Term
| Describe the procedure for heat fixation (agar) |
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Definition
a. Place ½ a drop a deionized water on the clean slide
b. Aseptically remove a small amount of culture and touch it 2-3 times to the water
c. Incinerate the remaining bacteria on the inoculating loop
d. Spread suspension over approx. half of the slide to form a thin film
e. Allow to completely air dry
f. Heat fix bacteria by waving it through fire 6 times then 6 more times |
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Term
| Describe the procedure for heat fixation (broth) |
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Definition
g. Make a circle about the size of a nickel on the slide
h. Aseptically place 2 or 3 loops of the culture within the circle
i. With inoculating loop spread over the circle area
j. Allow to completely air dry
k. Wave slide through fire 6 time then 6 more times |
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Term
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Definition
| if the color portion is in the negatively charged ion (nigrosin , congo red) |
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Term
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Definition
| if the dye resides in the positive ion (methylene blue, crystal violet , safranin) |
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Term
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Definition
| when the positively charged color portion of the basic dye mixing with the negatively charged bacterial cytoplasm |
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Term
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Definition
| when the negative ion portion of the acidic dye forms a deposit around the organism instead of mixing with negatively charged bacterial cytoplasm. The organism is colorless |
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Term
| 3) State in chemical and physical terms the principle behind direct staining and the principle behind indirect staining |
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Definition
a. Direct stain – when the positively charged color portion of the basic dye mixing with the negatively charged bacterial cytoplasm
b. Indirect stain – when the negative ion portion of the acidic dye forms a deposit around the organism instead of mixing with negatively charged bacterial cytoplasm. The organism is colorless |
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Term
| why is the dye not washed off during an indirect stain |
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Definition
| the cells and dye will wash off cause its not heat fixed |
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Term
| 1) State why the gram is said to be a differential stain |
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Definition
| It differentiates between gram positive and gram negative bacteria |
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Term
| 2) Describe the differences between a gram positive and a gram negative cell wall |
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Definition
a. Gram positive – appears as a broad , dense wall 20-80 nm thick and consisting of numerous interconnecting layers of PG 60%-90% of the cell wall is PG
b. Gram Negative – contains only 2-3 layers of PG and is surrounded by an outer membrane composed of phospholipids, LP, LPS and proteins. Only 10% - 20% of gram negative wall is PG |
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Term
| 3) Describe a theory as to why gram positive bacteria retain the crystal violet- iodine complex while gram-negatives become decolorized. |
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Definition
a. Gram positive – crystal violet and iodine combine to form a larger molecule that precipitates out within the cell. The alcohol mixture causes dehydration of the multilayered PG thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell
b. Gram negative – alcohol mixture being a lipid solvent dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the PG is attached. The single thin layer of PG is unable to retain the crystal violet-iodine complex and the cell is decolorized |
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Term
| 4) Describe three conditions that may result in a gram positive organism staining gram negatively |
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Definition
a. The method and techniques used – overheating, over decolorization, too much washing
b. The age of the culture – more than 24 hours old may lose their ability to retain the crystal violet-iodine complex
c. The organism itself – some gram positive bacteria are more able to retain the crystal violet-iodine complex than others |
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Term
| a. State the procedure for the gram stain |
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Definition
- Put a drop of water on a slide
- Aseptically remove organism
- Spread all over the slide
- Let completely air dry
- 6 times through fire and 6 times again
- Put on crystal violet and let sit for 1 minute
- Wash with water
- Stain with grams iodine for 1 minute
- Wash with water
- Flood with grams decolorizer (ethyl alcohol and acetone) for 10-15 seconds
- Wash with water
- Stain with safranin for 1 minute
- Wash with water
- Blot dry with paper |
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Term
| 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 |
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Definition
| - Gram positive is purple , gram negative is pink |
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Term
| 7) State the chemical nature and major functions of bacterial capsules |
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Definition
- Composed of polysaccharide , polypeptide or both
Function:
1. Resist phagocytosis by the host’s phagocytic cells
2. Also help prevent bacteria from being engulfed by protozoans
3. Also help adhere to surfaces –resist flushing |
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Term
| 1) Name two endospore- producing genera of bacteria |
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Definition
| - Bacillus and clostridium |
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Term
| 2) State the function of bacterial endospores |
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Definition
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Term
| 5) Define the following flagellar arrangements |
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Definition
a. Monotrichous – one flagella on one side
b. Lophotrichous – a few on each end
c. Amphitrichous – one on each end
d. Peritrichous – flagella all around
e. Axial filaments – wrap around the spirochete towards the middle from both ends |
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Term
| 6) State the function of bacterial flagella |
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Definition
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Term
| 7) Describe three methods of testing for bacterial motility and indicate how to interpret the results |
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Definition
a. Direct observation of motility using special purpose microscopes
i. Phase contrast microscopy – gives optical effect of direct staining. Bacterium appears darker than the background
ii. Dark – field microscopy – gives optical effect of indirect stain. Organism will appear bright against the dark background
b. Motility test medium – stab an agar. If there is motility it will grow horizontal if no motility it will only grow along the stab line
c. Flagella staining – stain the organism and then you can see the flagella and what type they have – they are dead in order to stain so they will not be moving |
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Term
| 1) State the chemical nature and function of enzymes |
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Definition
| protein , speed up reactions |
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Term
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Definition
| functions within the cell |
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Term
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Definition
| secreted by bacteria into the surrounding environment in order to break down larger nutrient molecules so they may enter the bacterium |
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Term
| 3) Describe a method of testing for starch hydrolysis and state how to interpret the results |
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Definition
| use sugar agar. if bacteria have a white ring around it once the idodine is added then it is positive for starch hydrolysis |
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Term
| 5) Describe a method of testing for casein hydrolysis and state how to interpret the results |
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Definition
| use skim milk agar. one that has a clear ring around it is positive for casein hydrolysis |
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Term
| 7) 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 Durham tube |
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Definition
- acid end products or acid and gas end products
- acid --> fermentation
red = no acid orange/yellow = acid
bubble = gas no bubble = no gas
need acid to have gas red never has gas |
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Term
| 9) State the pathway for the breakdown of tryptophan to indole |
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Definition
| tryptophan is broken down by tryptophanase ---> indole |
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Term
| 10) State the pathway for the detection of sulfur reduction in SIM medium |
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Definition
| add Kovac's reagent. is there if there is sulfur reduction - black. |
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Term
| 11) State three reactions that may be tested for in SIM medium and describe how to interpret the results |
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Definition
indole - if has a red layer +
sulfur reduction - black +
motility |
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Term
| 13) State the function of the enzyme catalase and describe a mthod of testing for catalase activity |
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Definition
| add hydrogen peroxide to the culture. if it foams then it is breaking down the hydrogen peroxide. |
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