LABORATORY





GENERAL INFORMATION



The laboratory for this course may differ from those that you have taken in other science courses. Most basic science laboratories have as their objectives the communication to the student of factual information and/or scientific methodology. While these are also goals of this laboratory, there is another goal which is of equal significance: the development of proper microbiological laboratory techniques. Much of elementary microbiology, especially the clinical applications, is concerned with the identification of a particular microorganism. Identification can only be accomplished by the use of certain standard laboratory techniques which must be mastered. Therefore, the development of proper technique will be a major laboratory goal. Chapter one of the lab manual deals with basic laboratory skills and should be studied carefully and referred to throughout the course.



GRADING



The final laboratory grade will be based upon two laboratory examinations and the unknown. Each lab exam will consist of questions and problems as well as slides derived from the assigned laboratory work. See the objectives at the end of this syllabus for more specific information. The examination format will be fill in the blank and short essay questions. Grading is on a 60 to l00 basis. The unknown which you will be required to identify will count the same as a laboratory examination. The minimum grade for the unknown is 60 while the maximum is l00. A detailed report on the unknown will be required. Numerical grades will be assigned to the unknown report based upon the quality of the report, the accuracy of identification, and the difficulty of the organisms. As a rule of thumb, successful identification of both organisms is a l00 score, correct identification of one organism is an 80, while incorrect identification of both yields a 60. Students should make every attempt to take the examinations at the scheduled time. Any student missing who has a valid excuse for missing an examination will be permitted to take a make up examination. Only one make up examination will be permitted. All make up examinations are given at the end of the semester or a at the instructor's convenience.



CLEANING UP



All students are required to maintain a clean work area. Old cultures, tubes, etc. should be placed in the appropriate receptacles. At the end of the term all materials must be removed from the incubators, refrigerators, and desks. One point will be removed from the student's average for every tube or plate left in the above mentioned areas.



ATTENDANCE



Laboratory attendance is mandatory. One point will be removed from the final laboratory average for each unexcused absence.



LABORATORY RULES



1. Absolutely no eating or drinking in the laboratory. Remember, your neighbor may be working with E. coli of unknown origin.



2. Gas burners are in use. Be extremely careful when reaching or leaning over the burners. In most cases the flame will be almost invisible. Individuals with long hair should be especially careful. Make sure you know the location and use of the fire blanket and shower.



3. Although we do not work with pathogenic organisms, it is always possible for contamination by a pathogen to occur. Treat every culture as if it were highly pathogenic. Exercise proper aseptic technique in all cases. Place old tube and plate cultures in the designated area. They will be sterilized before being disposed of. Never pour a live culture down the drain or throw it into a trash can.



4. This laboratory is crowded with incubators, refrigerators, and other items. Exercise care when moving around the laboratory.



5. At the end of each lab period you should swab you work area with the disinfectant provided.



6. Be sure you wash your hands at the end of each lab period before leaving.



7. Because of the numerous biological stains used in this laboratory you should wear old clothes that can be ruined without great loss.







Laboratory Schedule





WEEK DAY EXERCISE
1 1.

2.





Orientation and light microscope. Ex. 3.1, 3.2

Protozoa. Ex. 10.2

2. 1.



2.

Fungi. Ex. 10.1



Bacteria - Morphology and Environmental Organisms. Ex. 1.

3. 1.



2.

Smear preparation and simple staining. Ex 3.3, 3.4

Negative staining. Ex. 3.5

4. 1.



2.

Gram stain, Ex. 1.2, 3.6



Acid fast stain. Ex 3.7

5. 1.



2.

Review and catch up day.



EXAMINATION 1.

6. 1.



2.

Capsule and endospore staining. Ex.3.8, 3.9



Determining motility. Ex. 3.11, 5.27. Isolation of pure cultures. Ex. 4.1. Pour plates. Handout

7. 1.





2.

Gram stain isolates from previous exercise. Cultivation of anaerobes. Ex. 2.4, 2.5



Continue anaerobe exercise. Issue knowns. Streak for isolation.

8. 1.





2.

Gram stain known isolates. Colony morphology. Ex. 2.1. Prepare stock cultures. Ex.1.



Microbial metabolism. Ex. 5.2, 5.5, 5.8, 5.15, 5.23. Use knowns for inoculation.

9. 1.



2.

Analyze known results. Effects of UV light. Handout.

UV light results. Disinfectants. Handout. Each student should bring a disinfectant of interest.

10. 1.





2.

Disinfectant results. Antimicrobial sensitivity testing. Ex. 8.3.



Antimicrobial results. Hand washing experiment. Handout.

11. 1.





2.

Hand washing results. Water Analysis. Ex.8.5, 4.6, Handout.

Each student should bring in 50 ml water sample of interest.



Food analysis. Ex.7.1, Handout.

12. 1.



2.

Food results. Snyder test. Ex.8.7.



Snyder continued. Issue unknowns. Streak for isolation. Handout.

13. 1.





2.

Examine isolates for colony morphology. Prepare stock cultures. Continue with Snyder test.



Gram stain stock cultures, determine motility, metabolic tests.

14. 1.



2.

Read results of previous tests. Special tests if necessary.



Holiday

15. 1.



2.

EXAMINATION 2. Continue unknown if necessary.



Submit unknown report. Clean up.

OBJECTIVES - LABORATORY EXAMINATION I



1. Listed below is the generic (genus) name of a number of organisms which you viewed in lab. You should be able to state this name when shown the organism. You should be able to state the phylum (protozoa) or class (fungi) to which each one belongs. Be sure you can describe the morphology of each one.



Protozoa Fungi

Amoeba Cryptococcus Trypanosoma Aspergillus

Plasmodium Candida

Paramecium Cryptococcus Penicillium

Claviceps Rhizopus



Bacteria

Streptococcus Bacillus

Staphylococcus Neisseria Treponema Mycobacterium

Spirillum Borrelia

Streptomyces Escherichia



2. You should be able to identify the following structures below when shown them.



Endospores Capsules Zygospores

Sporangia Conidia Hyphae

Nucleus Cilia Asci



3. You should be able to identify the processes of conjugation and fission when shown them.



4. For exercise one, the light microscope, be able to accomplish the following.



a. Identify all of the parts listed in Figure 3.1, and state the function of each one.

b. Calculate the total magnification of various combinations of eyepiece and objective magnifications.



c. Explain the difference between resolving power and magnifying power. Relate numerical aperture to each.



d. Define index of refraction. Relate the concept to the use of oil to improve resolution.



e. Describe the effect of closing down the diaphragm on brightness, resolution, contrast, and spherical aberration.



f. Define parfocal.



5. For the protozoa, be able to accomplish the following.



a. List and describe the four major protozoan phyla. Describe motility in each. What kingdom do the protozoa belong to?



b. Define pseudopod, flagellum, cilium, cyst, and trophozoite



c. Which phylum contains the most pathogenic members? Which contains the least?



6. For the Fungi, be able to accomplish the following.



a. List the four major classes and state the type of sexual spore produced by each class.



b. Describe the asexual spore type found in each of the four classes.



c. Define yeast, mold, mycelium, hypha, spore, saprophytic, sporangium, sporangiospores, zygospores, ascus, ascospore, conidia, basidiospores, basidium. Relate all of these structures to the fungi which were observed in lab.



d. Explain what being dimorphic means to a fungus.



7. Describe how to prepare a smear. Be sure to include all of the relevant steps.



8. Describe all of the steps for each of the staining procedures done in the laboratory. Be sure you include stains, times, procedures, and results. Explain how KOH aids in differentiating gram positive from gram negative bacteria



9. For the acid fast stain know which genus this is diagnostic for and be able to list two pathogenic species in this genus.

OBJECTIVES - LABORATORY EXAMINATION II



1. Describe the steps for performing a capsule and endospore stain.



2. Describe how motility may be determined. Describe Brownian movement and its cause, and explain how true motility may be differentiated from Brownian movement in a hanging drop.



3. Describe two methods by which a pure culture may be obtained from a mixed culture. Explain the theory behind streaking for isolation.



4. Describe how to grow anaerobes. Explain what is meant by a reducing medium.



5. List and describe all of the metabolic and respiration test done in laboratory. Explain what each test is measuring. Explain how a positive test appears for each test.



6. Describe in order the steps that are necessary in order to identify an unknown bacterium.



7. Describe the UV experiment and summarize the results. Be sure you can explain the results.



8. Describe and summarize the disinfectant experiment. Be sure to include the effects of time on disinfectant activity. Explain why a control sector was required.



9. Explain how antibiotic sensitivity of microorganisms may be determined. When presented the results of sensitivity tests, be able to state which antibiotics were the most effective against the test organisms. Be sure you can differentiate broad spectrum results from narrow spectrum results. Be able to apply the results of tests to practical situations such as the antibiotic of choice for a particular infection when the nature of the organism is known.



10. Summarize the results of the hand washing experiment. Explain the results.



11. Describe in detail the analysis of water for bacterial contamination. Be sure you know what constitutes a presumptive positive test and what must be done to confirm the results of such a test. When presented the results of an analysis, be able to utilize the MPN chart in order to estimate the minimum number of organisms, the most probable number of organisms, and the maximum number of organisms that the sample contains.



12. Analyze the results of the Snyder test experiment as regards the susceptibility of an individual to dental caries. What type of bacterial activity does this test measure?



13. Summarize the results of the bacterial food count experiment. Describe how the numbers of bacteria can be estimated. Be sure you can explain how to perform serial dilutions. Given a sample plate, be able to estimate the number of bacteria in the test sample.