Chapter 3Observing Microorganisms Through A Microscope

Q&AAcid-fast staining of a patients sputum is a rapid, reliable, and inexpensive method to diagnose tuberculosis. What color would bacterial cells appear if the patient has tuberculosis?

A Glimpse of HistoryHans Christian Joachim Gram (18531938)Danish physician working at morgue in BerlinWorked for Dr. Carl FriedlanderAttempting to identify cause of pneumoniaGram was developing methods to stain bacteria for easier viewing under the microscopeWith one method, bacteria stained unequallySome retained dye, others did notRevealed two different kinds of bacteriaBasis for modern Gram stainIdentifies two major groups of bacteriaGram-positive and Gram-negative

Microscopy Reveals Two Cell TypesMicroscopy reveals two fundamental cell typesProkaryotic cells (Bacteria, Archaea)Smaller size gives high surface area to low volumeFacilitates rapid uptake of nutrients, excretion of wastesAllows rapid growthDisadvantages include vulnerability to threats including predators, parasites, and competitorsEukaryotic cells (Eukarya)Larger, more complex, many cellular processes take place in compartments

1.5. Size in the Microbial WorldCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1 micron () = 106 metermicrometer (m) = 106 meter = .000001 meterNucleusMitochondriaProteinsSmallmoleculesAtomsLipidsRibosomesSmallestbacteriaMostbacteriaMost eukaryotic cellsAdult roundwormHuman height10 m1 m0.1 m1 cm1 mm100 m10 m0.1 nm1 nm10 nm100 nm1 mThe basic unit of length is the meter (m), and allother units are fractions of a meter.These units of measurement correspond to unitsin an older but still widely used convention.1 angstrom () = 1010 meternanometer (nm) = 109 meter = .000000001 meter1 meter = 39.4 inchesmillimeter (mm) = 103 meter = .001 meterPrion fibrilVirusesElectron microscopeLight microscopeUnaided human eye

Observing MicroorganismsFigure 3.2

Figure 3.2Units of Measurement1 m = 106 m = 103 mm1 nm = 109 m = 106 mm1000 nm = 1 m0.001 m = 1 nm

3.1. Microscopic Techniques: The InstrumentsLight microscope can magnify 1,000xCommon, important tool in microbiologyElectron microscope (1931) can magnify more than 100,000xAtomic force microscope (1980s) can produce images of individual atoms on a surface

Figure 1.2bMicroscopy: The InstrumentsA simple microscope has only one lens

Light MicroscopyUse of any kind of microscope that uses visible light to observe specimensTypes of light microscopyCompound light microscopyDarkfield microscopyPhase-contrast microscopyDifferential interference contrast microscopyFluorescence microscopyConfocal microscopy

Figure 3.1aThe Compound Light Microscope

Figure 3.1bCompound Light MicroscopyIn a compound microscope, the image from the objective lens is magnified again by the ocular lensTotal magnification = objective lens ocular lens

Principles of Light MicroscopyLight passes through specimen and then series of magnifying lensesBright-field microscope is most common typeThree key conceptsMagnification: apparent increase in sizeModern compound microscopes have two lens types: objective and ocular

Magnification is product of objective (4x, 10x, 40x, and 100x) and ocular lens (10x)Condenser lens (between light source and specimen) focuses light on specimen, does not magnifyOcular lens(eyepiece)Magnifies theimage, usually10-fold(10).SpecimenstageCondenserFocusesthe light. Iris diaphragmControls theamount of lightthat enters theobjective lens.Objective lensA selection of lensoptions providedifferentmagnifications. Thetotal magnification isthe product of themagnifying power ofthe ocular lens andthe objective lens.Light sourceRheostatControls thebrightness of thelight.Courtesy of Leica, Inc., Deerfield, FLCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Compound Light MicroscopyResolution is the ability of the lenses to distinguish two pointsA microscope with a resolving power of 0.4 nm can distinguish between two points 0.4 nmShorter wavelengths of light provide greater resolutionMaximum resolving power of light microscope is 0.2 m

Compound Light MicroscopyThe refractive index is a measure of the light-bending ability of a mediumThe light may bend in air so much that it misses the small high-magnification lensImmersion oil is used to keep light from bending

Contrast determines how easily cells can be seenTransparent bacteria lack contrast, difficult to see against colorless backgroundStains increase contrast butkill microbesPrinciples of Light Microscopy(a)(b)Light sourceSlideObjective lensOilAir(a): Richard Megna/Fundamental PhotographsCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Figure 3.4aANIMATION Light MicroscopyBrightfield IlluminationDark objects are visible against a bright backgroundLight reflected off the specimen does not enter the objective lens

Figure 3.4bDarkfield IlluminationLight objects are visible against a dark backgroundLight reflected off the specimen enters the objective lens

Figure 3.4cPhase-Contrast MicroscopyAccentuates diffraction of the light that passes through a specimenMakes cells and other dense material appear darker

Figure 3.5Differential Interference Contrast MicroscopyAccentuates diffraction of the light that passes through a specimen; uses two beams of lightLight waves are out of phase when recombined, yieldthree-dimensional appearance of image

Figure 3.6bFluorescence MicroscopyUses UV lightFluorescent substances absorb UV light and emit visible lightCells may be stained with fluorescent dyes (fluorochromes)

Scanning Laser Microscopes (SLM)Obtains detailed views of interior of intact cellsSpecimens usually stained with fluorescent dyeTags bind specifically to certain internal compoundsMarks their location

Other Light Microscopes

Figure 3.7Confocal MicroscopyCells stained with fluorochrome dyes Short wavelength (blue) light used to excite the dyesThe light illuminates each plane in a specimen to produce a three-dimensional imageUp to 100 m deep

Figure 3.8Two-Photon (Multiphoton) MicroscopyCells stained with fluorochrome dyesTwo photons of long- wavelength (red) light used to excite the dyesUsed to study cells attached to a surfaceLight also penetrates deeper to give interior views of relatively thick structuresUp to 1 mm deep

Electron microscopy (continued)Can magnify images 100,000xOne drawback is that lenses and specimen must be in vacuumAir molecules would interfere with electronsResults in large, expensive unit and complex specimen preparationTwo major typesElectron MicroscopesElectron beamsElectromagnetElectromagnetElectromagnetViewing screenEyeGlassGlassLightraysGlassLampTransmission ElectronMicroscopeLight MicroscopeOcularlensImageObjectivelensSpecimenCondenserlensElectrongunCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Electron MicroscopyUses electrons instead of lightThe shorter wavelength of electrons gives greater resolution

Figure 3.10aTransmission Electron Microscopy (TEM)Ultrathin sections of specimensLight passes through specimen, then an electromagnetic lens, to a screen or filmSpecimens may be stained with heavy metal salts

Figure 3.10aTransmission Electron Microscopy (TEM)10,000100,000; resolution 2.5 nm

Figure 3.10bScanning Electron Microscopy (SEM)An electron gun produces a beam of electrons that scans the surface of a whole specimenSecondary electrons emitted from the specimen produce the imageUsed to observe surface detailsSurface coated with thin film of metalYields 3-D effect

Figure 3.10bScanning Electron Microscopy (SEM)1,00010,000; resolution 20 nm

Figure 3.11bScanned-Probe MicroscopyAtomic force microscopy (AFM) uses a metal- and-diamond probe inserted into the specimen.Produces three-dimensional images.Resolving power much greater than that of EMAvoid special preparation required for EMSharp probe moves across samples surfaceFeels bumps, valleys of atomsLaser measures motion, computer produces surface map

Copyright 2010 Pearson Education, Inc.Check Your Understanding

A.) Identify the type of microscopy used. B.) Explain the utility of each type of microscopy.

Preparing Smears for StainingStaining: Coloring the microbe with a dye that emphasizes certain structuresSmear: A thin film of a solution of microbes on a slideA smear is usually fixed to attach the microbes to the slide and to kill the microbes

Preparing Smears for StainingSamples can be immobilized, stained to visualizeLive or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens.Figures B and C

Preparing Smears for StainingStains consist of a positive and negative ionIn a basic dye, the chromophore is a cationBasic dyes (positive charge)Attracted to negatively charged cellular components

In an acidic dye, the chromophore is an anionAcidic dyes (negative charge)Negative staining: cells repel, so colors backgroundCan be done as wet mount

Staining the background instead of the cell is called negative staining

Negative Stainingan acidic (-) stain is utilized.results in only the background being stained.aids in observing the shape and size of a cellwww. smccd.net/.../case/ biol240/images/negstain.jpeg Useful for cells that are unable to be stained due to composition of the cell wall

Simple StainsSimple stain: Use of a single basic dyeA mordant may be used to hold the stain or coat the specimen to enlarge it

Differential StainsUsed to distinguish between bacteriaGram stainAcid-fast stain

Gram StainClassifies bacteria into gram-positive or gram-negativeGram-positive bacteria tend to be killed by penicillin and detergentsGram-negative bacteria are more resistant to antibiotics

Gram stain most common for bacteriaTwo groups: Gram-positive, Gram-negativeReflects fundamental difference in cell wall structure3.2. Microscopic Techniques: Dyes and StainingCrystal violet(primary stain)3412Cells stain purple.Cells remain purpleIodine(mordant)Safranin(counterstain)Gram-positive cellsremain purple;gram-negative cellsappear pink.Alcohol(decolorizer)Gram-positive cellsremain purple;gram-negative cellsbecome colorless.State of BacteriaAppearanceSteps in Staining(a)(b)10 mCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.b: Leon J. Le Beau/Biological Photo Service

Gram Stain

Color of Gram-positive cellsColor ofGram-negative cellsPrimary stain:Crystal violetPurplePurple

Mordant:IodinePurplePurpleDecolorizing agent:Alcohol-acetonePurpleColorlessCounterstain:SafraninPurpleRed

Figure 3.12bMicrograph of Gram-Stained Bacteria

Acid-fast staining used to detect MycobacteriumCell wall contains high concentrations of mycolic acidWaxy fatty acid that prevents uptake of dyesHarsher methods neededUsed to presumptively identify clinical specimens3.2. Microscopic Techniques: Dyes and Staining

Differential Stains: Acid-Fast StainCells that retain a basic stain in the presence of acid-alcohol are called acid-fast.Nonacid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them.Figure 3.11

Acid-Fast Stain

Color of Acid-fastColor ofNonAcid-fastPrimary stain:CarbolfuchsinRedRedDecolorizing agent:Acid-alcoholRedColorlessCounterstain:Methylene blueRedBlue

Acid-Fast StainStained waxy cell wall is not decolorized by acid-alcohol used to identify bacteria in the genus Mycobacterium Includes causative agents of tuberculosis and Hansens disease (leprosy)

MycobacteriumNocardia

Q&AAcid-fast staining of a patients sputum is a rapid, reliable, and inexpensive method to diagnose tuberculosis. What color would bacterial cells appear if the patient has tuberculosis?

Special StainsNegative staining is useful for capsules.

Heat is required to drive a stain into endospores.

Flagella staining requires a mordant to make the flagella wide enough to see.Figure 3.12a-c

Special Stains

Capsule stain both the cell and background are stained. (Negative stain)Endospore stain - heat is required to drive a malachite green stain into endospores.Flagella staining - requires a mordant to make the flagella wide enough to see.Figure 3.12a-c

Case Study

An outbreak of an infectious disease sent more than 25 children from a local day care to the hospital. Five of the children were hospitalized with an upper respiratory tract infection. The initial onset of the clinical signs and symptoms of the disease included fever, sore throat, and headache. Later, signs included creamy yellow pus produced on the tonsils (exudative tonsillitis). Some also developed infections in deeper tissue, or a high fever and bright-red rash.Stainings revealed a purple-stained, round shaped organism arranged in chains (figure 1A). .

Questions: 1. Is this a gram-positive or gram-negative bacterium? Explain your answer.2. What stain would help in identification of the species?3. Briefly outline the process of this ___________-stain.4. What bacterial genus does this organism belong?Figure 1a

A Bacterial Outbreak in a Prison Housing Inmates Infected with HIVIn early July 1999. a 34-year old HIV-infected man housed in a dormitory in the prison was taken to the prison hospital with a 2-week history of fatigue, fever, abdominal pain, and cough with blood. His chest radiograph was normal. The staff neglected to obtain a sputum specimen for culture and the prisoner was returned to his dormitory.

In mid-August 1999, the man was evaluated at a community hospital. A sputum sample was obtained and an acid fast stained was performed. Figure 1.Later that year, 31 inmates and a medical student who examined the patient exhibited signs of this infection. Of the 31 inmates, all 31 resided in the same dormitory as the original infected patient. Case Study #2:

Questions:1. What stain would help identify this organism?2. Describe the ____________ stain method.3. What color is the positive cells?4. What molecule is detected using this stain?5. What genus of bacteria is the cause of this disease outbreak?

Simple staining involves one dyeDifferential staining used to distinguish different types of bacteria3.2. Microscopic Techniques: Dyes and Staining

*****************Special optics amplify difference between refractive index of dense material and surrounding medium

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