Monday - Lecture Exam 1 Tuesday - Wednesday - Thursday - Quiz 4 Ch. 14 Homework Due. Fermented Food Feast. Parts A & B of Semester Project Due.Study Guide for Lab Exam 1 General Lab Procedures & Lab Safety 1. Know how/where to dispose of contaminated slides, streak plates, and glassware (beakers or test tubes). Top shelf - lab supplies, non contaminated materials, generally materials prepped for lab that day Middle shelf - contaminated glassware slides & test tubes Bottom shelf - used agar plates, 2. Explain why it is important to wait for instructions before proceeding with lab exercises. Make sure there are no changes to the lab, as it is suppose to be done, helps prevent wasted materials. 3. Define aseptic. Careful steps/procedures taken to prevent contamination. 4. List at least four things that can be done during lab to practice aseptic technique. Flame loops, clean counters, flame tubes/hold tubes properly, wash hands. 5. Explain what you should do if you accidently spill a broth culture on the bench or floor. 6. Explain what you should do if you accidently drop and break a non-contaminated beaker. 7. Explain what you should do if you accidently get a cut or burn during lab. 8. Know where the eyewash and body shower are located and how to use each. 9. Explain why it is important not to eat or drink in the lab. 10. List two things you should do to help prevent a fire or burns when using a Bunsen burner 11. Where is the fire extinguisher in our lab? front of room 12. Explain the procedure for evacuating the building, including the meeting location outside the building. with class to the left out doors,, stay together. Exercise 1: Effectiveness of Handwashing 1. Define normal microbiota (normal flora). Also explain why normal microbiota are important. Normal microbiota is the microobes that are always on our body that we have accumulated thoughout life, most have functions that are benificial to us. They are also important because they help crowd out transient microbiota and help maintain an equilibrium of microbiota on our bodies, not allow opportunistic pathogens take hold. 2. Define transient microbiota (normal flora). Transient microbiota are temporary foreign microbes on our body that generally cause harm. 3. Draw a diagram of the nutrient agar plate (quadrants) and explain how each quadrant was treated during lab. Be able to interpret the results shown in a picture or actual plate. 4. Explain how the widespread use of antibacterial soaps is affecting bacterial populations. We are wiping out weaker strains, leaving strong strains to keep reproducing. 5. What are the most effective handwashing agents/methods, based on what you learned during this lab? Inconclusive results. Exercise 2: Microscopic Technique & Measurement 1. Be able to identify parts of the microscope and explain the function of each part. Be sure you know the name, individual magnification, and total magnification of each objective lens. 2. When initially viewing microorganisms, you should start with the scanning objective lens. Always use the coarse focus knob first with this lens. 3. Define magnification. makes specimen appear larger 4. Define resolution. clarity, sharpness of the image 5. Define parfocal. when the specimen is in focus using one objective lens, it is in focus in ALL objective lenses. (with minor adjustments of fine focus knob) 6. Define working distance. Which objective lens has the greatest working distance? Space between your object lens & the slide; Scanning lens 7. What happens to depth of field as magnification is increased? Depth of field is decreased as mag is increased. 8. What happens to the field of view as magnification is increased? Decreased. 9. Explain how to add oil to a slide so that you do not get oil on lenses other than the oil immersion lens. go to the high power lens and lift it up, drop the oil on the slide & while high power is still lifted turn to oil immersion, gently lower into oil. 10. How should you clean the oil immersion lens after using oil? lens papers. 11. Memorize the ocular micrometer calibration values for each objective lens (recorded on the bottom of p. 37 in the lab syllathese calibration values. Scanner: 22µm Low: 10µm High: 2.2µm Oil: 1µm 12. List the things that should be done to your microscope before you put it away in the cabinet after using it. Clean oil immersion lens, turn light off, replace to scanner lens, wrap cord between ocular lenses and under stage. Exercise 3: Culture of Microbes & Media Preparation 1. Define culture media. Substance that contains nutrients for supporting the growth of microbes. 2. Who first discovered that agar is a useful medium for growing microorganisms? Fanny Hess 3. Define agar and explain why agar is a useful medium for culturing microbes. Agar is found in the cell walls of red algae, it is useful because it does not get digested/degrade by mircrobes, & it holds solid up to 45°C 4. List the benefits and limitations for each of these media: a. Broth media -Great for studying cell morphology because cell retain their shape, can store longer, easy to extract cells. Can not study colony morphology b. Agar tubes (deeps) Can't study colony morphology, but great for seeing the oxygen requirements of microbes. c. Agar slants (slopes) Does not dry out as quickly as streak plates, not as easily contaminated, but you cannot get isolated colonies. d. Streak plates Great for studying colony morphology, (isolating colonies) but dry out fast & cells don't retain natural shape. 5. Explain the difference between complex media and defined media. Complex media is media that has a crude (unknown) amount & types of nutrients, whereas defined media is a known/ calculated quantity of nutrients & types of nutrients. 6. Given a container of agar with instructions for making the agar, be able to calculate how much agar is necessary for making specific volumes (e.g. 50 ml, 100 ml, 500 ml).What? I think the directions where on the back of the agar she gave us Doubt this will need to be known. Exercise 4: Aseptic Technique & Streak Plate Preparation 1. List the steps for preparing a proper streak plate. Flame loop, get glob and spread on one half of plate, flame loop, cross through previous streak and drag into new quarter of plate, flame loop & repeat. This process is to dilute and isolate colonies. 2. Be able to recognize a properly streaked plate vs. improperly streaked plate. 3. Explain how to sterilize a wire loop. Flame until glowing orange & allow to cool before use. 4. Why is it important to allow the wire loop to cool briefly before obtaining bacteria with the loop? You do not want to kill the microbes with a hot loop. 5. List the items that should be included on the label of a streak plate. Name, microbe name, date, media type. 6. In what position should a streak plate be stored and why? Upside down to prevent condensation from dripping onto cultures causing them to run together. 7. List the scientific names of the bacteria in Mix A and be able to recognize each species growing on a streak plate. What type of media was used for Mix A? Staphylococcu aureus - circular, shiny, white, smaller colonies of 2. Klebsiella pneumoniae - irregular, off-white, larger colony of the 2. 8. List the scientific names of the bacteria in Mix B and be able to recognize each species growing on a streak plate. What type of media was used for Mix B? Bacillus aureus - filamentous, cloudy white. Serratia marcescans - Irregular, shiny, red. 9. List two things you should do to practice aseptic technique when obtaining cells from a broth tube. flame end of tube, flame loop. Exercise 5: Pure Culture Technique 1. Define morphology. Science or study of form or external appearance without regard to function. 2. Be able to describe the characteristics (using correct terminology) of a pure bacterial culture, including the following: a. Colony forms (bottom of p. 49) (overall shape) Punctiform, Circular, Irregular, Filamentous, Rhizoid. b. Colony margins (top of p. 50) (edges) Entire, Undulate, Lobate, Serrate, FIlamentous, Curled. c. Colony elevations (top of p. 50) (Heights) Flat, raised, convex, pulvinate, umbonate d. Optical character (p. 50) Opaque, translucent, iridescent, opalescent e. Pigment/ color (p. 50) literally refers to color. Exercise 6: Direct vs. Indirect Staining, including bacterial morphology and all stains completed during labs 1. Be able to describe or recognize the various cell shapes and arrangements of bacteria viewed with a microscope (p. 59) Cocci = spherical shapes (diplococci, streptococci, tetrad, staphylococcus, sarcinae, singles) Bacilli= ros-like or cylindrical shapes (diplobaccili, streptobacilli, palisading, snapping, single cells, coccobacilli) Spirilla = spiral shapes (Vibrio) 2. Explain the difference between a direct stain and an indirect stain. Know which type of stain is basic (contains cations) vs. acidic (contains anions) or neutral. Stains are salts w/ one ion being colored. In a direct stain, you are directly staining the cells, leaving the background colorless, you will use a basic stain. Indirect stain will color the background leaving the cells colorless, you will use an acidic stain to accomplish this. 3. Explain why basic stains attach to cell membranes. Cell membranes have a negative charge, and a basic stain is a cation therefor, it is attracted to the cell membranes. 4. List the steps for preparing a cell smear for direct staining. How does this differ from the steps for preparing a cell smear for indirect staining?
- Obtain bacteria glob & mix with two drops of water aseptically smearing to thin consistency. Air dry
- Heat fix
- Douse in methelyne blue, allow to sit 60 sec. Rinse.
These steps differ in the swipes of heat fixing, and rinsing the stain. 5. What are two functions of heat-fixing bacterial smears? Kill bacteria & adhere them to slide. 6. Indirect stains such as nigrosin provide a more accurate view of cell shape and size than direct stains. Explain why this is so. Because you gently heat fix the slide, it will not distort cell shape as much. 7. Define differential stain. used to differentiate different types and & characteristics of cells. 8. Define mordant. Used to increase the affinity of attraction between the cells & the dye. 9. Know the steps (and function of each step/ reagent) for performing following staining techniques: a. Nigrosin stain (indirect stain) Create smear, air dry, add 2 loops of nigrosin (nigrosin's anion will be rejected by the cell wall and dye the background) b. Gram stain - Differential stain used to determine content of cell walls: positive = thick peptidoglycan w/ teichoic acid; negative = thin peptidoglycan w/ outer membrane w/ lipolysaccharides (toxic to mammalian cells) Steps: make smear & heat fix primary stain - crystal violet for 60 sec. & rinse (binds to gram pos.) Mordant - grams iodine for 60 sec. (binds color to cell walls) Rinse Decolorizer - acetone-ethanol until runs clear & rinse. (removes crystal violet from gram neg.) Counterstain - Safranin for 60 sec. (stains cells pink; only in gram-neg.) Rinse, dry, done! c. Acid-fast stain Differential stain to differentiate cells w/ mycolic acid (wax-like substance) from cells w/o mycolic acid (hence acid-fast) Steps : prepare smear & gently heat fix set over steam (boiling water) and add tissue, drop carbol fuchsin & reapply for 15 min rinse slide Decolorizer - rinse w/ acid-alcohol until color runs clear & then rinse w/ water. countertain - apply methylene blue for 60 sec. (colors non-acid-fast cells blue) Rinse & air dry. d. Endospore stain - Structural stain make stain & gently heat fix place over steam and apply tissue w/ malachite green, keep moist w/ water. for 10-15 min. Decoloration - rinse slide Counterstain - Apply safranin for 60 sec (colors vegetative cells pink) Rinse. Done. e. Capsule stain - Structural stain! Used Klebsiella pneumoniae as control. Steps: Prep smear & while wet add 1 loop nigrosin. Air dry Apply 95% ethanol or isopropyl alcohol (we used this one) onto smear for 2 min. (used to bind cells to slide instead of heat fix) Dump off excess alcohol and air dry. Add crystal violet, sit 2 min. Rinse GENTLY w/ water & DONE. 10. Know how to interpret the final colors for each staining technique listed above. Direct - N/A Indirect - N/A Gram Stain: Gram-negative = pink colored cells & Gram-positive= purple colored cells. Acid-fast Stain: Non-acid-fast:colors cells blue & Acid-fast: Colors cells pink. Endospore stain: Edospores= colored green otherwise whole cell is pink. Capsule stain: Area around cells will be clear. 11. Explain the importance of using controls/standards when you are performing these staining techniques. Know which controls were used for each technique. Having a control allows you to better detemine your unknown results. Gram Stain: (+) Bacillus aureus & (-) Serratia marcescens Acid-fast stain: Mycobacterium smegmatis Endospore Stain: Bacillus cereus Capsule stain control : Klebsiella pneumoniae 12. Explain how to do the KOH test and be able to interpret the results of a KOH test. 13. Define endospore and list two genera of bacteria that produce endospores. 14. Define capsule and list two genera of bacteria that produce capsules. 15. List two different methods that were used to test for the presence of endospores in the unknown samples during lab. 16. List two characteristics of the cell walls of Gram-positive bacteria. 17. List two characteristics of the cell walls of Gram-negative bacteria. 18. List two characteristics of the cell walls of acid-fast bacteria. Exercise 7B- Introduction to Cyanobacteria 1. Know the general characteristics of cyanobacteria 2. To which domain do cyanobacteria belong? 3. Do cyanobacteria have prokaryotic cells or eukaryotic cells? 4. Are cyanobacteria Gram-positive or Gram-negative? 5. Be able to recognize and identify the following cyanobacteria in prepared slides or live samples or photographs (from the Sierra College Biological Sciences web site): a. Oscillatoria b. Nostoc c. Gloeocapsa d. Anabaena e. Spirulina 6. Define trichomes 7. Define heterocyst (and be able to recognize a heterocyst) Exercise 8- Fungus and Fungus-like Protista 1. Know the general characteristics of fungi 2. Define osmotrophic and saprophytic (methods by which fungi obtain nutrients) 3. To which domain do fungi belong? 4. Do fungi have prokaryotic cells or eukaryotic cells? 5. Describe the anatomy of a mold or fleshy fungus, including the thallus, hyphae, and mycelium 6. Define the terms teleomorph and anamorph 7. What is the difference between a vegetative mycelium and an aerial mycelium? 8. Be able to recognize descriptions of these specialized hyphae: a. Stolons b. Rhizoids c. Mycorrhizae d. Haustoria e. Sproangiophores (what kind of spores are found in these hyphae?) f. Conidiophores (what kind of spores are found in these hyphae?) 9. Be able to recognize and identify the sexual and asexual reproductive structures of the following fungi in prepared slides or photographs: Study Guide for Lab Exam 1 a. Phylum Oomycota (flagellated lower fungi or protists): Albugo bliti (white rust) and Saprolegnia (water mold) b. Phylum Zygomycota: Rhizopus stolonifer (bread mold) c. Phylum Ascomycota: Saccharomyces cerevisiae (brewer’s yeast), Morchella esculenta (morel), Penicillium (mold), Aspergillus (mold), and Claviceps purpurea (ergot) d. Phylum Basidiomycota: Coprinus comatus (fleshy fungus that produces mushrooms) 10. Also be able to identify the phylum to which each of these organisms belongs and note their common names or descriptions (in parentheses above)