Lab 127 Day 2

Labs #20-24 Identification of a Bacterial Unknown

Read

Review procedures for Gram stain, streak plate technique, aerobic and anaerobic growth, and

biochemical tests. Use these materials to help you identify your organism.

Learning Objectives

The student will be able to:

• Use techniques learned during this term to identify an unknown bacterial species based on morphological and biochemical characteristics

• Prepare a dichotomous key and use it as an identification tool

Procedure

You’ve been assigned one unknown bacterial species. Your goal is to identify the unknown

bacterium to the genus and species level.

As you begin, keep these things in mind:

• You will establish purity by streaking for isolation on an agar plate. You will also examine cell morphology, colony morphology, and anaerobic growth of your unknown organism.

• Formulate a strategy for how your testing will proceed. Consider your initial observations and data. Consider which tests will be most informative and which tests will be least

informative. Consider which tests might be done first and which tests might be done later, or

as confirmation.

NOTE: You will be given results for every biochemical test on your unknown organism.

However, these results will provide a lot of unusable (and potentially confusing) data. So, it’s

best that you create a strategy or plan for which tests will be most helpful in identifying your

organism.

• There are variants of bacterial species. Therefore, do not rule out an organism solely on the basis of one test that does not conform to the typical reaction for a species. The test that does

not conform might be due to faulty reagents, media, technique, or difficulty in reading the

test. Consider the weight of evidence in coming to a decision on genus and species.

Follow this general approach to identify your unknown organism:

1. Record the code number of the unknown culture that you’ve been assigned to.

2. Observe a Gram stain of your culture. Record the Gram reaction, cell shape, and cell arrangement of your organism and comment on relative cell size.

3. Observe and critique a streak plate of your culture. Then, examine for isolated colonies and determine if you have a pure or mixed culture.

4. Observe growth on a TSA plate that was incubated in a Gas-Pak jar to determine oxygen requirements of your organism.

5. Based upon the results of your Gram stain, determine what biochemical tests you will perform. You will construct a dichotomous key to guide your thinking. Do only those tests that are

necessary and helpful to make your identification. Avoid unnecessary or useless testing. Write

legibly or type the dichotomous key. The dichotomous key will be submitted for grading.

6. Use all of your data to determine the genus and species of your organism. Check your results against the class database to identify your organism.

Your unknown organism is taken from the following list of bacterial species:

Aeromonas hydrophila

Alcaligenes faecalis

Bacillus laterosporus

Bacillus megaterium

Bacillus subtilis

Citrobacter freundii

Corynebacterium diphtheriae

Corynebacterium xerosis

Enterobacter aerogenes

Escherichia coli

Klebsiella pneumoniae

Listeria grayi

Morganella morganii

Mycobacterium smegmatis

Proteus vulgaris

Pseudomonas aeruginosa

Salmonella pullorum

Available Resources

To help you identify your bacterium, the following resources should be used:

• Biochemical Test Student Database generated in Labs 17-19. Based on the number of positive and negative responses for a given test, you can judge how reliable or definitive a

test might be for a certain species.

• All other references and instructional materials provided throughout the semester.

• *IMPORTANT: Students are NOT permitted to utilize the tutors in the Biology Learning Center (BLC) for assistance during this project.

Important Deadlines & Requirements

Deadline #1: Students must complete the two Lab 20 Assessments by 9:00 PM EST on

Monday, Nov. 23, 2020.

Deadline #2: Students must upload the Dichotomous Key to Blackboard by 9:00 PM EST on

Monday, Nov. 30, 2020. Do NOT email your dichotomous key.

Irrespective of your Gram stain results, compose a dichotomous key for Gram positive

organisms and Gram negative organisms. Include all organisms and use correct

nomenclature.

Write out the full names of the staining procedure and biochemical tests. Do not use

abbreviations.

Submit the dichotomous key as a PDF file or Word file.

If you have multiple pages, submit them together as one file.

Use the following format to label your file: Lab section #, Last Name followed by First

Name, Dichotomous Key. Example: #1604, Kryger Kate, Dichotomous Key

GRADING: You will be graded on the dichotomous key that you submit on this day.

However, you are welcome to revise your plan (dichotomous key) as you progress through

this project. If you choose to revise your dichotomous key, please attach the revised version

to the end of your Written Laboratory Report.

Deadline #3: Students must upload the Written Laboratory Report to Blackboard by 9:00 PM

EST on Wednesday, Dec. 9, 2020. Do NOT email your written report.

Submit all pages of the Written Laboratory Report together as one file.

Submit the Written Laboratory Report as a PDF file or Word file.

Use the following format to label your file: Lab Section #, Last Name followed by First

Name, Written Report. Example: #1604, Kryger Kate, Written Report

IMPORTANT: Students are required to upload the Dichotomous Key and Written Laboratory

Report to Blackboard for submission. Do NOT email these items to the instructor. 10 points

will be deducted for each emailed file.

Written Laboratory Report

Your lab instructor will provide the due date for your report. You are welcome to submit your

report before the specified deadline.

Your complete Written Laboratory Report must be submitted through Blackboard. The written

report must be typed, with the exception of Table 1.

The Written Laboratory Report will comprise three or four pages (see below). However, all

pages much be submitted together as one file.

• Page 1: Complete Table 1 (attached). Table 1 is the first page of your written report.

• Fill in the code number of your unknown culture, along with the genus and species name of the organism you identified. Use correct nomenclature.

• Record your observations for cell morphology, streak plate, colony morphology and oxygen requirement on Table 1.

• Page 2: Prepare a typed, summary table (call it Table 2) that has three columns.

• Column 1: List all staining procedures and biochemical tests you performed. Write out the full names of the staining procedure and biochemical tests.

• Column 2: List the result you obtained (+ or -). Use special notation, such as +/+, +/-, -/- or A/K,H2S,G, etc., for carbohydrate fermentation tests and TSIA test results.

• Column 3: List the standard result for that procedure or test from the Biochemical Test Student Database using + or - notations. Use special notation, such as +/+, +/-, -/- or

A/K,H2S,G, etc., for carbohydrate fermentation tests and TSIA test results.

• Page 3: A one-page discussion. Your report must include a one-page discussion and interpretation of your results.

• Be sure to address all of the following: a. Your strategy; and why you chose to perform or not perform certain tests b. How the results helped you come to a conclusion about the identity of the

bacterium

c. How you explain any differences between your results and the standard results for this species

• Limit the discussion to one page in length. The discussion must be typed, using 10 font, and single-spaced.

• Page 4: Attach your revised dichotomous key, if applicable.

Software Note

Microsoft Office 365 Education is available for free for all GRCC students.

• Students can install Microsoft Office on up to five PCs or Macs and other mobile devices.

• Full versions of Microsoft Word, Excel, PowerPoint, OneNote, Outlook, Publisher and Access are included, along with Office Online and 1 TB of OneDrive cloud storage.

• To sign up, click on the link. Select the "Find out if you're eligible" box and enter your GRCC email address. https://products.office.com/en-us/student

Available Points Point value

Streak Plate and Anaerobic Growth Procedure Assessment (This score will be posted separately in Blackboard Grade Center.)

5

Gram Stain Assessment (This score will be posted separately in the Blackboard Grade Center.)

5

Dichotomous key is legible, correct (including nomenclature), includes all organisms, full names of stains

and biochemical tests, uploaded to Blackboard in proper format (This score will be posted separately in the Blackboard Grade Center.)

15

Written Laboratory Report (This score will be posted separately in the Blackboard Grade Center.) • Code number recorded 5

• Organism identified correctly 15

• Table 1 - fully completed with correct information, clearly and legibly presented 15

• Table 2 – typed, appropriate and accurate information, clearly presented 15

• Discussion is typed, contains the requested information, and is thoughtfully presented 15

• Overall report has correct grammar, spelling, nomenclature, and page order 5

• Files are submitted through Blackboard and are properly titled and formatted 5

Total Points for Identification of the Bacterial Unknown 100

IMPORTANT: Late submissions will not be accepted under any circumstances and will result in a score of zero.

Timeline: What to Expect

DAY 1

• Find your unknown code number: This is the bacterium that you will identify.

• Streak Plate & Anaerobic Growth Assessment

• Gram Stain Assessment

• Observe a Gram stain for you organism

DAY 2

• Examine streak plate results

• Examine aerobic & anaerobic growth results

• Biochemical Test Student Database: This is our “standard” for comparing your biochemical test

results to what is known for each organism.

• Formulate a testing strategy to identify your organism: Using the Biochemical Test Student Database,

you will create a dichotomous key for Gram positive organisms and Gram negative organisms. Your

dichotomous key must be uploaded to Blackboard no later than the specified deadline.

DAY 3

• Evaluate and interpret the biochemical test results for your unknown organism

DAY 4 & DAY 5

• Day 4 & 5 are free days to complete the Identification of the Bacterial Unknown Project

• Your completed Written Laboratory Report must be uploaded to Blackboard no later than the

specified deadline. You are welcome to submit your report before the deadline.

Table 1 (This is page 1 of your report.)

Name_________________________________

Unknown Code Number: ________

I have identified this organism as:

Table 1 Morphological, Colony, and Cultural Characteristics

Gram Stain Sketch your observations of the Gram Stain

Gram reaction:

Cell shape:

Cell arrangement:

Comments on cell size:

Magnification:

Streak for Isolation Sketch the streak plate provided by the

instructor. Critique the streak technique, and

examine for purity and well-isolated colonies.

Criteria for a “good” streak plate:

Is this a pure culture or mixed culture?

Colony Morphology

Comment on size:

Shape:

Color:

Other notable observations:

Sketch a few colonies

Cultural Characteristics

Growth under anaerobic

conditions:

Amount of

Growth (0 to +3):

Conclusion: Obligate aerobe

(circle one) Facultative anaerobe

Obligate anaerobe