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