Tarrant Country College Identification of Unknown Microorganism Chemistry Lab Report

A patient presents to the clinic and is diagnosed with an infection. A nurse practitioner takes a swab of the infected area and sends the sample to the lab to be analyzed.

Because of the current pandemic, staffing in your clinical microbiology lab has been affected. Your staff performs the necessary steps for isolation and finds that the infection involved two bacterial organisms. They document, with pictures, each step of their isolation, agar plate growth, gram stain, and pure culturing process. Additionally, they perform appropriate biochemical tests as well as Kirby Bauer susceptibility tests, and take pictures for the official record of those results.

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You, their manager, are working from home, and tasked with overseeing their work, as well as using their information to determine the identity of the organisms in question causing the patient infection, as well as the best antibiotics for use.

You will receive a PDF presentation with the unknown patient number. It will contain pictures that are representative of all the steps and observations that would need to be performed to isolate and identify two species from the same culture (patient clinical mixed polymicrobial specimen) as done by your staff. You will record the observations for both gram positive and gram negative organisms. Then, with the provided flowchart, you will determine the identity of both species. Finally, you will observe and interpret the Kirby-Bauer susceptibility results for both species, so that appropriate antibiotics may be prescribed to the patient.


  • Receive your powerpoint that has images of each step of isolation and species determination, including:
  • Images of dilution streak plate technique performed on an initial set of plates with the mixed specimen: TSA, PEA, and MacConkey media. Your images may also contain TSA blood agar or mannitol salt agar (MSA) result
  • Images of the Gram stain of the mixed specimen.
  • Images of pure subculture plates prepared for each organism.
  • Images of the gram stains performed of each presumptive pure culture.
  • Close up images of colony morphology of each subculture.
  • Images of the results of biochemical tests performed on each organism.
  • Images of the results of the kirby bauer test.
  • Begin recording observations for BOTH ORGANISMS on the Unknown Form. Record the appropriate notations according to each test as found in Lab 9 and other prior labs. Recording color or a short description is also advised. Use the Unknown Form to organize your information. Any tests or results you have images for must be recorded. There will be some tests that you will leave blank if you are not given images or data.

  • Use the posted Flow Charts (dichotomous keys) to determine the identity of your gram + and gram – organism. Be sure to use the correct flow chart for each organism. Do not use flow charts found anywhere else, and do not use resources elsewhere to confirm reactions on the flowchart. Some species identifying names with their reactions have been changed from established research.
  • Record the Kirby Bauer Antibiotic Sensitivity results in the Unknown Form. Images of plate(s) and measurements will be provided.
  • Interpret the sensitivity results using the provided chart of standards included with the Flow charts, and record results in the Unknown Form.
  • Once the identity of each organism has been obtained, a complete Unknown Report must be prepared according to the guidelines to thoroughly communicate your observations and results of isolation, pure culturing, biochemical testing, and KB tests.

    This Unknown Report will be turned in using a Turnitin link in this module.



    You should have your unknown PDF file sent to you or made available to you by your instructor. Check your email or announcements for instructions. It will include images that guide you through the isolation of your organisms from a mixed sample, gram stains and images of media used in isolation, and biochemical test result images.

    After you receive your Unknown file, you will record what you find from the data and images, and keep it organized using the Unknown Form. You will be required to turn in a copy of the Unknown Form by attaching it to your report. You may fill it in electronically, or by hand. If you fill it in by hand, you will need to scan it or take a picture of it when completed, and insert it into your unknown report. Make sure you refer to previous labs for the proper notations for each test. You will also include a brief note about a color or reaction you visibly see for each item, if applicable. Do not elaborate or go into details about your interpretation on the Form (instead, do so in the discussion of your Report).

    Once all your observations are recorded properly, use the attached copy of the Flowchart to help you determine your unknown organisms identities. The Antibiotic Susceptibility Chart is also there.Help with learning Lab 9, preparation for completing the project and studying:

    A document you can download to build flashcards for all the media and biochemical tests we’ve learned about is attached to this page. If you refer to these flashcards that you make while doing your project, you can get through it faster, and have a resource you can study for lab exam 3. These are NOT completed cards – use this page to know what to include on your cards.

    Help with learning how to read the flowchart or dichotomous key:

    Download the document here to get some practice using our flowcharts using data from biochemical tests. The answers to these scenarios are on the last page.


    Your organisms:you have exactly one gram positive, and one gram negative organism in your mix (A and B).

    Nitrate results:Often, the first step in evaluating the nitrate test is simply looking at the durham tube for the production of gas. This tells you definitively what result you have, and nitrate A and B do not need to be added.

    Some of the pictures in your file – the first few pictures in the presentation represent inoculation done with the mixed culture that had both organisms (A&B) on various types of media. Use these to help you determine (with other clues) which organism is your G+ (A or B), and which organism is your G- (A or B).


    Report forms: Your forms, where you can succinctly record the results of the tests you’ve been provided, need not have any information about test you weren’t given in your file. The forms should also have more than just +/- filled in the blanks or yes/no. Record colors or reactions that you see too. You CAN print them and fill them in by hand, and attach them as pictures in your report (or as screenshots). Regardless, they MUST be large and legible to grade.

    Discussion: Your discussion section for your G+ and G- organism MUST be typed, and should be separate for your G+ and G-. Be specific and thorough about the isolation steps that appear to have been performed on selective/differential media, and about the interpretation of the biochemical tests in your report (including what they MEAN).


    For Growth Media
    • Name and Type of medium
    • For G+/G- or both?
    • Selective? For what? Why? (ingredients that make it selective and
    • Differential? For what? Why? (ingredient and indicator and what the
    differences look like)
    • Important color changes/reactions and how to interpret
    • Special notes
    • PICTURE of the reactions
    For Biochemical Tests
    • NAME OF TEST and abbreviation
    • CULTURE MEDIUM – what is in it?
    o INDICATOR in media, or REAGENT added later that allows you to
    see an observable change
    • What are we testing for? An enzyme? A fermentation waste product?
    Be as specific as you can while understanding the concept
    • TEST FOR G+/G-/BOTH ??
    o Reagents added after incubation (if relevant)
    o how to read results
    o Biochemical interpretation and visual description
    What does a positive result MEAN? Negative result?
    • PICTURE of the results (be sure to use the pictures included in lab 9 or
    another similar one from online. DOUBLE CHECK! Don’t just use a
    random image from a quick google search. Images included in your lab
    are the most clear we could find or taken ourselves. I’m not
    responsible if you download an image for a test and it’s the wrong
    thing. You’ll be expected to be able to identify images of typical
    results for each test.
    Student Name:
    Concise, descriptive title
    (0.5 pt)
    Background of project,
    in your own words. (4
    Broken into weekly
    paragraphs (1 pt)
    Summary of project (4
    Weekly paragraphs
    include background
    explanation of tests
    received, with any
    additional reagents,
    indicators, etc
    explained. (20 pts)
    Title page formatted
    correctly (1 pt)
    Subheadings present (0.5
    Grand total
    Biology 2420 Unknowns Report Rubric – Last Updated Spring 2021
    Title Page
    ___ out of 2 pts
    Unknown # listed (1 pt)
    ___ out of 9 pts
    Observations of test results
    described. (15 pts)
    Summary of tests
    (10 pts)
    Description of result
    interpretations. (15 pts)
    Statement of
    unknown (1 pt)
    Correct unknown ID (5
    Explanation or
    background of
    bacteria identified
    (1-2) sentences.
    (3 pts)
    Weekly paragraphs
    include list of tests
    requested. (2 pts)
    *Must include all tests
    provided & requested. -2
    pts for from grand total
    for every missing test.
    *Must include all tests
    provided & requested. -2 pts
    for every missing test from
    grand total.
    ___ out of 23 pts
    ___ out of 30 pts
    ___ out of 100 pts
    Bibilography Page
    Minimum of 2 sources
    Correct gram nature / (lab manual must be
    morphology ID (5 pts)
    one) (2 pts)
    Grammar / Syntax
    Correct use of grammar (2
    In-text citations in
    introduction and
    methods formatted
    correctly (2 pts)
    Paper is written in past
    tense, third person (2 pts)
    Bibilography page
    formatted correctly (2
    Proper sentence structure
    used throughout paper. (2
    *Papers with no in-text
    citations in the
    introduction or
    methods will receive a
    zero for those
    sections. Papers with
    direct quotes in the
    introduction or
    methods will receive a
    zero for those
    ___ 14′ pts
    ___ out of 10 pts
    ___ out of 6 pts
    ___ out of 6 pts
    Rev Jan6 2021
    Students should not use Bergey’s to confirm reactions on this chart
    Gram Positive
    (Simple stains or negative stains can help determine shape)
    All are Starch –
    Starch +
    *Catalase –
    Starch * Catalase +
    *Catalase +
    Family Streptococcaceae
    *Catalase +
    *Catalase [***Anaerobic]
    Bile Esculin +
    Bile Esculin –
    ** Mann +
    PRGlucose A–
    ** Mann #gamma
    – NO3
    **Mann –
    $ Bacillus
    [VP+ , +NO2]
    PRGlucose A+
    Bacillus megaterium
    & irregular, beige
    [Mann+, VP-]
    $ Bacillus pumilus
    & white
    [VP+, Mann weak+]
    #weak beta
    #gamma/weak alpha
    [MR+, -NO3]
    VP# alpha (green)
    &small, translucent
    [&&Bacitracin S]
    [! Coag +]
    [&&Bacitracin R]
    [! Coag -]
    – NO3
    &Yellow Pigment
    [Methyl Red-]
    [VP – ]
    &Pink Pigment
    [Methyl Red+]
    Bacillus cereus
    Special Notes:
    &Colonies Spreading
    [Red] Confirmatory test can be performed if other results conflict, or if supplies available
    ** Use MS plate and/or Phenol Red Mannitol, (weak A+) means media may be orange at 48 hrs
    $ May grow on MAC plate
    $$ Might appear as diplococcus, pleomorphic or coccobacillus
    # Hemolysis on blood, streak pure culture; view after 48 hrs max; weak = reaction subtle, not obvious
    Observe isolated colony morphology on TSA plates
    NO3, NO2, N2, N2O, NH3 – Nitrate test
    && Observe Kirby Bauer results
    VP = Voges Proskauer PR = Phenol Red
    Special Stain; Endo=endospore
    Coag = coagulase MR = Methyl Red
    Observe arrangement on negative stain or gram stain
    talk to instructor for guidance, info may be used if available
    Perform stain from plate or culture left in incubator 3+ days
    Must be from solid agar growth grown in incubator *** Use FTM thioglycollate if needed
    [VP- , +NO2]
    Rev Jan 6 2021
    Students should not use Bergey’s to confirm reactions on this chart
    Gram Negative
    MAC Growth
    MAC No Growth
    Bacilli or Coccobacilli
    Oxidase + (See instructor for test) Oxidase –
    Cocci or Vibrio
    Spirilli or Spirochete
    PRGlucose A+
    Family Enterobacteriaceae [$Oxidase -]
    PRGlucose A- [$Oxidase +]
    #H2S – , P+/A-
    $$PRLactose A+
    PRGlucose +
    – NO3
    Sucrose +
    PRGlucose –
    Sucrose –
    $$ PRLactose ACitrate –
    Indole +
    Citrate +
    Indole [Sucrose +]
    aeruginosa %
    &green pigment
    %% obligate aerobe
    coli *
    [#A+/A+ H2S-]
    [MR+, Suc-]
    #H2S +
    &UV fluorescence- use UV light
    #H2S ##A+/A+
    Citrate –
    Citrate +
    #H2S+ P+/A+
    PA –
    PA –
    Urea Urea +
    [Urea- ] VP+
    [VP- ]
    Urea +
    #H2S+ P+/A+
    #H2S P+/A+
    [MR+ VP-]
    [@VP- @Urea-]
    [@MR- Suc+]
    [VP- Urea-]
    [MR+ Suc-]
    [Urea – VP -]
    [Suc- MR+]
    Urea –
    [VP- MR+]
    #H2S P+/A+
    #H2S +
    Urea +
    [VP- MR+]
    [Indole+, Suc-]
    [Indole -]
    [VP- MR+]
    [Urea +]
    [Indole + ]
    [Urea+ Suc -]
    [VP- MR+]
    Special Notes
    [Blue] – confirmatory tests are recommended only for conflicting results
    Use PR sugar test, check durham tube for gas+
    PA = Phenylalanine MR = Methyl Red VP= Voges-Proskauer
    Confirmation only, run from growth on blood agar
    @@ = perform capsule stain w/ litmus milk broth
    & Observe morphology on TSA plates
    @ S. marcescens pigment may give confusing reactions for MAC (lactose), urea. For VP, MR note color before adding reagents
    % P. aeruginosa may exhibit false glucose + reaction in broth
    %% Use thioglycollate to confirm
    # Kligler’s tube; A and P are fermentation/peptonization reactions, H2S+ or H2SNO3, NO2, N2, N2O, NH3 – Nitrate test
    ##Kligler’s tube, alkaline reversion known to occur on slants after 24 hrs; information on this chart is reliable at 48 hrs
    $$ Some lactose reactions, especially after 24 hrs, can have different colors in the durham tube vs. broth vs Kliglers. Use PR broth color. Check MAC to verify.
    Lab 9, Experiment 5: The Unknown Report. Report must be typed and submitted through the Blackboard
    Turnitin portal by the due date, in one WORD (.doc or .docx) or ADOBE (.pdf) file in your instructor’s
    blackboard page. The report MUST have
    A. Title Page:
    a. Student name; Course section number ; Unknown patient number;
    b. Correct spelling and format of your Gram + and Gram – (Genus & species name)
    B. Flow of Testing necessary to identify each organism according to your flow chart (put these on a page by
    themselves) (Ex. Gram negative → growth on MAC → Glucose + → Lactose – → etc…)
    a. (optional: include copy of Flow charts provided and highlight/circle the path you used to identify
    your Gram + & Gram – organism.).
    C. Unknown Form for Gram Positive and Negative
    a. The Unknown Form, important items to not forget (double check your notations with information
    from labs this semester. Include observations on color or reaction, not just ‘+’ or ‘-‘ for results.
    i. Microscopic Characteristics:
    1. Colored drawing of Gram stained organisms.
    2. Written Gram reaction, plus shape & arrangement of organisms
    ii. Cultural Characteristics:
    1. Colony morphology: size, pigment, configuration, elevation, margin as best you
    can determine from photos provided
    2. Selective/differential media: note presence of growth AND differential
    iii. Biochemical Characteristics:
    1. You MUST record ALL results for any images provided
    2. Refer to your instructors directions on the desired notation guidelines
    iv. Antibiotic Sensitivity: Kirby Bauer antibiotic assessment for each organism, with
    measurements and sensitive, intermediate, or resistant.
    D. Discussion: Typed interpretation and thorough walk-through of your conclusions
    a. Separate your conclusion by organism – have one section for your Gram Positive Conclusions
    and one section for Gram Negative Conclusions
    b. You should include all these points about each organism you identified:
    i. Genus, species name
    1. You should list an ALTERNATE possibility for identification (and why you
    chose this as your alternate) if you are unsure of the interpretation of a test or
    your conclusions.
    ii. A thorough description of the isolation and pure culture method and steps that were
    followed based on visual evidence in your Powerpoint file, to get pure cultures of each
    organism. Include likely staining and sub-culturing steps that were necessary and gram
    iii. Include summary and interpretations of biochemical tests you used to determine identity.
    Please be somewhat brief, but accurate. For each biochemical test it should be clear you
    understand: how to perform the test, its purpose, the result, and what that result means.
    iv. Discuss the antibiotic resistances and sensitivities of your organism, and which would be
    the most logical antibiotic to use against your organism.
    v. Research: What kind of infections/diseases does this organism usually cause, if any? Is
    there anything else we should know about this organism? What clinical relevance does
    this have, and what medical implications do we need to be aware of when treating an
    infection with this organism? Do some research!
    vi. Cite at least 2 sources using APA format (see guideline sheet) PER organism.
    E. Be sure to refer to guidelines and instructions in Blackboard for how to insert images if you wish to use
    handwritten versions of your Flowchart and Unknown Form, and insert them as images in your document.
    The report must be submitted as ONE document. The discussion MUST be typed.
    Unknown Report Guidelines
    When you have recorded all your results in the Unknown
    Form, and used the provided flowcharts to identify your
    organisms, you are ready to start constructing your
    Unknown Report.
    Briefly, your unknown report must include your completed:
    • Title Page
    • Flow of Testing or highlighted Flow Charts
    • Unknown Form
    • Discussion on Gram Positive Organism
    • Discussion on Gram Negative Organism
    The attached document gives more specifics for the expectations
    of these items needed for your report. The Guidelines are
    considered necessary in preparing your Report.
    If needed, a helpful document on APA format, and inserting images in
    your report is included. Contact your instructor if you have any questions
    about this assignment. Be sure to allow ample time for a response prior to
    the due date.
    Your report will be graded as follows:
    30pts – Gram Positive Organism
    • 4 pts – correct organism
    • 10 pts – correct recording of all tests and data on forms
    • 16 pts – discussion (4pts isolation and pure culture,
    6pts interpretation of selective, differential, biochemical tests,
    3pts antibiotics, 3pts research)
    30 pts – Gram Negative Organism
    • 4 pts – correct organism
    • 10 pts – correct recording of all tests and data on forms
    • 16 pts – discussion (4 pts isolation and pure culture,
    6pts interpretation of selective, differential, biochemical tests,
    3pts antibiotics, 3pts research)
    may be taken if required items are not included, such as title page,
    workflow, APA format, or if work is found to be plagiarized.

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