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.

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.

Procedures:

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.

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.

FIRST TWO ATTACHED ARE FOR THE FOLLOWING BELOW

BE SURE TO REVIEW ALL VIDEOS AND INFORMATION IN LAB 9 BEFORE BEGINNING YOUR UNKNOWN PROJECT!

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.

OTHER HELPFUL NOTES for the UNKNOWN:

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).

To determine your organism: ONLY USE THE POSTED FLOWCHART IN THE LAB 9 AREA TO DETERMINE WHAT YOUR ORGANISM’S IDENTITIES ARE! DON’T USE ANYTHING ELSE!

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).

https://drive.google.com/drive/mobile/folders/15JIJrPEgNN2wScEilTfT_koivuhYi8v4?usp=sharing&fromMobile=true

For Growth Media
• Name and Type of medium
• For G+/G- or both?
• Selective? For what? Why? (ingredients that make it selective and
how?)
• 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 ??
• WHAT TO OBSERVE
o Reagents added after incubation (if relevant)
o how to read results
• + RESULTS, – RESULTS
o Biochemical interpretation and visual description
What does a positive result MEAN? Negative result?
• SPECIAL INSTRUCTIONS
• 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:
Introduction
Concise, descriptive title
(0.5 pt)
Background of project,
in your own words. (4
pts)
Broken into weekly
paragraphs (1 pt)
Summary of project (4
pts)
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
pt)
Total
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
Methods
Results
Conclusion
Observations of test results
described. (15 pts)
Summary of tests
(10 pts)
Description of result
interpretations. (15 pts)
Statement of
suspected
unknown (1 pt)
Unknown
Correct unknown ID (5
pts)
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
pts)
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
pts)
Proper sentence structure
used throughout paper. (2
pts)
*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
sections.
___ 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
Bacillus
Cocci
(Simple stains or negative stains can help determine shape)
All are Starch –
Starch +
*Catalase –
Starch * Catalase +
*Catalase +
Family Streptococcaceae
*Catalase +
[Facultative,
***Aerobic]
*Catalase [***Anaerobic]
Clostridium
sporogenes
[@terminal
Endospores]
**Mann+
Bile Esculin +
VP+
+NH3/N2
Bile Esculin –
** Mann +
#beta
VP-
PRGlucose A–
##Tetrad/
** Mann #gamma
Sarcina
– NO3
**Mann –
$ Bacillus
subtilis
&white
#gamma
[VP+ , +NO2]
@%Endo
PRGlucose A+
Staphylococcus
Bacillus megaterium
& irregular, beige
[Mann+, VP-]
#alpha
@%Endo
$ Bacillus pumilus
& white
@%Endo
[VP+, Mann weak+]
#weak beta
Enterococcus
faecalis
#gamma/weak alpha
[Mann+]
[MR+, -NO3]
Streptococcus
pneumoniae$$
VP# alpha (green)
[Mann-]
[%capsules]
Streptococcus
pyogenes
&small, translucent
[&&Bacitracin S]
#beta
[!CAMP-]
VP+
Staphylococcus
aureus
[! Coag +]
[NO2+]
Streptococcus
agalactiae$$
#beta
[&&Bacitracin R]
[!CAMP+]
Staphylococcus
epidermidis
[! Coag -]
[NO2+]
– NO3
Micrococcus
luteus
&Yellow Pigment
[Methyl Red-]
#gamma
[VP – ]
+NO2
Kocuria
rosea
&Pink Pigment
[Methyl Red+]
#gamma
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
$$ Might appear as diplococcus, pleomorphic or coccobacillus
# Hemolysis on blood, streak pure culture; view after 48 hrs max; weak = reaction subtle, not obvious
&
Observe isolated colony morphology on TSA plates
NO3, NO2, N2, N2O, NH3 – Nitrate test
&& Observe Kirby Bauer results
VP = Voges Proskauer PR = Phenol Red
%
Special Stain; Endo=endospore
Coag = coagulase MR = Methyl Red
##
Observe arrangement on negative stain or gram stain
!
talk to instructor for guidance, info may be used if available
@
Perform stain from plate or culture left in incubator 3+ days
*
Must be from solid agar growth grown in incubator *** Use FTM thioglycollate if needed
@%Endo
#beta
[VP- , +NO2]
Rev Jan 6 2021
Students should not use Bergey’s to confirm reactions on this chart
Gram Negative
MAC Growth
MAC No Growth
Bacilli or Coccobacilli
Oxidase + (See instructor for test) Oxidase –
Cocci or Vibrio
Spirilli or Spirochete
PRGlucose A+
Family Enterobacteriaceae [$Oxidase -]
PRGlucose A- [$Oxidase +]
#H2S – , P+/A-
$$PRLactose A+
+N2/N2O/NH3
+NO2
PRGlucose +
– NO3
Sucrose +
PRGlucose –
Sucrose –
Campylobacter
jejuni
$$ PRLactose ACitrate –
Indole +
Citrate +
Indole [Sucrose +]
Pseudomonas
aeruginosa %
&green pigment
%% obligate aerobe
Escherichia
coli *
[#A+/A+ H2S-]
[MR+, Suc-]
#H2S +
A+/A+
Alcaligenes
faecalis
Pseudomonas
fluorescens
&UV fluorescence- use UV light
#H2S ##A+/A+
Helicobacter
pylori
Haemophilus
influenzae
Citrate –
Citrate +
PA+
#H2S+ P+/A+
PA –
PA+
PA –
Urea Urea +
Citrobacter
freundii
[Urea- ] VP+
[VP- ]
Neisseria
sicca
Acidophile
Urea +
#H2S+ P+/A+
#H2S P+/A+
Salmonella
enteritidis
VP[Mann+]
Proteus
[MR+VP-]
vulgaris
[Suc-]
Serratia
Enterobacter
[Mann-]
marcescens
aerogenes
Klebsiella
[MR+ VP-]
&red/white
[MR-]
pneumoniae
[Suc+]
pigment
[MR+]
[@VP- @Urea-]
[@@capsules]
[@MR- Suc+]
#H2S+
P+/A+
Shigella
boydii
[VP- Urea-]
[MR+ Suc-]
Salmonella
typhimurium
[Urea – VP -]
[Suc- MR+]
Urea –
Shigella
flexneri
[Indole-]
[VP- MR+]
#H2S P+/A+
#H2S +
P+/A+
#H2SP+/A+
Urea +
Proteus
mirabilis
&spreading
[VP- MR+]
[Indole+, Suc-]
Salmonella
arizoniae
[Indole -]
[VP- MR+]
[Urea +]
[Suc-]
Morganella
morganii
[Indole + ]
[Urea+ Suc -]
[VP- MR+]
Special Notes
[Blue] – confirmatory tests are recommended only for conflicting results
*
Use PR sugar test, check durham tube for gas+
PA = Phenylalanine MR = Methyl Red VP= Voges-Proskauer
$
Confirmation only, run from growth on blood agar
@@ = perform capsule stain w/ litmus milk broth
& Observe morphology on TSA plates
@ S. marcescens pigment may give confusing reactions for MAC (lactose), urea. For VP, MR note color before adding reagents
% P. aeruginosa may exhibit false glucose + reaction in broth
%% Use thioglycollate to confirm
# Kligler’s tube; A and P are fermentation/peptonization reactions, H2S+ or H2SNO3, NO2, N2, N2O, NH3 – Nitrate test
##Kligler’s tube, alkaline reversion known to occur on slants after 24 hrs; information on this chart is reliable at 48 hrs
$$ 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.).
b. THIS SHOULD NOT BE A PARAGRAPH OR COMPLETE SENTENCES. KEEP IT BRIEF.
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
characteristics
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
staining.
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.
10:47
Х
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)
Deductions
may be taken if required items are not included, such as title page,
workflow, APA format, or if work is found to be plagiarized.