm 709–711 flatworm flatworm 591 705–707 flatworm flatworm 326–327, 705–707 705–707 flatworm 705–707 flatworm 705–707 protozoan roundworm protozoan roundworm 747–749 330, 707 615 709 Toxocara sp roundworm 709 swimmer’s itch tapeworm infestation (taeniasis) roundworm 707 protozoan Agent prion toxoplasmosis trichinosis trichomoniasis trichuriasis (whipworm) visceral larva migrans 78793 ffirs.3d GGS 11/6/07 17:24 78793 ffirs.3d GGS 11/6/07 17:24 78793 ffirs.3d GGS 11/6/07 17:24 MICROBIOLOGY 7e PRINCIPLES AND EXPLORATIONS JACQUELYN G BLACK Marymount University, Arlington, Virginia Contributor: LAURA J BLACK Laura Black has been working on this book since she was ten years old She has now been brought on as a contributing author for the seventh edition Jacquelyn and Laura Black JOHN WILEY & SONS, INC 78793 ffirs.3d GGS 11/6/07 17:24 To Laura for sharing her mother and much of her childhood with that greedy sibling ‘‘the book.’’ Senior Acquisitions Editor Kevin Witt Associate Editor Merillat Staat Senior Production Editor Elizabeth Swain Executive Marketing Manager Clay Stone Text Designer Madelyn Lesure Cover Designers Madelyn Lesure and Merillat Staat Senior Illustration Editor Anna Melhorn Photo Editor Hilary Newman Photo Researcher Mary Ann Price Senior Media Editor Linda Muriello Editorial Assistant Alissa Rufino Cover Image: #Russell Kightley/Photo Researchers, Inc Author photos: Paul D Robertson This book was set in 10/12 Times Ten by GGS Book Services, Atlantic Highlands and printed and bound by R R Donnelley, Jefferson City The cover was printed by R R Donnelley, Jefferson City This book is printed on acid-free paper Copyright 2008 # John Wiley & Sons, Inc All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc 222 Rosewood Drive, Danvers, MA 01923, website www.copyright.com Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030-5774, (201)748-6011, fax (201)748-6008, website http://www.wiley.com/go/permissions To order books or for customer service please, call 1-800-CALL WILEY (225-5945) Library of Congress Cataloging-in-Publication Data Black, Jacquelyn G Microbiology : principles and explorations / Jacquelyn G Black – 7th ed p cm Includes index ISBN 978-0-470-10748-5 Microbiology I Title QR41.2.B58 2008 616.9’041–dc22 2007033363 Printed in the United States of America 10 78793 fpref.3d GGS 11/6/07 17:25 Preface The development of microbiology—from Leeuwenhoek’s astonished observations of ‘‘animalcules,’’ to Pasteur’s first use of rabies vaccine on a human, to Fleming’s discovery of penicillin, to today’s search to stop the spread of SARS and race to develop an AIDS vaccine is one of the most dramatic stories in the history of science To understand the roles microbes play in our lives, including the interplay between microorganisms and humans, we must examine, learn about, and study their world—the world of microbiology Microorganisms are everywhere They exist in a range of environments from mountains and volcanoes to deepseas vents and hot springs Microorganisms can be found in the air we breathe, in the food we eat, and even within our own body In fact, we come in contact with countless numbers of microorganisms every day Although some microbes can cause disease, most are not disease producers; rather they play a critical role in the processes that provide energy and make life possible Some even prevent disease, and others are used in attempts to cure disease Because microorganisms play diverse roles in the world, microbiology continues to be an exciting and critical discipline of study And because microbes affect our everyday lives, microbiology provides many challenges and offers many rewards Look at your local newspaper, and you will find items concerning microbiology: to mention a few, reports on diseases such as AIDS, tuberculosis, and cancer; the resurgence of malaria and dengue fever, or ‘‘new’’ diseases such as Avian Flu, HPV, West Nile fever, Monkey Pox, SARS, and those caused by the Ebola virus and the hantavirus; a bacterium that can cause ulcers and stomach cancer; technologies designed to increase food production; bacteriophages used to circumvent antibiotic resistance; microorganisms used to clean up toxic wastes and oil spills; and the Human Genome Project that identifies the complete set of genetic instructions within the cells of the human body yyy THEME yyy AUDIENCE AND ORGANIZATION The theme that permeates this book is that microbiology is a current, relevant, exciting central science that affects all of us I would like to share this excitement with you Come with me as I take you, and your students,on a journey through the relevancy of microbiology In countless areas—from agriculture to evolution, from ecology to dentistry—microbiology is contributing to scientific knowledge as well as solving human problems Accordingly, a goal of this text is to offer a sense of the history of this science, its methodology, its many contributions to humanity, and the many ways in which it continues to be on the cutting edge of scientific advancement This book meets the needs of students in the health sciences as well as biology majors and students enrolled in other science programs who need a solid foundation in microbiology It is designed to serve both audiences—in part by using an abundance of clinically important information to illustrate the general principles of microbiology and in part by offering a wide variety of additional applications In this edition, boxed essays have been newly organized to help students readily recognize the type of application being presented Each application is identified with an appropriate icon v 78793 fpref.3d GGS 11/6/07 17:25 vi z Preface The organization of the seventh edition continues to combine logic with flexibility The chapters are grouped in units from the fundamentals of chemistry, cells, and microscopy; to metabolism, growth, and genetics; to taxonomy of microbes and multicellular parasites; to control of microorganisms; to host-microbe interactions; to infectious diseases of humans; and finally to environmental and applied microbiology.The chapter sequence will be useful in most microbiology courses as they are usually taught However, it is not essential that chapters be assigned in their present order; it is possible to use this book in courses organized along different lines yyy STYLE AND CURRENCY In a field that changes so quickly—with new research, new drugs, and even new diseases—it is essential that a text be as up-to-date as possible This book incorporates the latest information on all aspects of microbiology, including geomicrobiology, phage therapy, deep hot biosphere vents, and clinical practice Special attention has been paid to such important, rapidly evolving topics as genetic engineering, taxonomy, lateral gene transfer, cervical cancer, mad cow disease, and immunology The rapid advances being made in microbiology make teaching about—and learning about—microorganisms challenging Therefore, every effort has been made in the seventh edition of Microbiology: Principles and Explorations to ensure that the writing is simple, straightforward, and functional; that microbiological concepts and methodologies are clearly and thoroughly described; and that the information presented is as accessible as possible to students Students who enjoy a course are likely to retain far more of its content for a longer period of time than those who take the course like a dose of medicine There is no reason for a text to be any less interesting than the subject it describes So, in addition to a narrative that is direct and authoritative, students will find injections of humor, engaging stories, and personal reflections that I hope impart a sense of discovery and wonder and a bit of my passion for microbial life Because students find courses most interesting when they can relate topics to their everyday life or to career goals, I have emphasized the connection between microbiological knowledge and student experiences One way that this connection is made is through the many boxed essays described previously Another is through the use of factoids, post-it type notes that are tidbits of information relating to the running text and that add an extra dimension of flavor to the discussion at hand 78793 c03.3d GGS 11/5/07 11:2 56 z x Microscopy and Staining Basketballs Tennis balls Jelly beans trated by the condenser lens and collected by the objective The formula for calculating resolving power is RP l/2NA As this formula indicates, the smaller the value of l and the larger the value of NA, the greater the resolving power of the lens The NA values of lenses differ in accordance with the power of magnification and other properties The NA value is engraved on the side of each objective lens (the lens nearest the stage) of a light microscope Look at the NA values on the microscope you use the next time you are in the laboratory Typical values for the objective lenses commonly found on modern light microscopes are 0.25 for low power, 0.65 for high power, and 1.25 for the oil immersion lens The higher the NA value, the better the resolution that can be obtained PROPERTIES OF LIGHT: LIGHT AND OBJECTS Beads A Figure 3.6 An analogy for the effect of wavelength on resolution Smaller objects (corresponding to shorter wavelengths) can pass more easily between the arms of the letter E, defining it more clearly and producing a sharper image improves, and the shape of the letter is revealed with greater and greater precision Microscopists use shorter and shorter wavelengths of electromagnetic radiation to improve resolution Visible light, which has an average wavelength of 550 nm, cannot resolve objects separated by less than 220 nm Ultraviolet light, which has a wavelength of 100 to 400 nm, can resolve separations as small as 110 nm Thus, microscopes that used ultraviolet light instead of visible light allowed researchers to find out more about the details of cellular structures But the invention of the electron microscope, which uses electrons rather than light, was the major step in increasing the ability to resolve objects Electrons behave both as particles and as waves Their wavelength is about Galileo (an astron0.005 nm, which allows resolution of omer) was the first separations as small as 0.2 nm person to record The resolving power (RP) of a microscopic observalens is a numerical measure of the retions of biological solution that can be obtained with nature—an insect’s that lens The smaller the distance eye between objects that can be distinguished, the greater the resolving power of the lens We can calculate the RP of a lens if we know its numerical aperture (NA), a mathematical expression relating to the extent that light is concen- Various things can happen to light as it travels through a medium such as air or water and strikes an object (Figure 3.7) Let us look at some of those things now and consider how they can affect what we see through a microscope Reflection If the light strikes an object and bounces back (giving the object color), we say that reflection has occurred (a) Reflection Transmission (b) Absorption (c) with fluorescence (d) Angle of refraction Refraction Light source Figure 3.7 Various interactions of light with an object it strikes (a) Light may be reflected back from the object The particular wavelengths reflected back to the eye determine the perceived color of the object (b) Light may be transmitted directly through the object (c) Light may be absorbed, or taken up, by the object In some cases, the absorbed light rays are reemitted as longer wavelengths, a phenomenon known as fluorescence (d) Light passing through the object may be refracted, or bent, by it 78793 c03.3d GGS 11/5/07 11:2 Principles of Microscopy z 57 For example, light rays in the green range of the spectrum are reflected off the surfaces of the leaves of plants Those reflected rays are responsible for our seeing the leaves as green Transmission Transmission refers to the passage of light through an object You cannot see through a rock because light cannot pass through it, as it does through a glass window In order for you to see objects through a microscope, light must either be reflected from the objects or transmitted through them Most of your observations of microorganisms will make use of transmitted light Absorption If light rays neither pass through nor bounce off an object but are taken up by the object, absorption has occurred Energy in absorbed light rays can be used in various ways For example, all wavelengths of the sun’s light rays except those in the green range are absorbed by a leaf Some of the energy in these other light rays is captured in photosynthesis and used by the plant to make food Energy from absorbed light can also raise the temperature of an object A black object, which reflects no light, will gain heat much faster than a white object, which reflects all light rays In some cases, absorbed light rays, especially ultraviolet light rays, are changed into longer wavelengths and reemitted This phenomenon is known as luminescence If luminescence occurs only during irradiation (when light rays are striking an object), the object is said to fluoresce Many fluorescent dyes are important in microbiology, especially in the field of immunology, because they help us visualize immune reactions and internal processes in microorganisms If an object continues to emit light when light rays no longer strike it, the object is phosphorescent Some bacteria that live deep in the ocean are phosphorescent Refraction Refraction is the bending of light as it passes from one medium to another of different density The bending of the light rays gives rise to an angle of refraction, the degree of bending (Figure 3.7d) You have probably seen how the underwater portion of a pole that is sticking out of water or a drinking straw in a glass of water seems to bend (Figure 3.8) When you remove the object from the water, it is clearly straight It looks bent because light rays deviate, or bend, when they pass from the water into the air as their speed changes across the water-air interface The index of refraction of a material is a measure of the speed at which light passes through the material When two substances have different indices of refraction, light will bend as it passes from one material into the other Light passing through a glass microscope slide, through air, and then through a glass lens is refracted Figure 3.8 Refraction The refraction of light rays passing from water into air causes the pencil to appear bent (Southern Illinois University Niomed/Custom Medical Stock Photo, Inc.) each time it goes from one medium to another This causes loss of light and a blurred image To avoid this problem, microscopists use immersion oil, which has the TRY IT A Life of Crime If you want to take some diamonds through customs without declaring them, here’s how to it Obtain an oil with the same refractive index as the diamonds Pour it into a clear glass bottle labeled ‘‘Baby Oil,’’ and drop the diamonds in The diamonds are invisible if their surfaces are clean This trick works because light is not bent when it passes from one medium to another with the same index of refraction Thus, the boundary between the diamonds and the oil is not apparent If larceny is not in your heart or the price of diamonds is not in your pocket, try this entertaining, but legal, activity Clean a glass rod and dip it in and out of a bottle of immersion oil Watch it disappear and reappear This experiment will help you understand what is happening when you use the oil immersion lens Immersion oil (Richard Megna/Fundamental Photographs) Water 78793 c03.3d GGS 11/5/07 11:2 58 z x Microscopy and Staining Microscope objective lens Refracted and reflected Cover light rays lost to lens glass Air Oil Unrefracted light rays enter lens Slide Object Light source Figure 3.9 Immersion oil Immersion oil is used to prevent the loss of light that results from refraction The focusing of as much light as possible adds to the clarity of the image Immersion oil may also be added between the top of the condenser and the bottom of the slide to eliminate another site for refraction same index of refraction as glass, to replace the air The slide and the lens are joined by a layer of oil; there is no refraction to cause the image to blur (Figure 3.9) If you forget to use oil with the oil immersion lens of a microscope, it will be impossible to focus clearly on a specimen Staining (dyeing) a specimen increases differences in indices of refraction, making it easier to observe details Immersion oil is nothing new Robert Hooke, an early microscopist, first mentioned using a form of it back in 1678 Diffraction As light passes through a small opening, such as a hole, slit, or space between two adjacent cellular structures, the light waves are bent around the opening This phenomenon is diffraction Figure 3.10 shows diffraction patterns formed when light passes through a small aperture or around the edge of an object Similar patterns occur when water passes through an opening in, or around the back of, a breakwater Look for these patterns the next time you are flying over water Diffraction is a problem for microscopists because the lens acts as a small aperture through which the light must pass A blurry image results The higher the magnifying power of a lens, the smaller the lens must be, and therefore the greater the diffraction and blurring it causes The oil immersion (100X) lens, with its total magnification capacity of about 1,000X (when combined with a 10X ocular lens), represents the limit of useful magnification with the light microscope The small size of higher-power lenses causes such severe diffraction that resolution is impossible CHECKLIST Which color of light would give you better resolution when using a microscope: red (wavelength 0.68 mm) or blue (wavelength 0.48 mm)? Why? If you built a light microscope having a total magnification of 5,000X, would it give you better, worse, or the same resolution as one that has a magnification of 1,000X? Why? Why would radio waves and microwaves be unsuitable for examining microbes? Would standard immersion oil placed on a plastic slide prevent refraction? Why or why not? Direction of travel of light (a) Figure 3.10 (b) (c) Diffraction Light waves are diffracted as they pass (a) around the edge of an object and (b) through a small aperture (c) Water waves being diffracted as they pass through an opening in a breakwater (Runk Schoenberger/Grant Heilman Photography) 78793 c03.3d GGS 11/5/07 11:2 Light Microscopy z 59 yyy LIGHT MICROSCOPY Light microscopy refers to the use of any kind of microscope that uses visible light to make specimens observable The modern light microscope is a descendant not of Leeuwenhoek’s single lenses but of Hooke’s compound microscope—a microscope with more than one lens (bChapter 1, p 9) Single lenses produce two problems: They cannot bring the entire field into focus simultaneously, and there are colored rings around objects in the field Both problems are solved today by the use of multiple correcting lenses placed next to the primary magnifying lens (Figure 3.11) Used in the objectives and eyepieces of modern compound microscopes, correcting lenses give us nearly distortion-free images Over the years, several kinds of light microscopes have been developed, each adapted for making certain kinds of observations We look first at the standard light microscope and then at some special kinds of microscopes Camera attachment tube Eyepiece (ocular lens) Light pathway Arm Coarse and fine focusing adjustment knobs Body tube Objective lens Stage Light source Specimen Condenser Illuminator Base THE COMPOUND LIGHT MICROSCOPE The optical microscope, or light microscope, has undergone various improvements since Leeuwenhoek’s time and essentially reached its current form shortly before the beginning of the twentieth century This microscope is a compound light microscope—that is, it has more than one lens The parts of a modern compound microscope and the path light takes through it are shown in Figure 3.12 A compound microscope with a single eyepiece (ocular) is said to be monocular; one with two eyepieces is said to be binocular Light enters the microscope from a source in the base and often passes through a blue filter, which filters out the long wavelengths of light, leaving the shorter wavelengths and improving resolution It then goes through a condenser, which converges the light beams so that they pass through the specimen The iris diaphragm controls the amount of light that passes through the specimen and into the objective lens The higher the Figure 3.11 Cutaway view of a modern microscope objective What we refer to as a single objective lens is really a series of several lenses, which are necessary to correct aberrations of color and focus The best objectives may have as many as a dozen or more elements Figure 3.12 The compound light microscope Yellow indicates the path of light through the microscope magnification, the greater the amount of light needed to see the specimen clearly The objective lens magnifies the image before it passes through the body tube to the ocular lens in the eyepiece The ocular lens further magnifies the image A mechanical stage allows precise control of moving the slide, which is especially useful in the study of microbes The focusing mechanism consists of a coarse adjustment knob, which changes the distance between the objective lens and the specimen fairly rapidly, and a fine adjustment knob, which changes the distance very slowly The coarse adjustment knob is used to locate the specimen The fine adjustment knob is used to bring it into sharp focus Compound microscopes have up to six interchangeable objective lenses that have different powers of magnification The total magnification of a light microscope is calculated by multiplying the magnifying power of the objective lens (the lens used to view your specimen) by the magnifying power of the ocular lens (the lens nearest your eye) Typical values for a microscope with a 10X ocular lens are: • • • • scanning (3X) (10X) 30X magnification low power (10X) (10X) 100X magnification high ‘‘dry’’ (40X) (10X) 400X magnification oil immersion (100X) (10X) 1,000X magnification Most microscopes are designed so that when the microscopist increases or decreases the magnification by changing from one objective lens to another, the 78793 c03.3d GGS 11/5/07 11:2 60 z x Microscopy and Staining Objective lens Specimen Slide Condenser Light rays Diaphragm (a ) Light rays (b) Figure 3.13 A comparison of the illumination in brightfield and dark-field microscopy (a) The condenser of the (a) bright-field microscope concentrates and transmits light directly through the specimen (b) The dark-field condenser deflects light rays so that they reflect off the specimen at an angle before they are collected and focused into an image specimen will remain very nearly in focus Such microscopes are said to be parfocal (par means ‘‘equal’’) The development of parfocal microscopes greatly improved the efficiency of microscopes and reduced the amount of damage to slides and objective lenses Most studentgrade microscopes are parfocal today Some microscopes are equipped with an ocular micrometer for measuring objects viewed This is a glass disc with a scale marked on it that is placed inside the eyepiece between its lenses This scale must first be calibrated with a stage micrometer, which has metric units engraved on it When these units are viewed through the microscope at various magnifications, the microscopist can determine the corresponding metric values of the divisions on the ocular micrometer for each objective lens Thereafter, he or she needs only to count the number of divisions covered by the observed object and multiply by the calibration factor for that lens in order to determine the actual size of the object (b) Figure 3.14 A comparison of bright-field and dark-field images (a) Bright-field and (b) dark-field microscope views of Saccharomyces cerevisiae (brewer’s yeast, magnified 975X) Darkfield illumination provides an enormous increase in contrast (Jim Solliday/Biological Photo Service) DARK-FIELD MICROSCOPY The condenser used in an ordinary light microscope causes light to be concentrated and transmitted directly through the specimen, as shown in Figure 3.13a This gives bright-field illumination (Figure 3.14a) In some cases, however, it is more useful, especially with lightsensitive organisms, to examine specimens that would lack contrast with their background in a bright field under other illumination Live spirochetes (spi0 ro-kets), spiral-shaped bacteria that cause syphilis and other diseases, are just such organisms In this situation darkfield illumination is used A microscope adapted for dark-field illumination has a condenser that prevents light from being transmitted through the specimen but Figure 3.15 A phase-contrast image Amoeba, a protozoan (160X) (Biophoto Associates/ Photo Researchers, Inc.) 78793 c03.3d GGS 11/5/07 11:2 Light Microscopy z 61 Figure 3.16 A Nomarski image The protozoan Paracineta is attached by a long stalk to the green alga Spongomorpha (magnified 400X) (Biological Photo Service) instead causes the light to reflect off the specimen at an angle (Figure 3.13b) When these rays are gathered and focused into an image, a light object is seen on a dark background (Figure 3.14b) PHASE-CONTRAST MICROSCOPY Most living microorganisms are difficult to examine because they cannot be stained by coloring them with dyes—stains usually kill the organisms To observe them alive and unstained requires the use of phasecontrast microscopy A phase-contrast microscope has a special condenser and objective lenses that accentuate small differences in the refractive index of various structures within the organism Light passing through objects of different refractive indices is slowed down and diffracted The changes in the speed of light are seen as different degrees of brightness (Figure 3.15) wavelength than that originally striking them (see Figure 3.7c) The different wavelengths produced are often seen as brilliant shades of orange, yellow, or yellow green Some organisms, such as Pseudomonas, fluoresce naturally when irradiated with ultraviolet light Other organisms, such as Mycobacterium tuberculosis and Treponema pallidum (the cause of syphilis), must be treated with a fluorescent dye called a fluorochrome They then stand out sharply against a dark background (Figure 3.17) Acridine orange is a fluorochrome that binds to nucleic acids, and it colors bright green, orange green, or yellow, depending on the filter system used with the fluorescence microscope It is sometimes used to screen samples for microbial growth, with live cells showing up in bright orange or green Fluorescent antibody staining is now widely used in diagnostic procedures to determine whether an antigen (a foreign substance such as a microbe) is present Antibodies—molecules produced by the body as an immune response to an invading antigen—are found in many clinical specimens such as blood and serum If a patient’s specimen contains a particular antigen, that antigen and the antibodies specifically made against it will clump together However, this reaction is ordinarily not visible Therefore, fluorescent dye molecules are attached to the antibody molecules If the dye molecules are retained by the specimen, the antigen is presumed to be present, and a positive diagnosis can be made Thus, if fluorescent dye-tagged antibodies against syphilis organisms are added to a specimen containing spirochetes and are seen to bind to the tagged organisms, those organisms can be identified as the cause of syphilis This technique is especially important in immunology, in which the reactions of antigens and antibodies are studied in great detail (see bChapters 17 NOMARSKI (DIFFERENTIAL INTERFERENCE CONTRAST) MICROSCOPY Nomarski microscopy, like phase-contrast microscopy, makes use of differences in refractive index to visualize unstained cells and structures However, the microscope used, a differential interference contrast microscope, produces much higher resolution than the standard phasecontrast microscope It has a very short depth of field (the thickness of specimen that is in focus at any one time) and can produce a nearly three-dimensional image (Figure 3.16) FLUORESCENCE MICROSCOPY In fluorescence microscopy, ultraviolet light is used to excite molecules so that they release light of a longer LM Figure 3.17 Fluorescent antibody staining The fluorescent dye-tagged antibodies clearly show live bacterial cells (green) and dead (red) cells (854X) (David Phillips/Visuals Unlimited) 78793 c03.3d GGS 11/5/07 11:2 62 z x Microscopy and Staining Figure 3.18 (a) (b) (c) (d) Images of the same organism (Paramecium, 600X) produced by four different techniques (a) Bright-field microscopy, (b) dark-field microscopy, (c) phase-contrast microscopy, (d) Nomarski microscopy One microscope can have the optics for all four techniques (David M Phillips/Visuals Unlimited) and 18, especially Figure 18.35 on the technique of fluorescent antibody staining) Diagnoses can often be made in minutes rather than the hours or days it would take to isolate, culture, and identify organisms Figure 3.18 shows the images produced by four different microscopic techniques (a) Figure 3.19 (b) CONFOCAL MICROSCOPY Confocal systems use beams of ultraviolet laser light to excite fluorescent chemical dye molecules into emitting (returning) light (Figure 3.19) The exciting light beam is focused onto the specimen (usually nonliving) either through a thin optical fiber, or by passing through a (c) (a) A confocal microscope system manufactured by Olympus Cell with microtubular fragments shown using (b) standard fluorescent microscopy, and (c) confocal microscopy (Courtesy Olympus Corporation, Scientific Equipment Division) 78793 c03.3d GGS 11/5/07 11:2 Electron Microscopy z 63 (b) Figure 3.20 (a) (a) A digital microscope system manufactured by Nikon Instruments, Inc (Courtesy of Nikon Instruments Inc.) (b) Cyanobacterium, Chroococcus, viewed by digital microscopy (ß Wim van Egmond) Electron source small aperture shaped as a pinhole or a slit Resultant fluorescent emissions are focused on a detector which also has a small aperture or slit in front of it The smaller the apertures used at both sites, the greater the amounts of out-of-focus light blocked from the detector A computer reconstructs an image from the emitted light with resolution that can be up to 40% better than with other types of light microscopy Because of the sharpness of focus, the image is like a very thin knifeblade cut through the specimen For thick specimens, a whole series of successive focal plane cuts can be recorded, and assembled into a three-dimensional model This is very helpful in studying communities of microbes without disturbing them, as in examining living biofilms Time-lapse images can also be collected Condenser lens Specimen Objective lens Intermediate image Projector lens Viewing Figure 3.21 Electron lenses microscopy (a) A cross-sectional diagram of an electron microscope, showing the Fluorescent screen or pathways of the electron photographic beam as it is focused by film (final electromagnetic lenses image) (b) A modern scanning electron microscope in use (Pascal Goetgheluck/ Photo Researchers, Inc.) DIGITAL MICROSCOPY Have you had frustrating moments in the lab when you just couldn’t get a slide into focus? Then you would like the auto-focus, auto-aperture, auto-light, motorized stage and magnification changers of a digital microscope (Figure 3.20) Not only that, but these microscopes also come with a built-in digital camera and preloaded software All you is plug in the unit, turn on the power, and use the mouse to view live or stained specimens on a monitor, or in a group situation on a screen through use of a projector It can also be integrated into a local or wide area network for distance learning or teleconferencing Imagine being able to show your cousin in Kansas live images of what’s swimming in your sample of pond water There are, however, some limitations: maximum magnification is quite limited, and price is high yyy (a) ELECTRON MICROSCOPY The light microscope opened doors to the world of microbes However, because it could not resolve objects (b) 78793 c03.3d GGS 11/5/07 11:3 64 z x Microscopy and Staining (a) LM more expensive than light microscopes They also take up much more space and require additional rooms for preparation of specimens and for processing of photographs Photographs taken on any microscope are called micrographs; those taken on an electron microscope are called electron micrographs Nothing else can show the great detail of minute biological structures that EMs can (Figure 3.22) The two most common types of electron microscope are the transmission electron microscope and the scanning electron microscope Both are used to study various life forms, including microbes The more advanced scanning tunneling microscope and atomic force microscope let us see actual molecules and even individual atoms TRANSMISSION ELECTRON MICROSCOPY (b) SEM Figure 3.22 Light and electron microscopy images compared (a) Light (160X) and (b) electron (425X) microscope images of a Didinium eating a Paramecium Notice how much more detail is revealed by the scanning electron micrograph (top: Eric V Grave/Photo Researchers, Inc.; bottom: Biophoto Associates/Photo Researchers, Inc.) separated by less than 0.2 mm, the view was limited to observations at the level of whole cells and their arrangements Few subcellular structures could be seen; neither could viruses The advent of the electron microscope (EM) allowed these small structures to be visualized and studied The EM was developed in 1932 and was in use in many laboratories by the early 1940s Germans invented the The EM uses a beam of electrons EM At the end of instead of a beam of light, and elecWorld War II, the tromagnets rather than glass lenses U.S confiscated are used to focus the beam (Figure Hitler’s personal 3.21) The electrons must travel physician’s EM through a vacuum because collisions U.S scientists studied with air molecules would scatter the it to learn to make electrons and result in a distorted imAmerican EMs age Electron microscopes are much The transmission electron microscope (TEM) gives a better view of the internal structure of microbes than other types of microscopes Because of the very short wavelength of illumination (electrons) on which the TEM operates, it can resolve objects as close as nm and magnify microbes (and other objects) up to 500,000X To prepare specimens for transmission electron microscopy, a specimen may be embedded in a block of plastic and cut with a glass or diamond knife to produce very thin slices (sections) These sections are placed on thin wire grids for viewing so that a beam of electrons will pass directly through the section The section must be exceedingly thin (70–90 nm) because electrons cannot penetrate very far into materials The specimens can also be treated with special preparations that contain heavy metal elements The heavy metals scatter electrons and contribute to forming an image Figure 3.23 Shadow casting Spraying a heavy metal (such as gold or platinum) at an angle over a specimen leaves a ‘‘shadow,’’ or darkened area, where metal is not deposited This technique, known as shadow casting, produces images with a three-dimensional appearance, as in this photograph of polio viruses (magnified 330,480X) You can calculate the height of the organisms from the length of their shadows if you know the angle of the metal spray (John J Cardamone Jr & B.A Phillips/Biological Photo Service) 78793 c03.3d GGS 11/5/07 11:3 Electron Microscopy z 65 Cell frozen in block of ice Fracture exposes interior surface to nuclear membrane Etched cytoplasm Detail of fractured membrane Cytoplasm Etched ice Etching exposes outer surface of organelles and plasma membrane Knife Ice Nucleus (a) Fracture Figure 3.24 Ice (b) Etching Freeze-fracturing and freeze-etching (a) In freeze-fracturing, a specimen is frozen in a block of ice and broken apart with a very sharp knife The fracture reveals the interiors of cellular structures and typically passes through the center of membrane bilayers, exposing their inner faces (b) In freeze-etching, water is evaporated directly from the ice and frozen cytoplasm of the fractured specimen, uncovering additional surfaces for observation Very small specimens, such as molecules or viruses, can be placed directly on plastic-coated grids Then a heavy metal such as gold or platinum is sprayed at an angle onto the specimen, a technique known as shadow casting A thin layer of the metal is deposited Areas behind the specimen that did not receive a coating of metal appear as ‘‘shadows,’’ which can give a threedimensional effect to the image (Figure 3.23) Electron beams are deflected by the densely coated parts of the specimen but, for the most part, pass through the shadows It is also possible to view the interior of a cell with a TEM by a technique called freeze-fracturing In this technique the cell is frozen and then fractured with a knife The cleaving of a specimen reveals the surfaces of structures inside the cell (Figure 3.24a) Freeze-etching, which involves the evaporation of water from the frozen and fractured specimen, can then expose additional surfaces for examination (Figure 3.24b) These surfaces must also be coated with a heavy metal layer that produces shadows This layer, called a replica, is viewed by TEM (Figure 3.25) The image formed by the electron beam is made visible as a light image on a fluorescent screen, or monitor (The actual image made by the electron beam is not visible and would burn your eyes if you tried to view it directly.) The electrons are used to excite the phosphors (light-generating compounds) coating the screen However, the electron beam will eventually burn through the specimen Therefore, before this happens, electron micrographs are made, either by photographing the image on the video screen or by replacing the screen itself with a photographic plate (Figure 3.26a) Electron micrographs can be enlarged, just as you would enlarge any photograph, to obtain an image magnified 20 million times! The micrographs are permanent records of specimens observed and can be studied at leisure The study of electron micrographs has provided much of our knowledge of the internal structure of microbes The ‘‘M’’ in TEM and SEM (next page) can refer to either microscope or micrograph Figure 3.25 A freeze-etch preparation The toxic cyanobacterium Microcystis aeruginosa (magnified 18,000X), showing details of large spherical gas vesicles (Biological Photo Service) 78793 c03.3d GGS 11/5/07 11:3 66 z x Microscopy and Staining (a) TEM (b) SEM Figure 3.26 TEM and SEM compared Colorized electron micrographs of Escherichia coli produced by (a) transmission electron microscopy (66,952X) and (b) scanning electron microscopy (39,487X) (a: Dennis Kunkel/Phototake; b: David M Phillips/Visuals Unlimited) SCANNING ELECTRON MICROSCOPY The scanning electron microscope (SEM) is a more recent invention than the TEM and is used to create (a) SEM (b) Figure 3.27 images of the surfaces of specimens The SEM can resolve objects as close as 20 nm, giving magnifications up to approximately 50,000X The SEM gives us wonderful three-dimensional views of the exterior of cells (Figure 3.26b) Preparing a specimen for the SEM involves coating it with a thin layer of a heavy metal, such as gold or palladium The SEM is operated by scanning, or sweeping, a very narrow beam of electrons (an electron probe) back and forth across a metal-coated specimen Secondary, or backscattered, electrons leaving the specimen surface are collected, the current is increased, and the resulting image is displayed on a screen Photographs of the image can be made and enlarged for further study Views of the three-dimensional world of microbes, as shown in Figure 3.27, are breathtakingly beautiful SCANNING TUNNELING MICROSCOPY In 1980, Gerd Binnig and Heinrich Rohrer invented the first of a series of rapidly improving scanning tunneling microscopes (STMs), also called scanning probe microscopes Five years later, they received the Nobel Prize for their discovery A thin wire probe made of platinum and viridium is used to trace the surface of a substance, much as you SEM (c) SEM Colorized SEM photos of representative microbes (a) The fungus Aspergillus, a cause of human respiratory disease (10,506X); (b) Actinomyces, a branching bacterium (5670X); (c) a radiolarian from the Indian Ocean (1761X); (d) the diatom Cyclotella meneghiniana, one of many that carry on photosynthesis and form the base of many aquatic food chains (1584X) (a: Visuals Unlimited; b: David M Phillips/Photo Researchers, Inc.; c: Manfred Kage/Peter Arnold, Inc.; d: Dr Anne Smith/Photo Researchers, Inc.) (d) SEM 78793 c03.3d GGS 11/5/07 11:3 Electron Microscopy z 67 would use your finger to feel the bumps while reading Braille Electron clouds (regions of electron movement) from the surfaces of the probe and the specimen overlap, producing a kind of pathway through which electrons can ‘‘tunnel’’ into one another’s clouds This tunneling sets up an observable current The stronger the current, the closer the top of the atom is to the probe Running the probe across in a straight line reveals the highs and lows of individual molecules or atoms in a surface (Figure 3.28) Even movies can be made using this technique The first one ever produced showed individual fibrin molecules coming together to form a blood clot Scanning tunneling microscopy also works well under water, and it can be used to examine live specimens, such as virusinfected cells exploding and releasing newly formed viruses The atomic force microscope (AFM), a more advanced member of this family of microscopes, allows three-dimensional imaging and measurement of structures from atomic size to about mm The AFM has been very useful in studying DNA because it enables investigators to distinguish between bases, such as adenine and guanine, from differences in their electron density states Atomic force microscopy has also been used underwater to study chemical reactions at living cell surfaces (Figure 3.29), which help to confirm chemical analyses of the cell wall material In addition to producing images, AFM can measure forces, for example, the force needed to unfold a protein located in a membrane One can also determine the flexibility of a polysaccharide molecule, that is, to know how far it can elongate before it ruptures This information is important in studying attachment of adjacent cells to form aggregations such as colonies and films, or for the ability to attach to host cells The various types of microscopy and their uses are summarized in Table 3.2 (a) Figure 3.29 (a) An atomic force microscope (b) Surface of a dormant spore of the fungus Aspergillus oryzae shows a covering of protein rodlets (c) A few hours later, the rodlets have disintegrated into a layer of soft material, beginning to reveal inner spore walls composed of polysaccharides The entire process can be watched live, underwater, and filmed with the atomic force microscope (Michael L Abramson/Time Life Picture/Getty Images, From B Jean and H Hoărben, Force Microscopy, Wiley Liss, 2006, Fig 5-4cd, page 77) CHECKLIST Figure 3.28 Scanning tunneling microscopy Individual atoms of the element xenon can be clearly distinguished by using a scanning tunneling microscope (STM) This chain of seven xenon atoms was built by IBM scientists moving each atom into position, one at a time The atoms are 20 billionths of an inch, or 0.5 nm, apart The atoms are bonded together; moving an end atom will relocate up to three of them at a time (Courtesy of International Business Machines Corporation Unauthorized use not permitted.) What does the electron microscope use instead of light beams and glass lenses? What does the fluorescence microscope use? How can you distinguish between TEM and SEM micrographs? Which type is Figure 3.25? Why are colored photos of electron micrographs referred to as ‘‘colorized’’ or ‘‘false color’’? Rank the following types of microscopes according to the wavelength of illuminating beam they use, beginning with the longest wavelength: fluorescent, TEM, bright-field What effect does wavelength have on the resolving power of these microscopes? 78793 c03.3d GGS 11/5/07 11:3 TABLE 3.2 Comparison of Types of Microscopy Type Bright-Field Special Features Uses visible light; simple to use, least expensive a Dark-Field Phase-Contrast Nomarski Fluorescence Confocal Digital Transmission Electron Scanning Electron Uses visible light with a special condenser that causes light rays to reflect off specimen at an angle Uses visible light plus phase-shifting plate in objective with a special condenser that causes some light rays to strike specimen out of phase with each other Uses visible light out of phase; has higher resolution than standard phase-contrast microscope Uses ultraviolet light to excite molecules to emit light of different wavelengths, often brilliant colors, because UV can burn eyes, special lens materials are used Uses laser light to obtain thin focallevel sections through a specimen, with 40 times greater resolution and less out-offocus light Uses computer technology to automatically focus, adjust light, and take photographs of specimens; can put directly online Uses electron beam instead of light rays and electromagnetic lenses instead of glass lenses; image is projected on a video screen; very expensive; preparation requires considerable time and practice Appearance Uses Colored or clear specimen on light background Observation of dead stained organisms or live ones with sufficient natural color contrast Bright specimen on dark background Observation of unstained living or difficult-to-stain organisms Allows one to see motion Specimen has different degrees of brightness and darkness Detailed observation of internal structure of living unstained organisms Produces a nearly threedimensional image Observation of finer details of internal structure of living unstained organisms Bright, fluorescent, colored specimen on dark background Diagnostic tool for detection of organisms or antibodies in clinical specimens or for immunologic studies Non-fuzzy thinsection image Observation of very specific levels of specimen Standard image Ease of operation plus online use Highly magnified, detailed image; not three-dimensional except with shadow casting Examination of thin sections of cells for details of internal structure, exterior of cells, and viruses, or surfaces when freezefracturing is used Threedimensional view of surfaces Observation of exterior surfaces of cells or internal surfaces Threedimensional view of surfaces Observation of exterior surfaces of atoms or molecules b c d e f g h Uses electron beam and electromagnetic lenses; expensive; preparation requires considerable time and practice i Scanning Tunneling Uses a wire probe to trace over surfaces, allowing electrons to move (tunnel), thereby generating electric currents that reveal highs and lows of specimen’s surface j (a: David M Phillips/Visuals Unlimited; b: Jim Solliday/Biological Photo Service; c: Biophoto Associates/Photo Researchers, Inc.; d: Biological Photo Service; e: David Philips/Visuals Unlimited; f: Courtesy Olympus Corporation, Scientific Equipment Division; g: ßWim van Egmond; h: Dennis Kunkel/Phototake; i: Manfred Kage/Peter Arnold, Inc.; j: Courtesy of International Business Machines Corporation Unauthorized use not permitted.) 78793 c03.3d GGS 11/5/07 11:3 Techniques of Light Microscopy z 69 yyy TECHNIQUES OF LIGHT MICROSCOPY Microscopes are of little use unless the specimens for viewing are prepared properly Here we explain some important techniques used in light microscopy Although resolution and magnification are important in microscopy, the degree of contrast between structures to be observed and their backgrounds is equally important Nothing can be seen without contrast, so special techniques have been developed to enhance contrast Depression (well) slide Coverslip Vaseline jelly (seal) Drop of liquid with specimen (hanging upside down from undersurface of coverslip) (a ) PREPARATION OF SPECIMENS FOR THE LIGHT MICROSCOPE Wet Mounts Wet mounts, in which a drop of medium containing the organisms is placed on a microscope slide, can be used to view living microorganisms The addition of a 2% solution of carboxymethylcellulose, a thick, syrupy solution, helps to slow fast-moving organisms so they can be studied A special version of the wet mount, called a hanging drop, often is used with dark-field illumination (Figure 3.30) A drop of culture is placed on a coverslip that is encircled with petroleum jelly The coverslip and drop are then inverted over the well of a depression slide The drop hangs from the coverslip, and the petroleum jelly forms a seal that prevents evaporation This preparation gives good views of microbial motility Smears Smears, in which microorganisms from a loopful of medium are spread onto the surface of a glass slide, can be used to view killed organisms Although they are living when placed on the slide, the organisms are killed by the techniques used to fix (attach) them to the slide Smear preparation often is difficult for beginners If you make smears too thick, you will have trouble seeing individual cells; if you make them too thin, you may find no organisms If you stir the drop of medium too much as you spread it on the slide, you will disrupt cell arrangements You may see organisms that normally appear in tetrads (groups of four) as single or double organisms Such variations lead some beginners to imagine that they see more than one kind of organism when, in fact, the organisms are all of the same species After a smear is made, it is allowed to air-dry completely Then it is quickly passed three or four times through an open flame This process is called heat fixation Heat fixation accomplishes three things: (1) It kills the organisms, (2) it causes the organisms to adhere to the slide, and (3) it alters the organisms so that they more readily accept stains (dyes) If the slide is not completely dry when you pass it through the flame, the organisms will be boiled and destroyed If you heat-fix (b) LM Figure 3.30 The hanging-drop technique (a) A drop of culture is placed on a coverslip, ringed with petroleum jelly and then inverted and placed over the well in a depression slide The petroleum jelly forms a seal to prevent evaporation (b) Dark-field micrograph of a hanging-drop preparation (2500X) showing the spiral bacterium Treponema pallidum, the cause of syphilis (A M Siegelman/Visuals Unlimited) too little, the organisms may not stick and will wash off the slide in subsequent steps Any cells remaining alive will stain poorly If you heat-fix too much, the organisms may be incinerated, and you will see distorted cells and cellular remains Certain structures, such as the capsules found on some microbes, are destroyed by heat-fixing, so this step is omitted and these microbes are affixed to the slide just by air-drying PRINCIPLES OF STAINING A stain, or dye, is a molecule that can bind to a cellular structure and give it color Staining techniques make the microorganisms stand out against their backgrounds They are also used to help investigators group major categories of microorganisms, examine the structural and chemical differences in cellular structures, and look at the parts of the cell In microbiology the most commonly used dyes are cationic (positively charged), or basic, dyes, such as methylene blue, crystal violet, safranin, and malachite green These dyes are attracted to any negatively charged cell components The cell membranes of most bacteria have negatively charged surfaces and thus 78793 c03.3d GGS 11/5/07 11:3 70 z x Microscopy and Staining TRY IT Are You Positive?—Or, No Strings Attached The Gram stain is not foolproof Some anaerobic Grampositive organisms decolorize easily and may falsely appear to be Gram-negative Gram-negative organisms such as Streptobacillus moniliformis can stain Gram-positive Is there some way to be sure of the organism’s correct Gram reaction? There are several ways One is the potassium hydroxide (KOH) test Place drops of a 3% solution of KOH on a slide Remove an inoculating loopful of the organism in question from a pure colony Add it to the KOH on the slide, and mix continuously for 30 seconds As you stir, occasionally lift the loop up or cm from the surface to see if ‘‘strings’’ of gooey material hang down If the organism is truly Gram-negative, the KOH will break down its cell walls, releasing its DNA and forming strings Gram-positive organisms will not form strings attract the positively charged basic dyes Other stains, such as eosin and picric acid, are anionic (negatively charged), or acidic, dyes They are attracted to any positively charged cell materials Two main types of stains, simple stains and differential stains, are used in microbiology They are compared in Table 3.3 A simple stain makes use of a single dye and reveals basic cell shapes and cell arrangements Methylene blue, safranin, carbolfuchsin, and crystal violet are commonly used simple stains A differential stain makes use of two or more dyes and distinguishes between two kinds of organisms or between two different parts of an organism Common differential stains are the Gram stain, the Ziehl-Neelsen acid-fast stain, and the Schaeffer-Fulton spore stain The Gram Stain The Gram stain, probably the most frequently used differential stain, was devised by a Danish physician, Hans Christian Gram, in 1884 Gram was testing new methods of staining biopsy and autopsy materials, and he noticed that with certain methods some bacteria were stained differently than the surrounding tissues As a result of his experiments with stains, the highly useful Gram stain was developed In Gram staining, bacterial cells take up crystal violet Iodine is then added; it acts as a mordant, a chemical that helps retain the stain in certain cells Those structures that cannot retain crystal violet are decolorized with 95% ethanol or an ethanolacetone solution, rinsed, and subsequently stained (counterstained) with safranin The steps in the Gramstaining procedure are shown in Figure 3.31 Four groups of organisms can be distinguished with the Gram stain: (1) Gram-positive organisms, whose cell walls retain crystal violet stain; (2) Gram-negative organisms, whose cell walls not retain crystal violet stain; (3) Gram-nonreactive organisms, which not stain or which stain poorly; and (4) Gram-variable organisms, which stain unevenly The differentiation between Gram-positive and Gram-negative organisms reveals a fundamental difference in the nature of the cell walls of bacteria, as is explained in bChapter Furthermore, the reactions of bacteria to the Gram stain have helped in distinguishing Gram-positive, Gramnegative, and Gram-nonreactive groups that belong to radically different taxonomic groups b(Chapter 9) Gram-variable organisms have somehow lost their ability to react distinctively to the Gram stain Organisms from cultures over 48 hours old (and sometimes only 24 hours old) are often Gram-variable, probably because of changes in the cell wall with aging Therefore, to determine the reaction of an organism to the Gram stain, you should use organisms from cultures 18–24 hours old The Ziehl-Neelsen Acid-Fast Stain The Ziehl-Neelsen acid-fast stain is a modification of a staining method developed by Paul Ehrlich in 1882 It can be used to detect tuberculosis- and leprosy-causing organisms of the genus Mycobacterium (Figure 3.32) Slides of organisms are covered with carbolfuchsin and are heated, rinsed, and decolorized with 3% hydrochloric acid (HCl) in 95% ethanol, rinsed again, and then stained with Loeffler’s methylene blue Most genera of bacteria will lose the red carbolfuchsin stain when decolorized However, those that are ‘‘acid-fast’’ retain the bright red color The lipid components of their walls, which are responsible for this characteristic, are discussed in bChapter Bacteria that are not acidfast lose the red color and can therefore be stained blue with the Loeffler’s methylene blue counterstain Special Staining Procedures Negative Staining Negative stains are used when a specimen—or a part of it, such as the capsule—resists taking up a stain The capsule is a layer of polysaccharide material that surrounds many bacterial cells and can act as a barrier to host defense mechanisms It also repels stains In negative staining, the background around the organisms is filled with a stain, such as India ink, or an acidic dye, such as nigrosin This process leaves the organisms themselves as clear, unstained objects that stand out against the dark background A second simple or differential stain can be used to demonstrate the presence of the cell inside the capsule Thus, a typical slide will show a dark background and clear, unstained areas of capsular material, inside of which are purple cells stained with crystal violet (Figure 3.33) or blue cells stained with methylene blue Flagellar Staining Flagella, appendages that some cells have and use for locomotion, are too thin to be seen easily with the light microscope When it is necessary to determine their presence or arrangement, ... 78793 fbrieftoc.3d GGS 11 /6/07 17 :23 Brief Contents 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Scope and History of Microbiology Fundamentals of Chemistry 28 Microscopy and Staining 52 Characteristics... Endocytosis and Exocytosis 11 0 Retracing Our Steps 11 1 / Terminology Check 11 2 / Clinical Case Study 11 3 / Critical Thinking Questions 11 3 / Self-Quiz 11 3 / Explorations on the Web 11 5 yyy Essential Concepts... migrans 78793 ffirs.3d GGS 11 /6/07 17 :24 78793 ffirs.3d GGS 11 /6/07 17 :24 78793 ffirs.3d GGS 11 /6/07 17 :24 MICROBIOLOGY 7e PRINCIPLES AND EXPLORATIONS JACQUELYN G BLACK Marymount University,