It is a standard part of most introductory microbiology labs to include an activity requiring students to apply concepts that have been learned in the course to identify unknown bacterial cultures. This is also an important activity in a clinical setting whereby a culture is sent to the lab for a culture and sensitivity. Typically the activity would assess your ability to perform the gram stain, describe cell morphology, grow the bacteria in pure culture so that colony morphology could be observed, make aseptic transfers, and conduct a series of selective and differential biochemical tests.

Unknown bacteria may be identified in the lab by microscopic analysis and completion of a series of biochemical assays, then using a dichotomous key (like the one shown here).

Flow chart.jpg

To complete this exercise, you will use your understanding of topics learned in the course, while still learning about the overall process of unknown bacterial identification. At the end of this unknown, you will write a report about your findings, and upload to Turnitin.

Objectives
Apply previous laboratory experiences to accurately interpret various biochemical tests conducted to identify an unknown bacterial organism
Describe cellular and colony morphology for bacterial unknowns
Identify Gram reactions of bacterial unknowns
Analyze and describe results for specific bacterial species on various selective and differential media
Analyze and describe results for the Kirby Bauer for unknown bacterial species
Use dichotomous keys with phenotypic evidence to determine unknown identity
Activities
Review all content including readings and video clips and experiment protocols prior to lab.
Download and print the following documents for use during the project: Unknown Form, Unknown Flowcharts, Guidelines, Help with APA and Images. Take notes while reviewing the content for this lab.
Unknown Form Download Unknown Form
having trouble? try downloading as a Unknown Form SP22.pdf Download Unknown Form SP22.pdf
Flow Charts Download Flow Charts
Unknown Report Guidelines Download Unknown Report Guidelines
Help with documents images APA Download Help with documents images APA
CLICK HERE Download CLICK HEREto open and review the protocols for LM 12.
Complete the following experiments in class:
Ex. 12.1 Streak isolation of mixed unknown
Ex. 12.2 Analysis of mixed streaks and TSA subculture
Ex. 12.3 Pure culture analysis
Ex. 12.4 Biochemical Testing
Ex. 12.5 Kirby Bauer Testing
Ex 12.6 Submit “Unknown Report” via Canvas Turnitin (graded activity, see syllabus)

LM 12: Unknown Project Introduction-2
A patient presents to the clinic and is diagnosed with an infection. A nurse practitioner takes a sample of the infected area and sends the sample to the lab to be analyzed.

Preliminary gram stain on the sample indicates that it is a polymicrobial infection, having two unknown species, one gram positive, and one gram negative.

You will receive a mixed broth with the unknown patient number that contains both organisms. You must perform the necessary steps for isolation of both of these species and identify them both. Start by following the understood typical steps for isolation, subculture, and evaluation of pure cultures. Document each step of your process. During and after isolation and testing, you will use a dichotomous key flowchart to identify both of the unknowns in the sample. Additionally, you will determine appropriate antibiotics to use for this infection.

Summary of Procedures – How do you do this?
Each student will be given a mixed broth, and use the dilution streak plate technique to create pure colonies of their two organisms from the mixed broth culture. Students will use TSA, PEA, and MacConkey media.
Following incubation, the student will check to see if pure colonies of each organism have been obtained by observing colony morphology and by gram staining. TSA pure subculture plates will then be prepared of each organism. If colonies are still mixed, additional TSA plates may be streaked until each organism is isolated. The use of selective media greatly improves the chances of successful separation, but their availability is limited – use resources wisely!