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Micro lab objectives


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Lab 5
Describe the procedure for heat fixation.
Place 1/2 drop of water on slide; remove small amount of culture from the agar and touch a it few times to water (if using broth culture simply place 2-3 drops on the slide). Flame the loop and use it to spread the suspention over the entire slide; let dry. Pass slide through flame several times (film side up) till uncomfortable to touch.
Lab 5
Be able to recognize direct and indirect staining.
Made you look!
Lab 6
State why the gram stain is said to be a differential stain.
It differentiates between gram-positive and gram-negative bacteria.
Lab 6
Describe the differences between a gram-positive and a gram-negative cell wall.
Gram-positive cell walls are 20-80 nm thick, consisting of 60-90% peptidoglycan in many interlinking layers with teichoic and lipoteichoic acids extending through the wall. Gram-negatives have few layers of peptidoglycan (10-20%) that are surrounded by an outer membrane of phospholipids, lipopolysaccharides and proteins.
Lab 6
Describe a theory as to why gram-positive bacteria retain the crystal violet-iodine complex while gram-negatives become decolorized.
In gram+ the combined crystal and iodine form a large molecule that enters the cell which is then trapped when the alcohol/acetone mixture dehydrates the peptidoglycan and causes it to be less permiable. Gram-can't contain the dye because the Alcohol/acetone dissolve the outer membrane and the thin peptidoglycan can't keep the dye in the cell.
lAB 6
Describe three conditions that may result in a gram-positive organism staining gram-negatively.
Poor technique, such as overheating, over decolorization, and too much washing; old cultures may not retain dye; the typeof organism, some retain stain better than others.
Lab 6
Determine if a bacterium is gram-positive or gram-negative when microscopically viewing a gram stain preparation and state the shape and arrangement of the organism.
Made you look again!
Lab 6
State the chemical nature and major functions of bacterial capsules.
Is usually composed of polysaccharide, polypeptide, or both. They serve to resist phagocytosis and adhere to surfaces.
Lab 6
State the procedure for the gram stain.
Heat fixate the bacterium to the slide and stain for 1 min. with crystal violet and wash. Then stain with gram's iodine solution and then his decolorizer for 1 min each, washing after every one. Finally stain with safranin for 1 min, wash and blot.
Lab 6
Recognize capsules as the structures observed when microscopically viewing a capsule stain preparation.
All your base are belong to us.
Lab 7
Name two endospore-producing genera of bacteria. State the function of bacterial endospores.
Bacillus and Clostridium. They are resistant dormant survival forms.
Lab 7
Recognize endospores as the structures observed in an endospore stain preparation. Identify a bacterium as an endospore-containing Clostridium by its "tennis racquet" appearance.
I'm not going to tell you.
Lab 7
Define the following flagellar arrangements: monotrichous, lophotrichous, amphitrichous, peritrichous, and axial filaments.
1. monotrichous - a single flagellum at one pole.
2. amphitrichous - single flagella at both poles.
3. lophotrichous - two or more flagella at one or both poles of the cell.
4. peritrichous - completely surrounded by flagella
5. axial filaments (endoflagella) wrap around the spirochete towards the middle from both ends and are located above the peptidoglycan cell wall but underneath the outer membrane.
Lab 7
State the chemical nature and function of bacterial flagella.
Noncontractile, semi-rigid, helical tubes composed of protein and anchored to the bacterial cytoplasmic membrane and cell well by means of disk-like structures. Allow many bacteria to propel themselves.
Lab 7
1. Recognize bacterial motility when using phase-contrast or dark-field microscopy.
2. Interpret the results of Motility Test Medium.
3. Recognize monotrichous, lophotrichous, amphitrichous, and peritrichous flagellar arrangements.
Yada yada yada..
Lab 7
Describe three methods of testing for bacterial motility and indicate how to interpret the results.
1. Direct observation by means of phase-contrast (bacterium will be darker than field) or dark field (bacterium will be lighter)microscopes. Simply watch for movement.
2. Motility Test medium: stab-tube agar is a soft gell that will permit motile bacteria to move but others will remain in origional location. Look for migration of inoculum.
3. Flagella staining: special staining of flagella so they become visible.
Lab 8
State the chemical nature and function of enzymes.
Enzymes catalyze all the various chemical reactions of which the organism is capable; they are composed of proteins.
Lab 8
Describe a method of testing for casein hydrolysis and state how to interpret the results.
Incoulate a divided Skim Milk agar plate with the two inoculums and incubate. If the protein casein was hydrolised by protease, them the agar will be clear, if not it will be opaque.
Lab 8
Name the general end products which may be formed as a result of the bacterial fermentation of sugars and describe how these end products change the appearance of a broth tube containing a sugar, the pH indicator phenol red, and a durham tube.
Acid and gas are the end products. If acid is produced then the indicator will turn yellow; gas production will create a bubble in the durham tube.
Lab 8
State the pathway for the breakdown of tryptophan to indole.
The enzyme tryptophanase converts the amino acid tryptophan into molecules of indole, pyruvic acid, and ammonia.
Lab 8
State the pathway for the detection of sulfur reduction in SIM medium.
Some bacteria are capable of breaking down sulfur containing amino acids to produce hydrogen sulfide (H2S). SIM contains S compounds that, if reduced will react with Fe to form black ferric sulfide as a visible incator of H2S production.
Lab 8
State three reactions that may be tested for in SIM medium and describe how to interpret the results.
Sulfur, indole, and motility. Black color indicates H2S, red indicates indole, migration of bacteria indicates motility.
Lab 8
State the function of the enzyme catalase and describe a method of testing for catalase activity.
Initiates the breakdown of hydrogen peroxide (H2O2) into water (H2O) and free oxygen (O2). Add hydrogen peroxide to each culture and look for the release of oxygen as a result of hydrogen peroxide breakdown.
Lab 8
Describe a method of testing for starch hydrolysis and state how to interpret the results.
Inoculate half a starch plate with one organism and the other half with the other to be tested. After incubation, add iodine; if the starch was hydrolized the iodine will not react and the area around the bacterium will be clear, otherwise the iodine will react with the starch and the agar will turn dark brown/black.
Lab 5
Define the following: acidic dye, basic dye, direct stain, and indirect stain.
Acid dyes contain the color portion in the negatively charged ion. Basic contain the color portion in a positive ion. Basic dye combines with the negatively charged cytoplasm to form direct stains. Acidic dye is repelled by the negatively charged cytoplasm resuting in an indirect stain.
Lab 5
Perform an indirect stain when given all the necessary materials. State why the dye is not washed off when doing an indirect stain.
Place small amount of nigrosin on slide and add organism. Use another slide to spread the mixture across the slide so there are both light and dark areas. The dye is not washed off because the goal is to stain the background of the slide.

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