What are the main parts of the Microscope?
Ocular lens, objective lens, stage, condenser, diaphragm, illuminator, coarse focusing knob, fine focusing knob and body.
Ocular Lens
Re-magnifies the image formed by the objective lens.
What is the magnification of the Ocular Lens?
10X.
Objective Lens
Primary lenses that magnify the specimen.
What are the different Objective Lenses?
Scanning Lens, Low Power Lens, High and Dry Lens, and Immersion Oil Lens.
What is the magnification of the Scanning Lens?
5X.
What is the magnification of the Low Power Lens?
10X.
What is the magnification of the High and Dry Lens?
40X.
What is the magnification of the Immersion Oil Lens?
100X.
What is Total Magnification?
The Ocular Lens x the Objective Lens.
Stage
Holds the microscope slide in position.
Condenser
Focuses light through the specimen.
Diaphragm
Controls the amount of light entering the condenser.
Illuminator
The light source for the microscope.
Coarse Focusing Knob
Moves the stage of the microscope up and down to focus the image.
Fine Focusing Knob
Focusing the image to fine detail.
Body
Transmits the image from the objective lens to the ocular lens using prisms.
What is the Path of Light in a Microscope?
Illuminator, Diaphragm, Condenser, Stage, Objective Lens, Body, Ocular Lens.
Reflection
Light that is lost.
Transmission
Light that passes through.
Refraction
Light that is a problem.
Fluoresence
A type of transmission.
Resolving Power
The ability to see fine details in a specimen.
What are the colours for the highest resolving power?
Blue and Violet.
What happens when light is lost?
Darker image and less resolution.
What is the purpose of Immersion Oil?
Prevents refraction of light.
What are the three basic cell shapes?
Coccus, Bacillus, and Coccibacillus.
Coccus
Cirlce in shape.
Bacillus
Rod shaped.
Coccibacillus
Circle/rod shaped.
Diplo
Two
Staphylo
Multiple Planes.
Strepto
Lines.
Spirochete
Flexible body.
Spirilum
Rigid body.
How do you make a smear?
- Shake culture before use.
- Sterilize the innoculating loop in bactcinerator.
- Insert the innoculating loop in the culture.
- Transfer the innoculating loop from the culture, to the center of the slide.
- Spread the culture from the innoculating loop o
What is the purpose of heat fixing?
Kills the MO, and the MO adhere to the slide.
How do you sterilize the innoculating loop?
Sterilize by placing the innoculating loop inside of the bactcinerator for 7 - 10 seconds.
How do you know the innoculating loop is sterile?
The loop glows red for less than a second after being in the bactcinerator.
What is the Procedure for the simple stain?
1. Start out by creating a smear.
2. Place smear('s) on a raco after air drying and heat fixing.
3. One slide stained with methylene blue for 1 - 1 1/2 minutes.
4. One slide stained with Carbolfuchsin for 5 - 10 seconds.
5. One slide stained with Crystal
What is the purpose of a simple stain?
To determine size, shape and arrangements of bacteria.
What happens if the simple stain is left too long?
It can overstain and distort the stain.
What happens if the simple stain is not left long enough?
The cells will not be seen.
What are the reagents for the simple stain?
Carolfuchsin, Methylene Blue, or Crystal Violet.
What is a differential stain?
The use of two or more dyes that react differently with different kinds/parts of bacteria.
What are examples of differential stains?
Acid-fast stain, Endospore stain, and Gram stain.
What is the procedure for a Negative Stain?
1. Create smear, do not heat fix.
2. Apply one to two drops of nigrosin, India ink, or eosin solution to the bacteria.
3. Slide another slide on top of the smear from one end to the other.
4. Air dry, do not heat fix.
When is Negative Staining used?
For bacteria that do not stain well like some spirochetes, or when it is desirable to confirm observations made on the shape and size of bacteria. It is also good for viewing capsules.
Why is heat fixing or harsh stains not used in the Negative Stain?
They can change the shape of the cells.
Why in the negative stain do the stains not penetrate the cell?
Because of the repulsion between the negative charge of the stains and the negative charge of the bacterial wall. Instead they produce a dark bacground so the bacteria appear as unstained cells with a clear area around them.
What are the Reagents of the negative stain?
Nigrosin, India Ink, or Eosin.
How do capsules influence virulence of a pathogenic bacteria?
The thicker the capsule on the bacteria, the higher the virulence the bacteria can hold. It varies with each bacterial species.
How does heat fixation effect the visibility of a capsule?
Heat fixing is not used in this procedure do to shrinkage, or destroying, is likely to occur and creates a clear zone around the bacterium, which can be mistaken for a capsule.
What is the Procedure for the Capsule Stain?
1. Smake smear.
2. Air dry ONLY.
3. Primary Stain: Crystal Violet. (stains cell purple, stains milk dark purple = dark background). 4 - 7 minutes.
4. Destain: Copper sulfate. (Do not remove dark purple background).
5. Blot with bibulous paper.
What are the Reagents for the Capsule Stain?
Crystal Violet Primary Stain, Destainer with Copper Sulfate which also acts as a counter stain.
What are the two uses for Copper Sulfate in the Capsule Stain?
A destainer as well as a counter stain.
What three items have been identified in Capsules?
Polysaccharides, polypeptides and glycoproteins.
What is the purpose of a Capsule for a bacteria?
Prevents the bacterium from dessecation (drying out), and protects it.
What are examples of bacterium that have capsules?
E. coli, K. pneumoniae, and b. megaterium.
What genera of bacteria are identified by the Acid-Fast Stain?
Mycobacteria and Norcardia.
What are two of the diseases caused by them bacteria that are identified in the Acid-Fast Stain?
Mycobacteria - TB/Leprosy, Norcardia - Rare Respiratory Infections, and Cryptosporidium.
What is the procedure for the Acid-Fast stain?
1. Make smear.
2. Add CarbolFuchsin - Primary Stain.
3. Heat fix to drive stain into the cell. (Carbolfucshin)
4. Decolorize with acid-alcohol drop by drop.
5. Rinse with water quickly.
6. Counterstain with Methylene blue for 2 minutes.
7. Rinse with wate
What will Acid Fast bacteria stain in colour?
Pink/Red.
What is Non Acid Fast bacteria stain in colour?
Blue/Purple.
What are the Reagents of the Acid-Fast stain?
Carbolfuchsin, Acid Alcohol, Methylene Blue.
What genera of bacteria produce spores?
Bacillus anthracis, Clostridium tetani, C. botulinium, C. difficile, and C. perfringens.
What are diseases caused by spore forming bacteria?
Bacillus anthracis - Anthrax, Clostridium tetani - Tetanus, C. botulinium - Botulism, C. difficile - C. dif, C. perfringens - Gangrene.
What is the function of spores?
A protective function in harsh living environments of some MO.
What causes the low permeability and high resistance of spores?
They have low permeability and high resistance due to multiple coats surrounding the spore.
What are the Reagents of Spore Stain?
Malchite Green - penetrates spore, Safranin - stains cell.
What is the procedure in the Spore Stain?
1. Make smear.
2. Primary stain - Malachite Green.
3. Heat over boiling water for ten minutes - forces M.G. into spore. (green spore at end of procedure).
4. Rinse with water.
5. Counterstain with Safranin. (60 - 90 seconds). (pink/red cells, spores stay
What is the procedure for the Gram Stain?
1. Make Smear.
2. Heat fix smears.
3. Primary Stain: Crystal Violet (C.V.) - Purple. (30 seconds).
4. Mordant: Grams Iodine (IKI) - Plugs P.G. layer in G+ cells, nothing in G-. (1 min)
5. Destain: Alcohol - acetone mix. MOST IMPORTANT. It too long G+ lose
What do Gram Positive cells appear in colour?
Blue.
What do Gram Negative cells appear in colour?
Pink/Red.
What cell structure takes up crystal violet to produce a Gram positive bacteria cell?
The cell wall is plugged by IKI (grams iodine), which keeps the C.V (crystal violet) in the cell wall in gram + bacteria, in gram - bacteria it has no effect.
What are the Reagents of a Gram Stain?
Crystal Violet, Grams Iodine (IKI), Alcohol Acetone mix, Safranin.
What is the most important step in the Gram Stain?
Decolorizing stage with Ethanol Acetone mix.
Why is the Decolorizing agent the most important step in the Gram Stain?
If done too long it can wash the Crystal Violet out of the Gram + cell wall, if too short it can leave the Crystal Violet in the Gram - cell.
Differential Media
Meda that distinguishes between different groups of bacteria and permit ID of MO based on their biological characteristics. i.e., colour change.
Selective Media
Favor the growth of a particular MO, while inhibiting the growth of another.
Mannitol Salt Agar (MSA)-What genus and species of bacteria is this media used to identify?
Staphylococcus, S. Aureus.
How does MSA media work and what are positive results?
This media can undergo fermentation, which lowers the pH of the media which causes a colour change from pink -> yellow (differential). It's high salt concentration inhibits the growth of many MO (selective).
Is MSA selective, differential, or both selective and differential?
Both selective and differential.
Eosin Methylene Blue Agar (EMB) and MacConkey Agar- What are the bacteria identified with these media?
Identifies G - bacteria, especially E. coli.
How does EMB media work and what are the results?
It inhibits the growth of G+ bacteria, allowing the G- bacteria to grow (selective). When E. coli grows, it produces a green metallic color (differential), which is due to the large amount of acids produced by fermentation of lactose.
Is EMB selective, differential, or both selective and differential?
Both selective and differential.
Anaerobic Bacteria
Do not like 02, 02 can be toxic depending on type of Anaerobe. Grow in bottom.
Aerobic Bacteria
Like 02, grow at top.
Facilitative Anaerobe
Grow throughout. Can be with and without 02.
What is the Special equipment required for growth of strict anaerobes?
They need to be placed in a anaerobic box, with the caps unscrewed 1 turn so that the air can be sucked out of it during the procedure. If the caps are left on then the air stays in the and test will be void.
Why is a Streak Plate used?
Used to get isolated, pure colonies by transfer in agar.
How do you perform a Streak Plate?
4 areas, start with one from liquid culture, zig through 1 - 2, then zig 2 - 3, then zig 3 - 4. Innoculating in between.
Where will the most and least bacteria be on the Streak Plate?
Most bacteria will be in zone 1, with most isolated colonies in zone 3 - 4.