Studying Microbes Seeing the InvisibleChapter 3

ObjectivesKnow how microbes are studied Know the different types of microscopesKnow how to distinguish between different types of microbes?Gram -/+, acid fast or non-acid fast bacteria, endospore forming or notGive specific examples of each type of bacteriaKnow the common stains in microbiologyGram, acid fast, endospore, and capsule stains

Units of MeasurementIf a microbe measures 10 m in length, how long is it in nanometers?

110 um = 10000 nm

Figure 3.2 Microscopes and Magnification.Tick Actual sizeRed blood cellsE. coli bacteriaT-even bacteriophages(viruses)DNA double helixUnaided eye 200 mLight microscope200 nm 10 mmScanningelectron microscope10 nm 1 mmTransmissionelectron microscope10 pm 100 mAtomic force microscope0.1 nm 10nmObserving Microorganisms

Light MicroscopyMicroscope that uses light to observe specimensExamples:Compound light microscopyDarkfield microscopyPhase-contrast microscopyFluorescence microscopy (use UV light)Confocal microscopy (use UV light)

Figure 3.1aCompound Light Microscope

Microscopy: The InstrumentsFigure 3.1bCompound microscopeLight rays come from illuminator condenser specimen Objective lenses Ocular lenses Image from objective lens is magnified again by ocular lensTotal magnification = Ocular mag. x Objective mag. Objective lens (4-100x)Ocular lens (10x)

Microscopy: The InstrumentsResolution: The ability of the lenses to distinguish fine detail and structure (two points)Microscope with 0.4 nm resolving power can distinguish two points 0.4 nm apartBest resolution of light microscopes = 0.2 mm (2,000x magnification)Shorter wavelengths of light provides greater resolution

Microscopy: The InstrumentsFigure 3.3Refractive index: Light-bending ability of mediumSpecimens can be made to contrast sharply with medium by stainingLight may bend in air so much that it misses the lensHigh magnification objective lens are smallImmersion oil prevents light from bending too much to miss specimenAchieve high magnification with good resolution

Light MicroscopyCyanobacteriaFungusBacteria

Brightfield MicroscopyFigure 3.4abDirect light enter objective lens

Dark objects visible against bright/white background

Light reflected off the specimen does not enter the objective lens

Staining increases contrast

Darkfield MicroscopyAn opaque disk placed between light and specimen

Only light refracted by specimen reaches eyepiece

Emphasizes edges of structures against a dark background

Examine unstained, live m/os suspended in liquid, i.e. spirochetes

Phase-ContrastTwo sets of light rays (direct and reflected or diffracted) brought together to form an image of a specimenUsed in detailed examination of living cellsView internal detailsNo staining required

Fluorescence MicroscopyUses UV light or other short wavelengths of lightSubstances absorb UV light and emit visible light (longer wavelengths)M/os are stained with antibodies combined with fluorescent dyes (fluorochromes) for diagnostic studies

Actin Rockets of Listeria The bacteria are green and the rockets are red (actin). Bacteria put actin (by polymerization) at one of their ends to propel themselves inside a cell and into the neighbor cellshttp://adollahite.wordpress.com/2010/10/21/microscopic-rockets/

Confocal MicroscopySpecimens are stained with fluorochromesUses laser to illuminate one plane of a small region of a specimen at a timeCan use a computer to construct a 3-D image to combine planes and regionsReconstructed images can be rotated and view in any orientationUp to 100 m deep

Other forms of MicroscopyTwo-photon microscopyCells are stained with fluorochrome dyesTwo photons of long-wavelength (red) light are used to excite the dyesUsed to study cells attached to a surfaceUp to 1 mm deepScanning acoustic microscopyMeasures sound waves that are reflected back from an objectUsed to study cells attached to a surfaceResolution 1 m

Electron Microscopy:Used for objects smaller than ~0.2mmBeam of electrons used instead of light (achieves greater resolution)Produce B & W images, but may be colored artificiallySpecimens may be stained with Metal salts (eg. Lead & Uranium salts)Immunostaining (antibodies coated with gold particles)TEM: Transmission electron microscopySEM: Scanning electron microscopy

Electron MicroscopyTEM: Ultrathin sections of specimensElectron beam through electromagnetic lens specimen electromagnetic lens screen or film2-D image of internal structuresTreatment may cause distortionsMagnifications of 10,000100,000 Resolution 2.5 nm

Electron MicroscopySEM: Electron gun produces beam of electrons that scans surface of whole specimenSecondary electrons emitted from specimen produce imageProvides striking 3-D viewUseful in studying surface structures1,00010,000Resolution 20 nm

TEMBacterial Flagella Viruses

SEM Biofilm on Contact Lens Case

TEM vs SEMCampellone et al. 2003. Cur Opin Microbiol 6:82-90.

Most organisms appear colorless under microscopeMust be stained to make visibleSmear: Organism spread on slide before staining

Fixing (pass slide through a flame) KillsAttachesPreserves (structures)Preparation of Specimens for Light Microscopy

StainingBacteria cells are slightly negative at pH 7Attract positive ionsStains are salts One of the ions is colored (chromophore)Basic dyes (simple stains) Most common dyesChromophore is a cation (+) Crystal violet, methylene blue, safraninAcidic dyes, (coloring the bg, not the organism, stain is repelled by the organism)Chromophore is an anion (-)Used in negative staining (colorless bacteria)

StainingSimple stainsStain the cell, increase contrastCrystal violet, methylene blue, malachite green, safraninDifferential stains Differentiate between organisms or structuresGram stain, Acid fast stainsSpecial stainsUsed to differentiate structuresCapsule (negative), endospore, flagella stains

Gram StainGram-positive bacteria : Stained purple by crystal violet/iodine Gram-negative: Stained red by safranin (counterstain)GramStain1GramStain2

Gram Positive Cell WallMany layers of peptidoglycanContains teichoic acidCross-linked to peptidoglycanProtects wall from breakdownProvides antigenic specificity of cells

Gram Negative Cell WallComposed of :Thin peptidoglycan, no teichoic acid, is bonded to lipoprotein, and in the periplasmOuter membrane contains Lipopolysaccharide (LPS) an endotoxin (lipid A, responsible for fever, shock & blood clotting)Protects cell from penicillin, lysozymeContains porins- allow passage of nutrients

Gram stainGram (+) positiveStain purple (or blue)Gram (-) negativeStain red (or pink)Blood smear w/ gonorrhea

Table 4.1 Some Comparative Characteristics of Gram-Positive and Gram-Negative Bacteria (Part 1 of 2)

Gram Staining Reactions

Some Important Gram (+) bacteriaCorynebacterium diphtheriaeCause diphtheria, small rod, often in pairs with pallisade (parallel) arrangementStaphylococcus aureusImportant cause of post-operative infections, boils, pimples, impetigo-Gram (+) coccus in clusters.Streptococcus pyogenesStrept- means chainsImportant cause of sore throats, tonsillitis, flesh-eating disease; Gram (+) coccus in chainsStreptococcus pneumoniaemajor cause of bacterial pneumonia; Gram (+) lancet-shaped coccus in pairsGardnerella vaginalisGram-variable rods

Gram Staining Reactions

Some Important Gram (-) bacteriaEscherichia coliNormal inhabitant of the gut, important cause of urinary tract infections, diarrhea, and hamburger disease, Gram-negative rodsNeissseria gonorhoeaeCause the sexually-transmitted disease (STD) gonorrheaGram-negative cocci in pairsGardnerella vaginalisMajor cause of bacteria vaginosis, Gram (-) to Gram variable rods

Acid-fast bacteria stain redThick outer lipid layer - acid fastMycolic acidHave slow nutrient exchange hence slow growthMycobacterium tuberculosis (TB)Myconacterium leprae (leprosy)Process Primary Stain- Carbolfuchsin (red dye),Decolorized by acid alcoholCounterstain- methylene blueNon acid-fast- stain blueAcid Fast StainMycolic acid

Other stainsCapsule stains Spore stainsBacillus cereusClostridium botulinumStreptococcus pneumoniae

****Electron beamers in electron microscopes have much shorter wavelength to increase wavelength************Capsules are used as a protective structure**Black lines are carbohydratesLipoteichoic acid is found in within the plasma membrane.Teichoic acid is outside*

TB is an infection of the lungs. Need treatment for about 3-6 months. **