CHAPTER 3Observing Organisms Through a MicroscopeUnits of MeasurementsMicroscopy: The InstrumentsPreparation of Specimens

We will be looking at very small things

Compound Light Microscope

Compound Light MicroscopeTotal MagnificationObjective lens power x ocular lens powerResolutionAbility of the lenses to distinguish fine detailResolution power of 0.2 m, Distinguish 2 points 0.2 m apartRefractive indexChange by staining specimensTwo different mediumsRays change more directionsOil ImmersionSame index as glassImproves resolution

STAINSSalts composed of a positive and a negative ionOne of which is colored (chromophore)Basic Dyes: positive ionAttracted to negatively charged bacteria cellEx: Crystal violet, methylene blue, malachite green and safraninAcidic Dyes: negative ionStain colors the background surfaceObserving overall cell shape, size and capsuleEx: fuchsin, nigrosinCalled Negative StainingThree types of staining techniquesSimple, differential and special

SIMPLE STAINAqueous or alcohol soln of a single basic dyeHighlight the entire microorganismApplied to fixed smear for length of time, washed, driedMordant (used to intensify) may be addedIncreases affinity of a stainCoat a structure to make it thicker and easier to see (flagella)Examples: methylene blue*, crystal violet, safranin

StainingFixed: kills/attaches org. to slideThin film spread over slide (smear) and allowed to dryPass through flame of Bunsen burner several times or cover with methyl alcohol for 1 min.Stain is applied and washed with waterBlot with absorbent paper

Differential StainsReact differently with different kinds of bacteriaUsed to distinguishGram Stain: one of most important staining techniques 1st used in 1884 by Hans Christian GramSearching for method to see cocci in lung tissueSeries of staining and decolorization steps According to cell wall compositionGram-positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of wall) PURPLEGram-negative bacteria have walls with thin layer of peptidoglycan (10% of wall) and high lipid content PINK

Differential StainsGram Stain:Heat-fixed smear covered with basic purple dyeprimary stain (crystal violet)2. Washed and covered with iodine (mordant), washed off3. Washed with alcohol (decolorizing agent). Removes purple from the cells of some spp4. Alcohol is rinsed and slide stained with safranin (basic red dye)5. Smear washed, blotted dry and examined

GRAM POSITIVEPurple dye and iodine combine in cytoplasm and color it dark violetThicker peptidoglycan cell wallTraps CV-I inside cellGRAM NEGATIVELose the dark violet after decolorizationSafranin applied to turn bacteria pinkLayer of lipopolysaccharideAlcohol disrupts outer lipopolysaccharide layer and CV-I complex washes out

E. coliStaphylococcus epidermidis

http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/keen/Gramstainkeen.htm

Special StainsUsed to color and isolate specific parts

Capsule of Klebsiella pneumoniaeare Endospore of Bacillus thuringiensis Flagella of Salmonella

Aseptic TechniqueMethod that prevents the introduction of unwanted organisms into an environment

Wear appropriate protective equipmentDisinfect working area before you startGather all necessary materialsDo any labelingNever light burner while wearing glovesProperly adjust the flame of the bunsen burner (small blue cone, not a large plume, nor is it orange)Once you have flamed your loop DO NOT lay it down or touch it to any surfaceAllow loop to cool before you pick up organismsEnsure you are transferring the correct organismsAlways keep caps and tops in your hand.Always tape inoculated plates together and incubate them upside downDiscard contaminated materials properly, return supplies to proper storage locations and clean up messDisinfect work area when done

Flaming loopHeat from the base of the wire first and slowly move towards the loop (tip). Heat the wire until it is red hotThe metal must glow orange-red before sterilization is considered complete

Test tubeRemove caps from liquid specimens and replace the caps of the test tubes with the same hand that holds the loop. The caps must be held during the entire procedure and not placed on the desktopFlame the neck of the tube. Remove a loop full of the culture with the cooled loop and briefly flame the opening of the tube again

Petri DishFlame the loop and then open the petri dish just enough to allow the entry of the loop.

Put in Biohazard BagContaminated gloves, paper towels, petri plates, swabs, other non-sharp items

Spilled Culture MaterialClean by saturating with alcohol and wiping with paper towels (dispose of in BioHazard)

WORKS CITEDhttp://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Bacterial_Smear_&_Staining/06_fix_specimen_P1092682.JPGhttp://www.biosynth.com/media/verschiedene/dyes1.JPGhttp://www.bigroom.org/images/Sally_MB.jpghttp://student.ccbcmd.edu/courses/bio141/labmanua/lab12/diseases/uti/images/gnrod.jpghttp://people.uleth.ca/~selibl/Biol3200/Morphology04/Btendo.jpghttp://bioinfo.bact.wisc.edu/themicrobialworld/S.typhi.Fla.jpg