Exercise 17: Unknown Identification

Introduction

Identifying an unknown organism is one of the hallmarks of microbiology. In the clinical microbiology lab identifying agents of disease from a patient specimen is a routine activity. A combination of biochemical, immunological, and genotypic tests are used to determine the most probable identification. It is common to have some variation in results. In other words, several different result combinations can lead to the same identification. It is up to the microbiologist to choose the right tests and analyze the results to determine the correct identification.

In this lab each student will be assigned an organism already isolated in pure culture. This is an independent project. Professors and other students are not to be involved in your project. Utilizing aseptic technique, staining, microscopy, and biochemical testing you will perform various tests to identify an unknown bacteria. The bacteria will be one of the organisms listed in Table 17.1 below. After you determine whether your organism is Gram positive or negative, you will select any 3 media choices to use for identification. Media will be checked out from the lab techs and cannot be returned. The list of available media will be posted by your instructor.

After testing is completed students will write a formal lab report that details your work including staining and biochemical testing results and your final identification. A dichotomous key (see figure 17.2 for example) detailing the steps followed in your identification should also be included. Your professor will provide additional instructions and details for your report.

Course Intended Outcomes

Identify an unknown microorganism using culture, microscopic, staining and biochemical techniques used in this laboratory throughout the semester.

Collect, organize and present data from an experiment, as well as draw appropriate conclusions and report finding in written from using the scientific method.

Materials Used

For this exercise students will work individually; however, some materials such as burners can be shared during the lab. You will need the following materials for this exercise.

• Bunsen burner
• Inoculation loop
• Glass slides, cleaned
• Test tube rack
• Microscope
• Additional materials and cultures will be assigned by your instructor

Procedure, Day 1

1. Your instructor will assign the culture to be used for your unknown identification.
2. Each student will start by performing a Gram stain on his/her unknown. View at 1000X total magnification to determine stain reaction and morphology. Record your results in your lab manual.
3. Each student will perform an isolation streak on TSA or NA medium to check for culture purity.
4. After completing Gram stain and isolation streak, the student will select ONE selective/differential or enriched medium to inoculate for next week. Students may use a combination of plates (BAP, EMB, MAC, MSA, XLD) and tubes (CIT, PDT, SIM, TSI). All students are required to use a total of 3 media for completion of testing and identification.
5. It is highly recommended that students inoculate S&D plates with isolation streak technique while working with their unknowns.
6. Tubes should be inoculated according to the written protocol in the manual.
7. Students may have the option to perform an oxidase or catalase test on their unknowns. Follow specific instructions from your professor. These enzyme assays do NOT count as media.
8. Clearly label tubes using label tape. Include your name, unknown number and section day/time.
9. Clearly label plates (on the bottom side) with your name or initials, unknown number, and section day/time.
10. Place inoculated plates and tubes in the rack labeled “to be incubated” on the back bench. Always place the plates with lids down and bottoms up.

Procedure, Day 2

1. Collect your plates and/or tubes from the back bench area labeled “last week’s lab”.
2. Examine your streak plate to check for culture purity.
3. Read and record the results for your selective/differential medium in your lab notebook. It is highly recommended that you photo document your results for future reference.
4. Review the results of your initial tests and determine what remaining tests/results are needed to identify your unknown.
5. Select and inoculate your 2^nd and 3^rd media for identification.

Procedure, Day 3

1. Collect your plates and/or tubes from the back bench area labeled “last week’s lab”.
2. Examine your plates and tubes.
3. Read and record the results for your selective/differential medium in your lab notebook. It is highly recommended that you photo document your results for future reference.
4. Review the results of your tests and use your key to determine to identify your unknown.
5. Document any errors or oversight that may have interfered with your project in your lab notebook.
6. Begin working on your lab report. Make sure that you include all required data and material.

Lab Clean-up

Wash slides with detergent, rinse and dry. Return slides to your slide box and store in the bin labeled for your section.

Return bacteria cultures to the front bench after inoculations are complete.

Remove labels from tubes and place in the tube rack ABOVE the biohazard container.

Plates are disposable and should be thrown away in the biohazard container when everyone in the group has completed the exercise.

Thoroughly clean your microscope and return it to the lab prep room.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

All results should be recorded in your lab notebook.

An example dichotomous key is provided below (figure 17.1).

Your professor will provide specific requirements for your lab report.

Fig 17.1