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PRE-LABS FOR MICROBIOLOGY LABORATORY Week of May 28 (Pre-lab #1) 1.

What should you do to your table at the beginning and at the end of each laboratory period?

2. What should you wear for each laboratory session when working with live organisms?

3. What four pieces of information should you always include on your label?

4. If you use a broth culture, what has to be done to it before taking a sample?

5. How should you always place petri dishes when not inoculating them?

6. List the four magnifications that the objectives have on the microscopes that you will use in lab. Include name and magnification number.

7. Name the ONLY type of paper that can be used to clean all microscope lenses.

Week of May 28 (Pre-lab #2) 1. What are cultures and what is used to solidify a liquid culture?

2. Where are cultures put in order to enhance their multiplication?

3. Which type of transfer instrument is used when taking a sample from a broth culture? To transfer a sample into a agar deep tube?

4. How do you sterilize the transfer instruments before and after each use?

5. List the two primary reasons to do a streak plate inoculation.

6. When doing a streak plate inoculation, do you sterilize your transfer instrument between each quadrant? If you answered no, state when you do not use sterilization.

7. List some of the cultural characteristics you can use to distinguish between colonies.

8. Explain what heat fixation can do to living organisms.

Week of June 4 (Pre-lab #3) 1. Do we use basic or acidic stains most often on bacterial organisms and why? 2. When making bacterial smears: what do you draw on your slide before placing your sample on it? when is it that you add water to your sample and why? why do you heat fix? what is the purpose of making the smears with different sample concentrations?

3. Which smears will you use to simple stain? 4. Where will you place your smear in order to stain it and how will you hold the slide in order to do this?

5. What paper will you use to blot dry the slide? 6. In negative staining, what type of dye will you use (basic or acidic) and what will you be actually coloring?

7. For the Gram Stain, give the name for each of the following: Primary stain Mordant Decolorizing agent Counterstain 8. What is the ideal age of cultures to be used for Gram Staining? 9. How many slides will you Gram Stain and what will be on each of these slides?

Week of June 4 (Pre-Lab #4) 1. What color will the G+ organisms be after Gram Staining? What color will the G- organisms be after Gram Staining? 2. For the Gram Stain, list the reagents, by specific name, in the order that you will use them.

3. How many reagents are used in differential staining techniques?

4. Name the genus of bacteria for which the acid fast stain is often used.

5. If an organism is said to be acid-fast, what do these organisms have that makes penetration by stains very difficult? What color are acid-fast organisms?

6. List the reagents, in the correct order, used for the acid-fast staining procedure.

7. List the two reagents used in the Schaeffer Fulton Method. List the decolorizing agent used in the spore stain.

8. After the Schaeffer Fulton Method, what color are the spores and what color are the cells?

9. Should you heat fix the slide when doing the capsule stain? Why or why not?

10. After the capsule stain, what color are the capsules and what color are the cells?

Week of Feb 13th (Pre-lab #5) 1. Name the most essential of the nutritional needs for microbial cells. 2. Name the nutritional need that is essential for formation of the nucleic acids and ATP. 3. What is the function of selective media? List the media in your lab book that have a selective property and for each, list what they are selective for.

4. What is the function of differential media? List the media in your lab book that have a differential property and for each, list what they are differential for (be very specific) and describe what you will look for (after incubation) to determine if the media was differential or not for each of the organisms.

5. For Selective/Differential media, how many of each type of media plates will your table work with? So, you will have a total of _______________ (give a number) plates per table. 6. How many organisms will you inoculate onto EACH plate? How will you know which organisms to put on which plate?

7. Will you inoculate each of the plates in the same manner? If no, which plate will be inoculated differently and how will it be done?

8. Name the specific temperature category in which organisms that infect humans are put into. 9. For your table, how many Trypticase soy agar plates will you have and which organism(s) will be inoculated onto these plates? Each of these plates will be incubated at what temperatures?

Week of Feb 13--continued

Per table, how many Sabouraud broth tubes will you have and which organism(s) will be inoculated onto these plates? Each of these tubes will be incubated at what temperature?

What is the function of the inverted Durham tubes in the Sabouraud broth tubes?

10. Which group of bacteria can survive only a narrow pH range? Which group of bacteria can survive a broad pH range?

11. Which group of organisms prefer an acidic pH?

12. For your table: How many Trypticase soy broth tubes at pH 5 will you have? How many at pH 7? How many at pH 10?

Name the organisms that you will inoculate into each group of pH tubes.

13. Will all pH tubes be incubated at the same temperature? If no, list which will go into different incubation temperature and what that temperature will be.

Week of Feb 20th (Pre-lab #6) 1. Be able to give a description of the five categories into which organisms are classified according to their oxygen requirements. In addition, be able to label a diagram such as Figure 15.1 in your lab book.

2. Will you be inoculating and incubating tubes/plates for Experiment 15? 3. What are exoenzymes? What are endoenzymes? What are these enzymes used for by cells?

4. For each of the macromolecules listed (starch, lipid, casein, gelatin) be able to list the enzyme(s) needed to break the molecule down and be able to list the building blocks that can then be transported into cells.

For each of the plates, describe what you will be looking for to determine if each organism produced the appropriate enzyme(s) to catabolize the specific macromolecule.

For your table, how many of each of the media plates will you work with? After incubation, will you add any reagents to any of the plates before reading the results? If you answered yes, to which plates and what will you add? 5. In the carbohydrate lab, what are the two results that you are looking to see if they occur or not?

For the two results you listed above, describe what you will actually see after incubation that will tell you that those results did occur.

What is included in the media that will enable you to determine if carbohydrate catabolism did occur? If it is a positive resultwhat color will you see and what does this indicate as far as pH? If it is a negative resultwhat color will you see and what does this indicate as far as pH? If carbohydrate fermentation did not occur, what else is in the media that could be an energy source for the organisms and, after incubation, what would you see and what does this result indicate as far as pH?

How many tubes, of each carbohydrate, will your table inoculate? What method of inoculation will you use and what inoculating instrument will you use? What is the incubation time and temperature for this type of media?

Week of Feb 27th (Pre-lab #7) 1. What carbohydrates are in the TSI media? With the results, can you determine if each carbohydrate was utilized by the organism? If you answered yes, explain how you determine which carbohydrate was used.

If you answered no, explain which carbohydrate(s) you cannot determine if they were used or not and why.

2. What other result may be observed (other than carbohydrate utilization) in the TSI media? 3. What has to be in the media in order to determine if a result is (+) or (-)? This will determine color of the media.

4. What do the initials IMVIC stand for? 5. How many different tests are included in Experiment 20? 6. IMVIC lab Part A What will you determine with a (+) result? (Two possible results) What enzyme (if any) does the organism have to produce for a (+) result? What media will you use? What do you need to do before reading your result? What will you see, after incubation, if your result is (+)? IMVIC lab Part B What will you determine with a (+) result? (Two possible results) What enzyme (if any) does the organism have to produce for a (+) result? What media will you use?

What do you need to do before reading your result? What will you see, after incubation, if your result is (+)? IMVIC Part C What will you determine with a (+) result? (Two possible results) What enzyme (if any) does the organism have to produce for a (+) result? What media will you use? What do you need to do before reading your result? What will you see, after incubation, if your result is (+)?

IMVIC Part D What will you determine with a (+) result? (Two possible results) What enzyme (if any) does the organism have to produce for a (+) result? What media will you use? What do you need to do before reading your result? What will you see, after incubation, if your result is (+)? 7. For Experiment 21, what media will you use? What are the two possible results from this test and how will you determine if these results did occur?

8. List all the possible results you can obtain on the Litmus milk experiment and for each, describe what you would see after incubation.

For each of your results, explain the reasons you would see different results, in other words, describe the mechanism of action for each type of result.

9. For Experiment 24, what enzyme must the organisms produce in order to reduce nitrate?

What does NO3- represent? What does NO2- represent? What are the three possible end products produced if nitrate was reduced?

When reading your results, what do you add first? These solutions will interact with _________________ if it is in the tube. Would this indicate a (+) or a (-) result? What will you see?

Why would you add zinc? After zincwhat indicates a (+) result? Zinc interacts with _________________ if it is in the tube. Would this indicate a (+) or a (-) result? Does a red color indicate a (+) or a (-) result? If red could be both a (+) and a (-) result explain how this happens. What will you see?

Week of March 5th (Pre-lab #8) 1. For Experiment 22, what enzyme does your organism have to produce in order to get a (+) result? What will you see for a (+) result? What has to be in the media so you can determine a (+) result? What does this indicate as far as pH? When is the only time you can use this media when working on your unknowns?

2. When performing the catalase test, from which type (solid, broth) of media will you take your sample? Note: Your media with the organism MUST be a fresh sample = just taken out of incubation. What enzyme(s) must your organism produce in order to break down H2O2? Why do organisms have to break down H2O2? Where will you put your sample before you add H2O2? What will you see for a (+) result? Will you use this test for Gram (+) or Gram (-) organism or for both? Look at page 142 of your lab book. When performing the oxidase test, from which type (solid, broth) of media will you take your sample? Note: Your media with the organism MUST be a fresh sample = just taken out of incubation. What enzyme(s) must your organism produce in order to get a (+) result? Where will you put your sample in order to perform this test? Will you add anything to your sample? What will you see for a (+) result for this test? Will you use this test for Gram (+) or Gram (-) organism or for both? Look at page 142 of your lab book.

Part Two Pre-Labs for Week of March 5th = Flow Charts (Pre-lab #9) Look at pages 146-149 for Examples of Flow Charts Using the tests you have performed in lab and using the 10 organisms from which your unknowns will come, start trying to build your own flow chart. You will start your flow chart with a Gram Stain. Start building your flow chart as follows: Gram Stain (+) List the 5 organisms that are G+ here (-) List the 5 organisms that are G- here

Decide which test you will use next for the organisms you listed above Name of Test (+) List the organisms that are (+) here (-) List the organisms that are (-) here

Decide which test you will use next for the organisms you listed above Name of Test (+) List the organisms that are (+) here (-) List the organisms that are (-) here

Once you have performed the test (either the one for G+ organisms or the test for G- organisms (depending on what your result was on the Gram Stainyou will either follow the flow chart for G+ or for G-

From the previous test, list the Organisms Eliminated Organisms Remaining

For example, if your actual test turned out (+), then your organisms remaining would be the ones you had listed under the (+) results and the ones eliminated would be the ones listed under the (-) result

Continue to build your flow chart only for the organisms remaining until you have only one organism remaining. This is your unkown organism.

Week of April 2nd (Pre-lab # 10) 1. List the four major characteristics of parasitic protozoa.

2. List the four taxonomic classes and for each, state what the adult organisms in each class use for locomotion.

For the organisms listed on page 156 of your lab book under At the Bench, under prepared slides, answer questions #1-#4 under procedure on page 157.

Week of April 2nd (Pre-Lab # 11) 1. List the four classes into which fungi are classified and for each class, state what the sexual mode of reproduction is.

2. List some of the beneficial effects that fungi have on humans.

3. List some of the detrimental effects that fungi have on humans.

4. What are superficial mycoses?

5. What are systemic mycoses?

6. Explain the following: Mycelium Hyphae Vegetative mycelium Aerial mycelium

7. Can the same media be used for bacteria and molds? Yes or no and explain why.

8. What environment (as far as pH) do molds prefer?

9. What temperature do molds prefer for growth?

10. Do bacteria and molds grow at the same rate? Explain.

11. List some of the characteristics of yeasts.

12. Which organisms have aerial hyphae and sporangia?

13. List and explain the two most common methods of yeast asexual reproduction.

14. Can yeast undergo sexual reproduction? Yes or no and explain.

15. List some of the beneficial effects of yeasts on humans.

16. List some of the detrimental effects of yeasts on humans.

Week of April 23rd (Pre-lab #12) Bacterial Transformation/Immunofluorescence You will perform this experiment as a table, not by pairs. 1. List the two groups of genes that the pVIB plasmid contains and explain what each of the groups will code for.

2. What is a big disadvantage of an organism being able to luminesce?

3. In the handout, it talks about four control plates. Each table will do all four plates. List what each plate will have in the media and what you will add to each plate. Control #1

Control #2

Control #3

Control #4

For the four plates listed above, list yes or no growth AND if there is growth, state what type of growth the plate should have and why.

4. What is the purpose of using calcium chloride?

5. What is the purpose of doing an abrupt ice/heat/ice shock?

6. Why should you transfer cells to the + plasmid tube before transferring cells to the plasmid tube?

7. Why is it important to resuspend the cells quickly after transferring them into the calcium chloride?

8. Why is it essential that the cells be spread quickly on the plates?

9. What will you use to spread the cells on your plates? What motion(s) will you use to make sure the cells are spread throughout the plates?

10. How many tubes will each table work with and how will you label each tube?

11. Name the bacterium that you will be adding to your plates. 12. How much time will you keep both tubes in ice after the plasmid is added? 13. How much time will you keep both tubes in the 42 degree water bath? 14. After the water bath, what will you do next to your tubes?