Lab 6 HW & Handout Sum 20 copy

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Lab 6_(Acid-Fast and Spore Stains)______________ _________ Name:___________________________ Ex. 3-7: Acid-Fast Stain (Ziehl-Neelsen Method) Terms: 1. Acid-Fast Bacilli (AFB): rod shaped bacteria that retain the primary fuchsia-colored stain; they tend to clump together because of their hydrophobic (water-repelling) cell surface. 2. Mycolic acid: A waxy substance that gives Acid-Fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution. Procedure: Review the steps in the procedural diagram of Figure 3-58 on page 178. (Note that we will not use the serum in step 1) You can use one of your extra smears you made for gram staining if you have any or just make a new smear using your unknown organism - remember to heat fix once dry before staining. Do Not Use the steaming set up & don’t use the filter paper! Just use your staining tray 1. Apply Zn Carbolfuchsin for five minutes then rinse with water 2. Decolorize with acid alcohol drop wise over a paper towel until drops are clear 3. Rinse with water over staining tray 4. Counter stain with Methylene blue one minute then rinse with water 5. Blot dry with bibulous paper 6. Observe under oil immersion and record your results

Lab 6_(Acid-Fast and Spore Stains)______________ _________ Name:___________________________ Questions: 1. How does heating the bacterial smear during a ZN stain promote entry of carbolfuchsin into the Acid-Fast cell wall? (What happens to lipids when they are heated?) Heating melts the waxy mycolic acid in Acid-Fast positive cells. Since the carbolfuchsin is lipid soluble, it enters the “liquid” mycolic acid. Acid-Fast negative cells are also stained with carbolfuchsin. 2. Are Acid-Fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain? What makes this a differential stain is the fact that the carbolfuchsin is easily removed from the walls of Acid-Fast negative cells, but it is “locked into” the mycolic acid of Acid-Fast positive cells. As with the Gram stain, this is a differential test not because of the stains, but rather how the different cell types respond to decolorization. 3. Why do you suppose the Acid-Fast stain is not as widely used as the Gram stain? When is it more useful than the Gram stain? The Gram stain divides the microbial world up into two main groups, with the Gram-negatives outnumbering the Gram- positives (about 60–40). A Gram stain result for an unknown organism considerably narrows the field of possible organisms it could be regardless of the result. The vast majority of cells are Acid-Fast negative, so an Acid-Fast result for an unknown is not likely to eliminate many organisms. It is most useful when a sample is brought in from a patient with symptoms consistent with a disease caused by an Acid-Fast organism. (That is, when the percentages are good that an AFB—Acid-Fast Bacillus—is present.) Then the staining result can support or eliminate AFBs as the causative agent of the condition. Ex. 3-9: Endospore Stain (Schaeffer-Fulton Method) (*Define & Draw a picture for the terms with an *) Terms: 1. Endospore: A dormant form of bacterium that allows it to survive poor environmental conditions such as nutrient depletion, dehydration, suboptimal temperatures and altered O2 availability. The spore coat ultimately protects the organism’s genetic information (its DNA) so that the genome can be expressed later to produce a vegetative cell when growth conditions are more favorable. 2. Keratin: A family of fibrous structural proteins. It is found in the skin, hair and fingernails of humans, and a stronger version is found in the hooves, horns and shells of other animals. It is the main protein that makes up the outer covering of bacterial endospores. 3. Vegetative cells: An actively metabolizing cell 4. Spore mother cells: Produces spores *5. Central: Spores located in the middle of the cell *6. Terminal: Spores located at the end of the cell *7. Subterminal: Spores located at the between the end and middle of the cell. 8. Spherical: Spores also may be differentiated based on shape, spherical (round). 9. Elliptical (oval): the shape of the spore.

Lab 6_(Acid-Fast and Spore Stains)______________ _________ Name:___________________________ 10. Saprophyte: A heterotroph that digests dead organic matter; a decomposer Procedure: Write out the steps in the procedural diagram of Figure 3-68 on page 189. 1. Begin with a heat-fixed emulsion. As many as four small emulsions can be put on one slide. 2. Cover the smear with a strip of bibulous paper. Apply malachite green stain. Steam (as shown in Figure 3-69) for 5 minutes or more, if necessary. Note: the slide needs to steam for 5 minutes. If the water isn’t boiling yet, wait until it is. Keep the paper moist with stain, but don’t overdo it. That’s messy and a waste. Perform this step with adequate ventilation, gloves, and eye protection. 3. Grasp the slide with a slide holder. Remove the paper and dispose of it properly. Gently rinse the slide with water. 4. Counterstain with Safranin stain for 1 minute. Rinse with water. 5. Gently blot dry in a tablet of bibulous paper or paper towels. (Alternatively, a page from the tablet can be removed and used for blotting.) Do not rub. When dry, observe under oil immersion. What color are the spores going to be? GREEN (bluish green) Questions: 1. Why does this exercise call for an older (5-day) culture of Bacillus ? (Why or when do bacteria produce endospores?) Spores are produced by Bacilli when the organisms are deprived of nutrients. Older cultures are more likely to have depleted nutrients in the medium and begun sporulation. 2. What does a positive result for the spore stain indicate about the organism? What does a negative result for the spore stain indicate about the organism? (Again, think about why or when bacteria produce endospores.) If you get a positive result for the spore stain, it indicates (barring contamination) that the organism produces spores. A negative result for the spore stain might mean the organism cannot produce spores, or it might mean the organism CAN produce spores but just ISN’T because the conditions were not right to induce sporulation. 3. Why is it not necessary to include a negative control for this stain procedure? (What is stained by the safranin dye?) Vegetative cells act as controls. They will stain red, whereas any spores present should stain green. Frankly, the hard part is getting the spores to stain, not the vegetative cells. 4. Spores do not stain easily. Perhaps you have seen them as unstained white objects inside Bacillus species in other staining procedures. If they are visible as unstained objects in other stains, of what use is the endospore stain? (Are endospores the only structures inside a bacterial cell that do not stain?) When the structure is an unstained white object, it might be a spore, or it might be a storage granule or other cellular inclusion of some sort. The spore stain technique provides good evidence that the structure stained is, in fact, a spore.

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Lab 6_(Acid-Fast and Spore Stains)______________ _________ Name:___________________________ ******This part of the handout will be used in class, read over before class****** Materials Per table : 1. ______2 Test tubes w/ water 2. ______Hot plate 3. ______Large beaker filled half-way with Deionized water 4. ______Staining screen 5. ______Box of slides Per individual : 1. _____Staining tray 2. _____Loop 3. _____ glass slides 5. ______1 precut bibulous paper (for slide) 6. ______Grease pen Per lab pair : 1. _____Water bottle (deionized water in sink) 2. _____Bibulous paper (for blotting) 3. _____Light box 4. Dye(s) _____ZN carbolfuchsin _____Acid Alcohol _____Methylene Blue _____Malachite Green _____Safranin 4. _____ incinerator w/tray Ex. 3-9: Endospore Stain (Schaeffer-Fulton Method) Each student will make heat-fixed bacterial smear. Draw one circle and a square on the bottom of your slide using a grease pen. (This procedure does not require a negative control. Do you know why?) Flip the slide over and write your unknown number on it. Add a loop full of water to the circle and square. 1. Add a very small amount of your unknown from the plate or slant to the square mixing with the water and spreading within the square. 2. Add a very small amount of the Endospore positive stock culture provided mixing with the water and spreading with in the circle. +

Lab 6_(Acid-Fast and Spore Stains)______________ _________ Name:___________________________ Name of organism________________________Shape______________________ Color of cells should be _________________ Color of endospores should be_________________________ 3. Allow slide to air dry. 4. Heat fix slides using light box (2 minutes should be adequate) Ex. 3-9: Endospore Stain (Schaeffer-Fulton Method) Did the control come out as expected? _______If NO you can’t trust your unknown results. Unknown results: Draw and *****label******on your unknown report Do you see endospores? _____________What color are they? ________________ Do you see cells? ________________What color are they? ________________ Ex. 3-7: Acid-Fast Stain (Ziehl-Neelsen Method) Each student will make heat-fixed bacterial smear. 1. Add a very small amount of your unknown from the plate or slant to the square mixing with the water and spreading within the square. 2. Allow slide to air dry. 3. Heat fix slides on light box before staining Unknown results: Also record on your unknown report. Shape: __________________Arrangement: _______________________ Color: _______________________________ Acid-fast result: ____________________________ *Special note: if your unknown has an endospore it is NOT Acid Fast Positive***** Each student will observe a prepared slide of Mycobacterium tuberculosis. 1. Describe, draw what you see under the microscope:

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