CHAPTER 3
Observing Organisms
Through a Microscope
Units of Measurements
Microscopy: The Instruments
Preparation of Specimens
We will be looking at very small things…
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
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: fuchsin, nigrosin
– Called Negative Staining
• Three types of staining techniques
– Simple, differential and special
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
see (flagella)
Examples: methylene blue*, crystal violet, safranin
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
Differential Stains
• React differently with different kinds of bacteria
• Used to distinguish
• Gram Stain: one of most important staining techniques 
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1st used in 1884 by Hans Christian Gram
Searching for method to see cocci in lung tissue
Series of staining and decolorization steps
According to cell wall composition
Gram-positive bacteria have cell walls that contain thick layers
of peptidoglycan (90% of wall)
PURPLE
– Gram-negative bacteria have walls with thin layer of
peptidoglycan (10% of wall) and high lipid content PINK
Differential Stains
•
Gram Stain:
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
GRAM NEGATIVE
• Purple dye and iodine
combine in cytoplasm
and color it dark violet
• Thicker peptidoglycan
cell wall
• Traps CV-I inside cell
• 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
Staphylococcus epidermidis
E. coli
http://www.microbelibrary.org/mi
crobelibrary/files/ccImages/Articl
eimages/keen/Gramstainkeen.htm
Special Stains
Used to color and isolate
specific parts
Endospore of Bacillus thuringiensis
Capsule of Klebsiella pneumoniaeare
Flagella of Salmonella
Aseptic Technique
• Method that prevents the introduction of
unwanted organisms into an environment
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Wear appropriate protective equipment
Disinfect working area before you start
Gather all necessary materials
Do any labeling
Never light burner while wearing gloves
Properly 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 surface
Allow loop to cool before you pick up organisms
Ensure you are transferring the correct organisms
Always keep caps and tops in your hand.
Always tape inoculated plates together and incubate them upside
down
Discard contaminated materials properly, return supplies to proper
storage locations and clean up mess
Disinfect work area when done
Flaming loop
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QuickTime™ and a
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• Heat from the base of the wire first and slowly move
towards the loop (tip). Heat the wire until it is red hot
• The metal must glow orange-red before sterilization is
considered complete
Test tube
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• Remove 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 desktop
• Flame 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 Dish
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• Flame the loop and then open the petri dish
just enough to allow the entry of the loop.
Put in Biohazard Bag
• Contaminated gloves, paper towels, petri
plates, swabs, other non-sharp items
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Spilled Culture Material
• Clean by saturating with alcohol and wiping
with paper towels (dispose of in BioHazard)
WORKS CITED
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http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Bacterial_Smear_&_Staining/06_fix_specimen_P1092682.JP
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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