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Tools and Techniques in Biology
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Centrifuge Instrument used to separate solutions according to density
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Least dense material collects on top
Most dense parts settle to the bottom
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Chromatography Thin Layer Chromatography (TLC) or paper chromatography
Procedure used to separate pigments based on density
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Least dense pigments are drawn farther up the paper
More dense pigments remain towards the bottom of the paper
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Uses of TLC Used to identify unknown solutions
The Rf value of a particular compound will always be the same
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Tools For Detection of Substances
Indicators show a reaction (usually a color change) to signify the presence of a substance Many indicators are used to test pH level
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pH Scale used to determine if a substance is an acid or a base
Ranges from 0 to 14 7 is neutral Less than 7 is an acid Greater than 7 is a base
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Testing pH Litmus paper - contains a substance that detects the presence of acids or bases
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Two types: 1. Red litmus - if it turns blue It’s Base!
2. Blue litmus – if it turns red It’s Acid!
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2)
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat**
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat**
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat** Orange in presence of sugar
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat** Orange in presence of sugar Phenol Red
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat** Orange in presence of sugar Phenol Red Red
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat** Orange in presence of sugar Phenol Red Red Magenta in the presence of base
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat** Orange in presence of sugar Phenol Red Red Phenolpthalein
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat** Orange in presence of sugar Phenol Red Red Phenolpthalein Colorless
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Color of Positive Reaction
Other indicators Indicator Original Color Color of Positive Reaction Lugol’s Iodine Brown Dark blue / Black in presence of starch Bromthymol Blue Blue Yellow in presence of acid (also tests for CO2) Benedict’s Solution **Must heat** Orange in presence of sugar Phenol Red Red Phenolpthalein Colorless Pink in presence of base
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Benedict’s Solution Bromthymol Blue Lugol’s Iodine
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Tools For Detection of Microscopic Objects
Dissecting Microscope Compound Microscope Electron Microscope
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Dissecting Microscope
Enlarges the image - Low magnification Has 2 optical lenses and 2 objective lenses Used to study the surfaces of solid specimens Useful in performing dissections Also called stereo microscope
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Compound Microscope Used to observe small objects on a slide
Specimen must be thin enough to let light pass through Has 1 optical lens and 2 or 3 objective lenses Can view living specimens When looking through microscope the image will appear: Enlarged Reversed Flipped upside down
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Care and Handling of the Compound Microscope
There are only a few ABSOLUTE rules to observe in caring for the microscopes you will use. Taken care of these instruments will last many decades and continue to work well. Please report any malfunctions immediately. 1. ALWAYS use two hands to carry the scope - one on the arm and one under the base - NO EXCEPTIONS! NEVER carry the scope upside down, for the ocular can and will fall out.
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2. Always use the proper focusing technique to avoid ramming the objective lens into a slide - this can break the objective lens and/or ruin an expensive slide. *Do not use course adjustment knob when using high power objective lens!**
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When finished using the microscope…
…Always turn off the light and move objective lens back to low power
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Viewing an object under the microscope
1. Plug in microscope and switch light on 2. Switch to the lowest power objective lens 3. Adjust the coarse focus to raise the nose piece 4. Place slide on stage. Secure with stage clips if desired. 5. Look at the slide and place it so the specimen is over the light aperture in the stage.
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5. Lower objective lens to lower limit (close to slide).
6. Look through eyepiece and slowly raise the lens using the coarse focus knob until you see the image come into focus 7. Adjust fine focus similarly. 8. Center the image and adjust the light using the diaphragm.
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**Do NOT use coarse adjustment
9. Now switch objectives to a higher power. 10. Readjust fine focus and light (diaphragm) as needed. **Do NOT use coarse adjustment in high power. **
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Preparing a Wet-mount slide
Place a clean slide on the lab table. Handle slides at the ends, not the center, to avoid getting fingerprints in the viewing area of the slide. Add specimen to the slide. For liquid samples, place one small drop in the center of the slide. For solid samples, place the sample in the center of the slide and add one drop of water.
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Hold the coverslip by the edges to avoid fingerprints.
Lower coverslip on specimen from a 45˚ angle. This helps to avoids air bubbles. Never view a slide without a coverslip. The coverslip protects the objective lens from the liquid on the slide.
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Stains Stains are chemicals that dye parts of cells (nuclei, cytoplasm, cell membranes and cell walls) for viewing under the microscope Natural cheek cells Stained cheek cells
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Observations may be improved by using a biological stain.
Microbial cytoplasm is usually transparent so it is difficult to view cells using the microscope Observations may be improved by using a biological stain. Natural cheek cells Stained cheek cells
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1. Lugol's Iodine Solution
Commonly used to stain plant cells – turns dark in the presence of starch and cell walls contain a lot of starch
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2. Methylene blue Commonly used to stain dead animal cells
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Staining technique Prepare a wet mount slide.
Add one drop of stain on one side of cover slip. Gently touch a paper towel to the other side of the coverslip. The paper towel will draw the water and the stain out from under the coverslip. Technique for Adding a Stain when making a Wet Mount
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Lab #13
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Other types of Microscopes
TRANSMISSION ELECTRON MICROSCOPE SCANNING ELECTRON MICROSCOPE
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Electron Microscope Used to observe very tiny objects
Can produce magnifications up to 500,000X Use a narrow beam of electrons instead of light Specimens must be embedded in plastic and cut into thin slices ONE DISADVANTAGE IS THAT SAMPLES MUST BE VIEWED IN A VACUUM – SO NO LIVING SAMPLES CAN BE STUDIED! Can see inside cells
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Electron Microscope Uses electrons for energy source
Specimen must be killed Transmission electron microscope (TEM) – take pictures of slices of specimen It is powerful enough to view all cell organelles such as mitochondria
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TEM Examples
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Scanning electron microscope (SEM) –
take pictures of surface of specimen
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E. COLI BACTERIA HEAD OF A SMALL BLACK ANT POLLEN
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SEM Examples
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