Microscope Part for Lab Final

What is TM?

Total Magnification

Ocular Lens (10x) x Objective Lens

Ex.

• Scan 10 × 4 = 40x

• Low Power 10 × 10 = 100x

• High Power 10 × 40 = 400x

• Oil Immersion 10 × 100 = 1000x

What does the Condenser do in Microscopes?

Condenses the light from the illuminator onto the specimen.

What is Simple Stain? Theory?

The stain is made of solvent and a colored molecule (chromagen). Chromagen has chromophore that provides color and auxochrome which is the charged part. The Stain binds to the cells through ionic or covalent bonds, often positively charged and attracted to negatively charged bacterial surface.

Steps of Simple Stain?

1. Place a small drop of water on slide

2. mix bacteria well with the water on slide

3. air dry

4. pass smear on flame until dry

5. stain!

What is Negative Stain? Theory?

• to examine morphology and cellular arrangement. (some bacteria are too delicate to withstand heat fixation.)

• Useful for determining accurate size and measurements.

• Produces minimal cell shrinkage.

Theory

• Chromagen in negative stains is ACIDIC. Negative charge on the surface of the bacteria repels negatively charged chromagen. Cell is unstained, Background becomes stained.

What are the steps of Negative Stain

1. Add acidic stain on one end of the microscope slide.

2. Add organism and emulsify with a loop

3. Take a clean slide and place on surface of first slide and draw it back into the drop

4. when drop flows across width of the spreader slide

5. push the spreader slide to the other end of the first slide.

6. air dry NO HEAT FIX!

What is Gram Stain? Theory?

• To distinguish between Gram positive cells, and Gram negative cells.

• Also for Morphology, Size, Arrangement.

Gram Negative:

• Higher lipid content

• thinner peptidoglycan layer

• The Alcohol/Acetone extracts lipids

  • makes cell wall more porous then incapable of retaining crystal violet iodine complex

Gram Positive:

• Thicker cell wall with more cross-links

• The stain sticks to it

• Less susceptible to decolorization

What are the steps of Gram Stain?

1. Heat fix emulsion

2. Cover smear with crystal violet for 1 minute

3. Rinse with Distilled Water → Clear runoff

4. Cover smear with Gram’s Iodine for 1 minute

5. Rinse with Distilled Water → Clear runoff

6. Decolorize with 95% ethanol or acetone let it tickle down slide until clear, rinse with distilled water

7. Counterstain with safranin for 1 minute and rinse with distilled water.

What is Endospore Stain? Theory??

• Certain bacteria can produce endospores

  • Dormant form of bacterium

  • Resistant to heat and chemicals

  • Tough outer layer of the protein keratin

• May be located..

  • Central (middle)

  • Terminal (end of the cell)

  • Sub terminal (end of middle of the cell)

• Can be Spherical or Elliptical

Steps to Endospore Staining?

1. Heat fixed

2. Cover smear with strip of bibulous paper and Apply Malachite Green Stain

3. Steam for 6 minutes (Paper must be moist with stain)

4. Grasp slide with slide holder remove paper and dispose. Gently rinse with water

5. counterstain with Safranin for 1 minute and rinse with water.

Know the bacterias morphology and gram reaction for these bacterias.

• E. coli

• Staphylococcus aureus 

• Micrococcus luteus 

• Bacillus subtilis 

• Enterobacter aerogenes

• E.coli → gram negative rod

• Staphylococcus aureus → gram positive cocci

• Micrococcus luteus → gram positive cocci

• Bacillus subtilis → gram positive rod

• Enterobacter aerogenes → gram negative rod