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microbiology result UNKNOWN INSTRUCTIONS DAY 1: Today you will be given a broth tube containing two unknown microorganisms: a Gram-positive coccus and a Gram-negative rod.…

microbiology result
UNKNOWN INSTRUCTIONS
DAY 1:
Today you will be given a broth tube containing two unknown microorganisms: a Gram-positive
coccus and a Gram-negative rod. Your ultimate task will be to separate the two organisms and
determine the identity of each organism by performing various biochemical tests previously
learned in this lab course. During the whole unknown project, you will be able to refer to your
notes and consult your classmates for assistance. (However, remember that this is an individual
project – a student should not rely completely on another student to tell them what to do!) You
will also get to ask your instructor 3 “free” questions without any adverse effect on your grade.
For TODAY you will be doing 4 things:
1. Streaking out the broth culture onto a Nutrient Agar plate using a 4-way or T-streak
isolation streak.
2. Streaking out the broth culture onto a MacConkey agar plate using a 4-way or T-streak
isolation streak.
3. Streaking out the broth culture onto a C-CNA agar plate using a 4-way or T-streak
isolation streak.
4. Performing a Gram stain on the culture to verify the presence of both a Gram-positive
coccus and a Gram-negative rod.
**Incubate all plates at 37°C. There will be Aneropak box on the front table if you wish to
incubate one of your plates in an aerobic environment enriched with CO2.
Helpful hints:
Think . . . why are we using 3 different kinds of plates to separate and isolate the two unknown
organisms?
Label, label, label! Label the type of media, your unknown #, your name and class period, etc.
Be sure you write down your unknown number!!
One of your Gram stains will be graded, so if you’re happy with the results on this one, it’s a
good idea to get this hurdle out of the way!
Your isolation streaks will also be graded, so make sure your lab instructor can identify which
plates belong to you!
DAY 2:
Today you will be doing the following:
1. Examining your streak plates from the previous lab period for the presence of isolated
colonies.
2. Using a sterile needle, pick a small part of one putative Gram-positive colony and
perform a Gram stain to confirm its identity.
3. Once you’ve confirmed the organism is a Gram-positive coccus, transfer the remainder of
the above colony to a nutrient agar slant. This is going to be your working stock culture
from which to inoculate the rest of your Gram-positive tests. Make sure you label this
slant with your unknown # AND with “Gram+” so you don’t get it mixed up with your
Gram-negative!
4. Using a sterile needle, pick a small part of one putative Gram-negative colony and
perform a Gram stain to confirm its identity.
5. Once you’ve confirmed the organism is a Gram-negative rod, transfer the remainder of
the above colony to a nutrient agar slant. This is going to be your working stock culture
from which to inoculate the rest of your Gram-negative tests. Make sure you label this
slant with your unknown # AND with “Gram-” so you don’t get it mixed up with your
Gram-positive!
6. Incubate all nutrient agar slants at 37°C.
Helpful hints:
Label, label, label!
If you didn’t have your Gram stain graded during the previous lab period, select one of your
Gram stains from today to be graded.
DAY 3
Today you will be inoculating your isolated Gram-negative and Gram-positive unknowns onto
the appropriate microbiological media in order to determine their identity.
1. Perform a Gram stain on bacterial growth from each nutrient agar slant to confirm that
your cultures are pure.
2. Determine which genus your Gram-positive isolate belongs to: is it Staphylococcus or
Streptococcus/Enterococcus?
3. Once you determine the genus, inoculate your Gram-positive isolate onto either the Staph
ID media or the Strep ID media. DO NOT INOCULATE BOTH!!
4. Inoculate your Gram-negative isolate onto the 7 pieces of Gram-negative ID media.
Helpful hints:
Label, label, label!
Antibiotic disks (Novobiocin, SXT, Bacitracin) are available on the front cart.
An Anaeropak box is available at the front table if you wish to incubate anything aerobically in
the presence of CO2.
DAY 4
Today you will be reading the results of the tests you inoculated during the previous lab period.
Helpful hints:
Don’t forget that some Gram-negative tests require you to add additional reagents – Read your
notes!
Rulers are available.
Don’t discard anything until you are confident that you have interpreted all of your results
Correctly
Name:___________________________________________ Unknown #:____________
Record the results of your unknown tests on this worksheet. Be sure to include this worksheet
in your unknown report!
Gram Stain of Mixed Culture: (Observations)
Isolation Streaks:
Media used for
Isolation
Observations Gram Stain Results
(if applicable)
Nutrient Agar
MacConkey
Columbia CNA
Agar
Gram Positive Identification:
Catalase Result: ______________
Result Result
MSA fermentation _______ Hemolysis _______
DNase _______ Bacitracin _______
Novobiocin _______ SXT _______
Bile esculin _______
MSA growth _______
Gram Negative Identification:
MacConkey: Fermentation ________
TSI: Slant _______ Butt______ Gas ______ H2S ______
Urease: ________
Citrate: _______
SIM: Indole _______ H2S ______
MR/VP: MR _______ VP _______
Nitrate: + or –
LIA: Deamination________ Decarboxylation ______
Name________________________ Unknown #__________
Unknown Deductions
Ded. Instr.
You may be deducted points for the following: Pts Initial
1. Incorrect 4-way streak procedure (5 pts) ______ _____
2. Gram stain (0-5 pts) ______ _____
3. Microscopy (5 pts) – can’t focus microscope ______ _____
4. Using the wrong media ______________ (10 pts) ______ _____
5. Using extra media b/c of inoculation errors (8 pts) ______ _____
6. Omitting or using the wrong disc on Staph or Strep ID (5 pts) ______ _____
7. Not labeling tubes and plates correctly (1 pt per piece) ______ _____
8. Incorrect ID of microbe (10 pts) ______ _____
9. Each additional question after your 3 permitted (2 pts each) ______ _____
10. Forgetting your Unknown Work sheet (5 pts) ______ _____
11. Not bringing your instructions (5 pts) ______ _____
12. Picking up extra media without instructor’s permission (5 pts) ______ _____
13. Viewing organism under wrong objective (5 pts) ______ _____
14. Not cleaning oil off of microscope objectives (5 pts) ______ _____
15. Not cleaning table at the end of the lab period (5 pts) ______ _____
16. Failing to invert agar plates during incubation (5 pts) ______ _____
17. Incorrectly interpreting results _____________ (__ pts) ______ _____
18. Incorrectly inoculating media ______________(3 pts each) ______ _____
19. ______ _____
20. ______ _____
NOTES TO REMEMBER:
1. Each student is allowed to ask 3 questions throughout the Unknown Identification process,
whether you ask a lab instructor or a lab assistant. After that, for every question asked, you are
penalized 2 pts.
2. You must refer to your notes for directions and procedures. You may also ask your fellow
students if you have questions. Be sure to bring your protocols to each class.
3. You are also required to bring this Unknown Identification Work Sheet each time you come to
lab. Points will be deducted if you do not.
4. You must be able to independently find your microbes under the microscope.
5. Instructors will NOT interpret results for you or tell you the identity of your unknowns. It is
your responsibility to correctly interpret the results of each test and to determine the identity of
each unknown once you have obtained your test results.
NOTES:
Appendix B: Unknown Reports Cain et al 129 Fall 2017
GENERAL:
Unknown reports in microbiology are written in scientific format. Scientific writing is different from other types of writing in that the results of the exercise or experiment are being showcased, not the writing. The purpose of scientific writing is not to entertain, but to inform. The writing should be simple and easy to understand. There is a specific style that must be followed when writing scientific reports. Scientific writing is typically written in the passive voice. The pronouns “I”, “We” and “They” are not used. For example, instead of writing “I used a TSA agar plate to isolate my unknown,” it is customary to write, “A trypticase soy agar (TSA) plate was used to isolate the unknown.” It is also customary to write in the past tense for most of the report. This includes the introduction, the summary, the description of the materials and methods, and the results. The present tense is reserved for the conclusions about the results.
Microbial nomenclature: The name of the bacterium should be written out and spelled correctly. The name should be italicized, e.g. Staphylococcus aureus. The genus is capitalized but the species is not. After the full genus name is given in the paper, it can be abbreviated as S. aureus, but it is still italicized. This is as long as there is no other genus in the paper that starts with the same letter. (For example, if you have a Staphylococcus species and a Salmonella species as your unknowns, you cannot abbreviate!)
MECHANICS:
The lab report should be typed using a 12 point normal font such as Times New Roman or Arial, and should be single-spaced. The lab report should be presented bound in a report folder. Reports will be graded on content as well as spelling, grammar, punctuation, and organization.
SECTIONS OF THE UNKNOWN LAB REPORT:
(Note: Other than the title page, the pages of the report must be numbered) TITLE PAGE
The title page should include the following information:
· UNKNOWN NUMBER
· YOUR NAME
· DATE (the due date)
· LAB INSTRUCTOR’S NAME
· COURSE NAME / SECTION NUMBER
· SEMESTER / YEAR
INTRODUCTION
This section introduces the reader to the study and why the study was done. This should only be a few sentences long. Example: “There are many reasons for knowing the identity of microorganisms. The reasons range from . . .[explain 2-3 reasons why you may want to identify an unknown bacterium]. This study was done by applying all of the methods that have been learned in the microbiology laboratory class for the identification of an unknown bacterium.”
MATERIALS & METHODS
This is where the details of the study are listed. The materials and methods section should contain three paragraphs: (1) a description of how the two unknowns were separated and isolated (2) a description of the identification of the Gram-positive isolate and (3) a description of the identification of the Gram-negative isolate. Cain et al 130 Fall 2017 Be specific, but concise: do not rewrite the lab manual! The most common way is to mention the names of the materials used and why they were used, and reference the lab manual for the procedure or method. See example 1.
Example 1: “An unknown labeled number 37 was selected. The methods that have been learned in microbiology laboratory were applied to identify this unknown. Procedures were followed as stated in the course laboratory manual (Cain et al, 2015) unless otherwise noted. The first procedure that was performed was streaking the unknown out on a nutrient agar plate, using the T streak method described in the lab manual. This was done in order to . . . [explain why]. After the plates were incubated and grown, the morphology was observed and recorded and a Gram stain was performed. After determining the Gram reaction, specific biochemical tests were performed. The biochemical tests were chosen from the lab manual. Since unknown 37 was determined to be a Gram-positive rod, an endospore stain was also performed. The following types of media were inoculated with unknown 37: starch, lipid, and casein agars, mannitol salt agar, a gelatin deep, a bile esculin agar slant, and a nitrate broth. Table 1 lists the test, purpose, reagents and media components, and results.”
RESULTS
This is where the results are summarized. The results should be presented in 2 tables, using a separate table for each unknown, i.e. one table for your Gram-positive, and another table for your Gram-negative (see example below). This is also where the flow charts showing how you arrived at your answers are presented. The flow charts included in the report should trace the route you followed on your flow chart to identify your unknown. A short paragraph explaining how the results are presented should also be included. Example: “Unknown 37 appeared as a large, mucoid, cream-colored colony on the nutrient agar plate. A Gram stain revealed that the organism was a Gram-positive rod, and an endospore stain demonstrated the presence of subterminal endospores. A stock of unknown 37 was grown on a nutrient agar slant and used to inoculate the tests listed in Table 1. The results of all tests are presented in Table 1 and shown in flow chart form.
DISCUSSION / CONCLUSION
This section interprets the meaning of the results. Again, you should have a separate paragraph
for each identified isolate. The following questions should be answered here:
How did the test results lead to identification? What problems were encountered, if any? In this
section, you should also include 1-2 paragraphs of background information on each bacterium
you identified, including its characteristics and significance.
Example of a discussion: “After several differential tests, it was concluded that the Grampositive organism in unknown 37 was Bacillus polymyxa. After performing the Gram stain, it
was determined that the unknown was a Gram-positive rod. The results of the endospore stain
showed that the organism produced subterminal endospores, indicating it belongs in the genus
Bacillus. The organism was grown on a TSA slant for use in inoculating the rest of the
biochemical tests. All of the biochemical tests worked well except for the test for gelatin
hydrolysis. It gave a false negative result at first, which was inconsistent with the rest of the
results. Using the Bacillus identification dichotomous key, it was concluded that the unknown
was Bacillus polymyxa
Bacillus polymyxa is a Gram-positive endospore-forming bacillus (Bauman, 2006). It is a
motile bacterium, and possesses peritrichous flagella (Todar, 2005). B. polymyxa can be found
in a wide range of environmental habitats, including acidic environments. It is commonly found
in soil, water, and decaying vegetables (Stedman, 2006). Because it can grow in both moderate
and cold temperatures, B. polymyxa is classified as a psychrotroph. B. polymyxa produces a
thick polysaccharide capsule, and is also a facultative anaerobe (Todar, 2005). Although B.
polymyxa does not cause disease in animals or humans, it is of medical significance because it
produces the antibiotics polymyxin and colistin (Madigan and Martinko, 2006).
REFERENCES:
Note: the minimum number of references is three, which can include the lab manual and the
textbook. You may not use wikis (such as Wikipedia or MicrobeWiki) as sources! References
should be listed in APA format, and parenthetical in-text citations must be used where
appropriate. Note: Any information presented that is not your original idea or your own data
must be credited with an in-text citation!
Use this format to cite the lab manual on your reference page:
Cain, D., Hanks, H., Weis, M., Bottoms, C., and Lawson, J. (2017) Microbiology Laboratory
Manual B2420. Collin County Community College District, McKinney, TX.
The corresponding in-text citation would be (Cain et al., 2017).
UNKNOWN WORKSHEET
Attach your completed unknown worksheet.
Modified from: http://www.sci.sdsu.edu/classes/biology/bio210/lab/demers/Howtowriteanunknownlabrepor.pdf

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