Observing organisms through a Microscope Flashcards
(TEM) Transmission Electron Microscope
Beam of electrons pass through a specifically prepared ultrathin section of the species.
Can produce magnification of 10,000-10,000,000x
Electron Microscope
Observes objects smaller than 0.2 micrometers (viruses & bacteria)
Beam of electrons used instead of light
Images are always black & white
Cant be used to view living specimens
Coarse Focusing Knob
Only to be used with Scanning Lens (4x)
Moves stage up and down
Big knob on arm Inner to fine focus
Scanning Objective
4x
Always start with this power to focus on object
Fine Focusing Knob
Ued to bring specimen into sharp focus
Smaller knob on arm
Use only after fine coarse knob is used to focus
Ocular
Eyepiece
10x
Total Magnification=(10x) times objective
Oil-Immerson
100x
Must use Immerson Oil
Technique used to increase resolving power of microscope
Low Power
10x
Scanning Electron Microscope
Provides striking 3-D image of specimen
Useful in studying surface structures of intact cells & viruses
Magnification is from 1,000-500,000x
Acid Dyes
Negative charge bind to positively charged cell
structures
Acid Fast Staining
Is a differential stain used to identify acid-fast organisms
Used to find Members of Mycobacterium (T.B. & leprosy)
Waxy material in cell walls of bacteria.
1)Apply stain carbo fuchsion for 30 sec.
2)Heat fix cells to slide using flame.
3)Decolorize w/acid alcohol for 15-20 sec
4)Apply countestain of methylene blue for 30 sec
5)Rince access stain.
6)Observe under microscope.
Gram Negative
Bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin.
E-coli
Gram Positive
Bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet.
Staphylococcus epidermidis
Bacillus subtilis
Differential Stain
Staining process which uses more than one chemical stain.Used for microbe identification
Gram-stain & Acid Fast stains are most frequent used.
Simple Staining
1) Perform a bacterial smear,
2) Saturate the smear with basic dye for approximately 1 min. Use crystal violet, safranin, or methylene blue.
3. Rinse the slide gently with water.
4. Carefully blot dry with bibulous paper.
5. Observe the slide under the microscope,
Gram Stain
Apply crystal violet to a heat-fixed smear of a bacterial culture.
Apply iodide, (mordant)
Decolorization with ethanol or acetone.
Counterstaining with safranin.
Observe under oil immersion under microscope
Either gram + or gram -
Smear
Bacterial cells, spread on a slide for microscopic examination or on the surface of a culture medium.
Heat fixing
Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the sample to more readily take up stains.
Allow the smear to air dry.
After the smear has air-dried, hold the slide at one end and pass the entire slide through the flame of a Bunsen burner two to three times with the smear-side up.
Now the smear is ready to be stained.
Negative staining
The background is stained, leaving the actual specimen untouched, and thus visible.
1) Remove a small amount of culture with an inoculating loop and disperse it in the drop of stain without spreading the drop.
2) Use another clean slide to spread stain
3) Allow to air dry without heat
4) Observe under oil immersion
Staining
Coloring microorganisms with dyes.
Kills microorganisms and attaches them to slide.
Preserves various parts of microbes in natural state with minimal distortion.
Basic dyes
This dye have positive charge to negatively charged molecules.
Crystal Violet, Methylene Blue, Safranin , basic fuchsin.
Acidic Dyes
Dye which has negative charge so they bind to positively charged cell structures like some proteins.
