Chapter3ObservingMicroorganismsThroughaMicroscope

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Chapter 3: Observing Microorganisms Through a Microscope,1,Microscopy:,The technology of making very small things visible to the naked eye.,Units of Measurement,: The,metric system,is used to measure microorganisms.,Metric system:,Basic unit of length:,Meter,.,All units are related to each other by factors of 10.,Prefixes are used to indicate the relationship of a unit to the basic unit (e.g.: meter).,2,Metric Units of Length and U.S. Equivalents,:,MetricRelationship to U.S.,Unitbasic unit (meter)Equivalent,kilometer (km) 1 km = 1000 m1 mile = 1.61 km,meter (m) 1 m = 39.37 in,decimeter (dm) 1 dm = 0.1 m = 10,-1,m1 dm = 3.94 in,centimeter (cm) 1 cm = 0.01 m = 10,-2,m 2.54 cm = 1 in,millimeter (mm) 1 mm = 0.001 m = 10,-3,m,micrometer (um) 1 um = 0.000001 m = 10,-6,m,nanometer (nm) 1 nm = 0.000000001 m = 10,-9,m,picometer (pm) 1 pm = 0.1 m = 10,-12,m,3,Instruments of Microscopy:,1. Simple Microscopes:,Only have one lens, similar to a magnifying glass.,Leeuwenhoecks,simple microscopes allowed him to magnify images from 100 to 300 X.,They were so difficult to focus, he built a new one for each specimen, a total of 419.,He did not share his techniques with other scientists. Even today, his source of lighting is unknown.,His daughter donated 100 of his microscopes to the Royal Society shortly before his death in 1723.,4,Instruments of Microscopy:,2. Compound Light (CL) Microscopy,History of CL Microscopes:,First developed by,Zaccharias,Janssen, Dutch spectacle maker in 1600.,Poor quality,Could not see bacteria,Joseph Jackson Lister (Listers father) developed improved compound light microscope in 1830s.,Basis for modern microscopes,Use,visible light,as a source of illumination.,5,Instruments of Microscopy:,2. Compound Light Microscopy,Have,several lenses,:,1.,Light originates from an illuminator and passes through,condenser lenses, which direct light onto the specimen.,2.,Light then enters the,objective lenses, which magnify the image. These are the closest lenses to the specimen:,Scanning,objective lens: 4 X,Low power,objective lens: 10 X,High power,objective lens: 40-45 X,Oil immersion,lens: 95-100 X,3.,The image of the specimen is magnified once again by the,ocular lens,or,eyepiece,(10 X).,6,Instruments of Microscopy:,2. Compound Light Microscopy,Total magnification,: Obtained by multiplying,objective,lens power by,ocular,lens power. (Condenser lenses do not magnify image).,LensMagnificationOcular,Mag,.Total,Mag,.,Scanning,4 X 10 X =,40 X,Low power,10 X 10 X =,100 X,High power,45 X 10 X =,450 X,Oil immersion,100X,10 X =,1000 X,Highest possible magnification with CL microscope is about 2000 X.,7,Instruments of Microscopy:,2. Compound Light Microscopy,Resolution (Resolving power):,Ability of microscope to see two items as separate and discrete units.,The smaller the,distance,between objects at which they can be distinguished as separate, the greater the resolving power.,Light must pass between two objects in order for them to be seen as separate.,Depends on light,wavelength,. If wavelength is too long to pass between objects, they will appear as one.,White light,has a relatively long wavelength (550 nm), and cannot resolve structures less than,220 nm (0.2 um),apart.,Ultraviolet (UV) light,has a shorter wavelength (100 to 400,nm,), and can resolve distances as small as 110,nm,.,8,Instruments of Microscopy:,2. Compound Light Microscopy,Refraction:,Bending of light as it passes from one medium to another of different density.,Index of refraction:,A measure of the speed at which light passes through a material.,Can be changed by,staining, which increases,contrast,between specimen and surrounding medium.,When two substances have a different index of refraction, the light will,bend,as it passes from one material to another.,As light passes through a glass slide, air, and the objective lens, it bends each time, causing loss of light and a blurred image.,Immersion oil,has the same index of refraction as glass slide, preventing light loss from refraction.,9,Instruments of Microscopy:,3. Darkfield Microscopy,Useful to examine,live,or,unstained,specimens.,Light sensitive organisms,Specimens that lack contrast with their background.,Spirochetes which cause syphilis.,Darkfield,condenser with,opaque disc,blocks light that would enter objective lens directly:,Light reflects off specimen at an angle.,Only light reflected by specimen enters objective lens.,No direct background light.,Image,:,Light specimen,against,dark background,.,10,Instruments of Microscopy:,4. Phase Contrast Microscopy,Useful to examine,live,specimens:,Doesnt require,fixing,or,staining, which usually kill and/or distort microorganisms.,Permits,detailed,examination of,internal,structures,.,Special objective lenses and condenser with ring shaped diaphragm accentuate small differences in refractive indexes of internal structures.,Image:,Direct rays and reflected light rays come together, forming an image with many shades of gray to black.,11,Instruments of Microscopy:,5. Fluorescence Microscopy,Fluorescence:,Ability of substances to absorb short wavelengths of light (ultraviolet light) and emit them at a longer wavelength.,Natural Fluorescence,: Some microorganisms fluoresce naturally under UV light (,Pseudomonas,).,Fluorochrome,: Fluorescent dye.,Acridine,orange: Binds to nucleic acids, colors cells orange, green, or yellow depending on light source.,Immunofluorescence,: Fluorescent antibodies can be used to detect specific antigens. Very useful for the rapid diagnosis of specific diseases (e.g.: syphilis).,Image:,Luminescent bright object against a dark background.,12,Instruments of Microscopy:,Limitations of light microscopy:,Magnification,: Up to 2000 X.,Resolving Power,: Up to 0.2 um.,Because of the limits of magnification and resolving power,viruses and most internal structures of cells cannot be seen with a light microscope,.,13,Instruments of Microscopy:,6. Electron Microscopy,Electron microscopes were first developed in 1932, and became widely available in 1940s.,Use a,beam of electrons,instead of a beam of light.,Wavelength of electron beam is about 100,000 times,smaller,than visible light.,Used to examine structures too small to be resolved with a light microscope.,Two types of electron microscope:,A. Transmission Electron Microscope (TEM),B. Scanning Electron Microscope (SEM),14,Instruments of Microscopy:,6. Electron Microscopy,A. Transmission Electron Microscope (TEM),Gives excellent view of,internal structures.,Magnification:,100,000 X or more.,Resolving power:,2.5 nm or better.,Two dimensional,image.,Drawbacks of TEM:,Due to limited penetrating power of electrons, can only view very thin slices (70-90 nm) of specimen.,Must slice, fix, dehydrate, and view specimen under a vacuum. Staining may be used to enhance image contrast.,Treatments kill specimen and may cause shrinkage and distortion of cells (,artifacts,).,15,Instruments of Microscopy:,6. Electron Microscopy,B. Scanning Electron Microscope (SEM),Gives excellent view of,external surface.,Magnification:,10,000 X or more.,Resolving power:,20 nm or better.,Three dimensional,image.,More recent invention than TEM. Used mainly to observe the,surfaces of cells and viruses,.,Specimens are covered with a layer of heavy metal (gold or palladium).,A narrow beam of electrons (,primary electron beam,) is swept across specimen surface.,Electrons on the specimen surface are knocked out, creating a,secondary electron beam,which is collected and amplified to produce an image.,16,7. Scanning Tunneling Microscopy and Atomic Force Microscopy (AFM),Developed in the 1980s.,Used to observe structure and surface of biological molecules and silicon computer chips.,A. Scanning Tunneling Microscope (STM),Uses a thin,metal probe,that scans the,surface,of a specimen.,B. Atomic Force Microscopy (AFM),Uses a,diamond and metal probe,that scans surface of specimen.,Advantages of both microscopes:,Higher resolving power,than electron microscopes,No special specimen preparation required,17,Preparation of Specimens for Light Microscopy,1.,Smear:,Spread a thin film of material containing microorganisms over slide surface. Allow to air dry.,2.,Fixing,:,Process that kills microorganisms and attaches them to a microscope slide. Fixing preserves and minimizes distortion of cells.,Two main methods of fixation:,Heat fixation,: Pass over Bunsen burner flame several times.,Chemical fixation,: Cover with methanol for 1 minute.,18,Preparation of Specimens for Light Microscopy,3.,Staining:,Coloring microorganisms with a dye that emphasizes certain structures. Before staining a sample, it must be,fixed,.,Stains are,salts,composed of a positive ion (cation) and a negative ion (anion).,The colored ion is called the,chromophore,.,Two types of dyes:,A. Basic dyes,B. Acidic dyes,19,Preparation of Specimens for Light Microscopy,A.,Basic dyes,:,Chromophor,is in,positive,ions.,Most commonly used dyes.,Bacteria are slightly negatively charged at pH 7, therefore they stain with basic dyes.,Examples,:,Crystal violet,Methylene,blue,Saffranin,20,Preparation of Specimens for Light Microscopy,B.,Acidic dyes,:,Color is in,negative,ions.,Stain the background:,negative staining,.,Bacteria do not stain with acidic dyes.,Used to observe cell shape, size, and capsules.,Minimal distortion because heat fixing is not necessary an dye is not taken up by cells.,Examples:,Eosin,Nigrosin,India ink.,21,Preparation of Specimens for Microscopy,1. Simple Stains,Aqueous or alcohol solution of a,single basic dye,.,Primary purpose is to stain entire microorganism to view cell shape and basic structures.,Procedure:,Stain is applied for a certain time, and then washed off.,Slide is dried and examined.,Mordant:,May be used to increase stain intensity. Increases affinity of stain for specimen.,Examples:,Safranin,methylene,blue, crystal violet, and,carbolfuchsin,.,22,Preparation of Specimens for Microscopy,2. Differential Stains,React differently to different types of bacteria.,Can be used to distinguish among different groups of bacteria.,There are two important differential stains used in microbiology:,A.,Gram stain,B.,Acid-Fast stain,23,Preparation of Specimens for Microscopy,2. Differential Stains,A.,Gram Stain,Developed in 1884 by Hans Gram, a Danish microbiologist.,The most useful staining procedure in medical microbiology.,Distinguishes bacteria of two large and medically important groups:,Gram-positive bacteria,Gram-negative bacteria,Provides useful information for disease treatment.,24,Preparation of Specimens for Microscopy,2. Differential Stains,Steps of,Gram Stain,1.,Primary stain,: Cover a heat fixed smear with a basic dye (crystal violet).,All cells, gram-positive and gram-negative, are,stained with crystal violet,(appear,purple,).,2.,Mordant:,After smear is rinsed with water, an,iodine mordant,solution is applied.,Crystal violet-iodine CV-I complex forms,25,Preparation of Specimens for Microscopy,2. Differential Stains,Steps of,Gram Stain,3.,Decolorizing:,Slide is washed with alcohol, which will remove stain from Gram-negative cells but not from Gram-positive cells.,Gram-negative,cells will be,decolorized,.,Gram-positive,cells will remain,purple,.,4.,Counterstain,:,Alcohol is rinsed off.,Safranin,is applied, which will stain cells that were decolorized.,Gram-negative,cells are stained,pink,.,Gram-positive,cells remain,purple,.,26,Preparation of Specimens for Microscopy,2. Differential Stain,What accounts for the differential staining between,Gram-positive and Gram-negative cells?,Gram-positive cells have very thick,peptidoglycan,cell walls, whereas gram-negative cells have very thin cell walls. Crystal violet easily penetrates both cell types.,Because of its larger size, the crystal violet-iodine complex CV-I is not easily removed from gram-positive cells, due to their thick cell wall. The CV-I complex is readily washed out of gram-negative cells with alcohol.,Counterstain,only colors gram-negative cells.,27,Preparation of Specimens for Microscopy,2. Differential Stain,Applications and Limitations of the Gram stain,Chemotherapy,:,Gram-positive cells with their very thick,peptidoglycan,cell walls, are susceptible to,penicillins,and,cephalosporins,.,Gram-negative cells with their thin cell walls and,lipopolysaccharide,layer are resistant to these antibiotics.,Limitations,:,Not all bacterial cells stain well with the Gram-stain.,Gram-stain only works well on young bacterial cultures, that are actively growing. Therefore it is best to use cultures that are 18 to 24 hours old.,Older cultures (over 24-48 hours), are often gram-variable.,28,Preparation of Specimens for Microscopy,2. Differential Stains,B.,Acid-Fast Stain (Ziehl-Nielsen Stain),Modification of a method developed in 1882 by Paul Ehrlich.,Used to detect tuberculosis and leprosy causing organisms of the genus,Mycobacterium,and pathogens of the genus,Nocardia,.,These bacteria have waxy cell walls, which makes them difficult to stain.,29,Preparation of Specimens for Microscopy,2. Differential Stains,Steps of,Acid-Fast Stain,1.,Primary stain,:,Cover a heat fixed smear with,carbolfuchsin, a red basic dye.,Gently heat for several minutes to increase penetration and retention of dye.,Allow to cool and rinse with water.,30,Preparation of Specimens for Microscopy,2. Differential Stains,Steps of,Acid-Fast Stain,2.,Decolorizing:,Slide is washed with,acid-alcohol,.,Non acid-fast,cells will be,decolorized,.,Acid-fast,cells will remain,red,.,3.,Counterstain,:,Acid-alcohol is rinsed off.,Methylene,blue is applied, which will stain cells that were decolorized.,Non acid-fast,cells are stained,blue,.,Acid-fast,cells remain,red,.,31,Preparation of Specimens for Microscopy,3. Special Stains,Used to color and isolate specific parts of microorganisms such as:,Endospores,Capsules,Flagella,32,Preparation of Specimens for Microscopy,3. Special Stains,A. Endospore Stain,Endospores,are extremely resistant, dormant structures that are formed by some gram-positive bacteria to protect them from harsh environmental conditions: heat, drought, chemicals, radiation, etc.,Ordinary staining methods cannot penetrate the thick,endospore,wall.,Most commonly used method is,Schaeffer-Fulton,endospore,stain.,33,Preparation of Specimens for Microscopy,3. Special Stains,A. Endospore Stain,Steps for Schaeffer-Fulton,Endospore Stain,1. Primary stain:,Malachite green is applied to heat fixed smear and steamed for about 5 minutes.,Malachite green will penetrate,endospore,.,2. Wash,: Rinse with water for 30 seconds.,Removes green dye from rest of the cell, except for,endospore,3.,Counterstain,:,Safranin,will stain rest of the cell.,Appearance of cell with,endospore,:,Pink cell with green,endospore,.,34,Preparation of Specimens for Microscopy,3. Special Stains,B. Capsule Stain,Capsules are gelatinous covers on top of the cell wall, which are important virulence (disease) factors.,Capsules are difficult to stain,because they repel most stains, are water soluble, and are easily disrupted with harsh treatment.,Negative stain is used to obtain a dark background (E.g.: India ink or,nigrosin,).,Cell is stained with a basic dye (E.g.:,safranin,).,Capsule appearance,: Light halo around stained cell, dark background.,35,Preparation of Specimens for Microscopy,3. Special Stains,C. Flagella Stain,Flagella are appendages used for locomotion that are too thin to be seen easily with a light microscope.,Staining procedures are difficult. Usually involve using a mordant and coating the,flagellar,surface with a dye or metal (e.g.: silver).,The number and arrangement of flagella can be used as diagnostic aids.,36,