chapter 3 observing organisms through a microscope units of measurements microscopy: the instruments...
TRANSCRIPT
CHAPTER 3Observing Organisms Through a Microscope
Units of Measurements
Microscopy: The Instruments
Preparation of Specimens
We will be looking at very small things…
Com
pound Light M
icroscope
Compound Light Microscope• Total Magnification
– Objective lens power x ocular lens power
• Resolution– Ability of the lenses to
distinguish fine detail– Resolution power of 0.2 μm, – Distinguish 2 points 0.2 μm
apart
• Refractive index– Change by staining specimens– Two different mediums– Rays change more directions
• Oil Immersion– Same index as glass– Improves resolution
Staining• Fixed: kills/attaches org. to slide
– Thin film spread over slide (smear) and allowed to dry
– Pass through flame of Bunsen burner several times or cover with methyl alcohol for 1 min.
– Stain is applied and washed with water
– Blot with absorbent paper
STAINS• Salts composed of a positive and a negative ion
– One of which is colored (chromophore)• Basic Dyes: positive ion
– Attracted to negatively charged bacteria cell– Ex: Crystal violet, methylene blue, malachite
green and safranin• Acidic Dyes: negative ion
– Stain colors the background surface– Observing overall cell shape, size and capsule– Ex: acid fuchsin, nigrosin
SIMPLE STAIN• Aqueous or alcohol sol’n of a single
basic dye• Highlight the entire microorganism• Applied to fixed smear for length of
time, washed, dried• Mordant (used to intensify) may be
added– Increases affinity of a stain– Coat a structure to make it thicker
and easier to seeExamples: methylene blue*, crystal violet, safranin
Differential Stains• React differently with different kinds of bacteria• Used to distinguish• Gram Stain: one of most important staining techniques
1. Heat-fixed smear covered with basic purple dye-primary stain (crystal violet)
2. Washed and covered with iodine (mordant), washed off
3. Washed with alcohol (decolorizing agent). Removes purple from the cells of some spp
4. Alcohol is rinsed and slide stained with safranin (basic red dye)
5. Smear washed, blotted dry and examined
GRAM POSITIVE• Purple dye and iodine
combine in cytoplasm and color it dark violet
• Thicker peptidoglycan cell wall
• Traps CV-I inside cell
GRAM NEGATIVE• Lose the dark violet after
decolorization
• Safranin applied to turn bacteria pink
• Layer of lipopolysaccharide
• Alcohol disrupts outer lipopolysaccharide layer and CV-I complex washes out
E. coliStaphylococcus epidermidis
Special StainsUsed to color and isolate
specific parts
Capsule of Klebsiella pneumoniaeare
Endospore of Bacillus thuringiensis
Flagella of Salmonella
file:///E:/Chapter_03/A_PowerPoint/a_Lecture_Outline/staining.html
WORKS CITED• http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Bacterial_Smear_&_Staining/06_fix_specimen_P1092682.JP
G
• http://www.biosynth.com/media/verschiedene/dyes1.JPG
• http://www.bigroom.org/images/Sally_MB.jpg
• http://student.ccbcmd.edu/courses/bio141/labmanua/lab12/diseases/uti/images/gnrod.jpg
• http://people.uleth.ca/~selibl/Biol3200/Morphology04/Btendo.jpg
• http://bioinfo.bact.wisc.edu/themicrobialworld/S.typhi.Fla.jpg