Unit 13

Unit 13: Identification of Unknown BacteriaBy Karen Bentz, Patricia G. Wilber and Heather FitzgeraldCopyright Central New Mexico Community College, 2015

Introduction

For this project, you will be separating and identifying two bacteria, one a Gram(+) and one a Gram(-), that are mixed together in your unknown tube. Each student will do their own unknown identification of the two bacteria that are in the broth that they choose. Your initial goal is to separate the Gram(+) from the Gram(-). Once you have the two species separated and growing on different media, you will use your pure cultures to inoculate new media in order to identify the organisms to species. You will have several days to isolate the two species by using techniques and performing various biochemical tests you have learned in this lab. Then you will be able to name the two species of bacteria in your unknown broth.

Your instructor will provide a format for the lab report that you will write and turn in for this unknown identification process. Your lab work and written lab report will count as the final exam for this course. (Formats do vary a bit by instructor, so follow the rubric provided by your instructor, only.)

Tips for Success

Carefully perform streak isolations so that you can identify Gram(+) versus Gram(-) colonies.

Use good aseptic technique to ensure your cultures remain pure. Take detailed notes and photos of your procedures and results. These will make it

easier to write a detailed lab report. You may perform two tests and/or inoculate two media per lab session. Usually you will

choose one test and/or media for your Gram(+) identification, and one test and/or media for your Gram(-) identification. Take your time and choose wisely. Once you check out your media from your instructor, you cannot exchange it.

You may consult your lab manual, other students, or other resources regarding your bacterial identification project. You may not consult your instructor or other instructors.

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Materials and Media (per student)

1 each of MacConkey, Blood and Chocolate Agar plates (for Day 1 inoculation) Gram stain materials Additional media/tests as needed to identify both bacterial species. No student will use

all of the media/tests available, but the possible media/ tests will include: Catalase, Oxidase, T-soy, MacConkey, Chocolate Agar, Blood Agar, MR-VP, Lysine, TSI, Lactose, Sucrose, SIM, PA, Urease, Coagulase, Novobiocin, Bacitracin, Optochin.

Bacterial Cultureso Your instructor will mix 0.05 ml (50µl) of one Gram(-) species into a 5ml broth

containing one Gram(+) species. Thus, you will have two unknown species mixed together in your unknown tube; one Gram(-) and one Gram(+).

o The Gram(-) will be one of these: Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Citrobacter freundei, Klebsiella pneumonia, Serratia marcescens, and Shigella flexneri.

o The Gram(+) will be one of these: Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus mitis, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecalis

Procedure

Day 1

1. Pick up one of the broth tubes of unknowns. Each numbered tube contains both a Gram(+) and a Gram(-) bacterial species mixed together.

2. Record the tube number on your instructor’s sign-out sheet, and also on your notes. 3. Label a MacConkey Agar, Chocolate Agar and Blood Agar plate with your full name and

unknown number, as well as the standard label information. 4. STIR your mixed broth thoroughly with your sterilized loop. The Bacillus species grow

on the top and the Streptococcus are on the bottom of the tube, so if you have one of those, you will want them to be mixed in really well with your sterilized loop before streaking!

5. Tap your loop to reduce bacteria before performing the streak isolation.6. Use the streak isolation method to inoculate each of the three plates with your

unknown bacteria (because your goal IS isolation.)7. Put your plates in the proper rack/candle jar for incubation. Return your unknown tube

to the original test tube rack.8. Take careful notes of your procedures today, as these will be included in your lab report.9. You should BEGIN WRITING your report and write as you go.

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Day 2

1. Check your plates for growth, isolation of colonies, and metabolic results. 2. Record your observations from each plate (colony appearances, lactose fermentation,

hemolysis, if you can tell) on the data collection sheet. 3. GRAM POSITIVE:

If you have two types of isolated colonies on your TSA Blood or your Chocolate agar, you will probably be able to isolate your Gram(+). Your instructor will help you.A. Gram stain the colonies you think are Gram(+). Once you have identified the Gram

(+) colonies: Record an accurate description of these colonies; be sure to include which plate

they are on. DO NOT assume the colonies look the same on the other plate with Gram(+) growth. To determine that, you will need to Gram stain off that plate as well and keep accurate records of those colonies.

Record cell shape. DO NOT worry about arrangement. Estimate and record the size of your cells. You may wish to perform a catalase test (or you may not). If you do the catalase

test, carefully record which colonies and from which plate you sampled. Your instructor may wish to observe your catalase test.

Record your results. After the catalase test (if you needed it) choose a medium upon which to grow

more of your Gram(+) to aid in identification of your Gram(+). Record the name of the new test. PUT YOUR FULL NAME, Sample # and G+ on the label. Incubate in the proper location.

B. If you have two species on your Chocolate or TSA Blood plates, but they are not isolated: You must re-streak isolate from your Chocolate or TSA Blood plate. Your

instructor will help you determine if you have two species on your plate(s) and which plate to use as your source for the re-streak.

Choose a plate type for the re-streak. Do you want to learn more about hemolysis, perhaps?

PUT YOUR FULL NAME, Sample # and “G+” on the label Put your re-streak in the proper location for incubation.

C. If you only have one type of colony (= 1 species) on your TSA Blood and Chocolate agar, Gram stain to determine if the colonies are G+ or G-. If they are Gram(+), GREAT!

o Record the cell shape and estimated size.o Record a good description of the colonies and the plate they are on.o You may wish to do the catalase test. If so, your instructor may wish to

observe your test.

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o Record your results and a good description of the colony that you used for the test.

o Choose your next media to grow more of your G+. o PUT YOUR FULL NAME, Sample # and “G+” on the label.o Put your plate in the proper location for incubation.

If the colonies are Gram(-), o Choose a plate type for your re-streak.o Re-streak isolate from your original broth. o PUT YOUR FULL NAME, Sample #, and “re-streak” on the label.

4. GRAM NEGATIVE:A. The MacConkey’s selects for Gram __________. You do not need to Gram stain

off your MacConkey’s plate. B. Your instructor may ask you to Gram stain to find your Gram(-) colonies on your

TSA blood and/or your chocolate agar plates. If so, record cell shape and size in your data sheet.

C. Carefully observe your results on all your plates, especially your MacConkey’s. Compare to your colleagues.

D. Record your observations.E. Based on your interpretation of the results of your MacConkey’s, choose a next

test for your Gram(-) that will help you identify the Gram(-) species. F. PUT YOUR FULL NAME, Sample # and “G-” on the label.G. Place the new test in the proper place for incubation.

5. Sign for your new tests/media on your instructor’s sign-out sheet at the front of the lab. Your instructor will then give you any media/materials required.

A. Once you have signed for and received your media/materials, it cannot be returned, so choose wisely.

B. If you are choosing one of the antibiotic discs, you will also need to specify which media to use it with.

C. Reagents and Gram staining materials will be out in the lab for you to use, but you will need to sign the instructor sheet for these as well.

D. Some instructors may require you to ask them for each media or reagent instead of using a sign out sheet.

6. If you are performing a test, complete the test and record your results. Your instructor may wish to see you perform the test.

7. You may keep two of your three Day 1 plates as back up. These two plates will be stored in the refrigerator, and you can use them any time during this identification project as a source of bacteria. Place the two media you wish to save in the designated rack and discard your third plate.

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8. You should start writing your Unknown Lab Report TODAY. Use the instructions provided by your professor (the instructions ARE NOT the same from instructor to instructor) to set up the format for your paper. At the end of today’s lab you should have enough information to write the Day 1 and Day 2 materials and methods section of your lab report, as well as the Day 1 results and interpretation sections. DO NOT wait until the end to start writing your paper. If you write it as you go, you will not be rushing at the end to finish your paper.

Day 3

1. Collect your media and record your observations on the data collection sheet. You may need to add reagents to some media before observing results. The reagents are available as part of the original test but you may still need to get them from your instructor. Your instructor may wish to see the results or watch you perform the test.

2. If you are still working on isolating your Gram(+) on Day 3, refer to the Day 2 instructions and consult with your instructor. Your instructor may provide you with a pure culture plate at this time. Or they may not. You will lose points.

3. Choose two new media and/or tests as needed to further identify your two unknowns. Sign for this media at the front of the lab. On the label put PUT YOUR FULL NAME, Sample # and “G+” or “G-“ as appropriate.

4. Inoculate your two new media and place them in the appropriate rack for incubation. If you are performing a test, complete the test and record your results.

5. You may keep only two of your old media. These may be the two you saved on Day 2, or two from today. Discard all other old media.

6. If you have identified your bacteria, you do not need to run any more tests.7. Continue writing your lab report, adding the details of today’s inoculations and results.

Day 4

1. Last day to inoculate. No one needs to do all of the available tests or media inoculations. In most cases, a relatively small number of carefully selected tests will be enough to identify your organism.

2. Choose two new media and/or tests if needed. If you have identified both of your bacteria, you do not need to run any new tests. On the label put PUT YOUR FULL NAME, Sample # and “G+” or “G-“ as appropriate.

3. Inoculate your media and place it in the proper rack for incubation. If you are performing a test, complete the test and record your results.

4. Discard all old media including your “saved” media.5. Continue/finish writing your lab report.

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Day 5:

1. Last day to observe results. Collect your media and record results. 2. Discard all media when finished.

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No

MacConkey

Unknown Bacteria Identification Project

YesGram StainChoose new media to inoculateDay 3 -5 Choose new media as needed 

No

YesGram StainChoose new media to inoculateDay 3 -5 Choose new media as needed 

Unit 13

Results and Interpretation

Your streak isolations performed on MacConkey Agar, Blood Agar and Chocolate Agar on Day 1 will hopefully produce obvious results you will see clearly on Day 2. The streak isolation, the Gram stain and perhaps the catalase test results should be used as the basis for your decision making process on Day 2. Once you have narrowed the possible choices to a few organisms, the additional tests you do should only be ones that help you narrow it down still further. Tests where all of your possible organisms would give the same result (for instance, all positive or all negative) are of no value.

If you did not get isolation of your Gram(+) review the directions above and consult with your instructor.

Take detailed notes on the results of your tests. Interpret the results of all of the media and/or tests that you conduct. Explain in detail what the results indicate about the cell wall structure and metabolism of your unknown. Also discuss the evidence for or against your bacteria being a possible pathogen. Always record the dates of your inoculations and observations.

Your Unknown Bacteria tube number: ________________

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Final Unknown Project Data Collection Table

Day Media Inoculated / Test performed and date performed

From Where?

Results: Describe what all results looked like and date observed.

Interpretation: What do results indicate about organism’s size, shape, cell wall, metabolism, pathogenicity

1/2Date:MacConkey Agar

Date:Describe the colonies.

Is a G+ or G- growing on this plate? What is a reason to save this plate?

1/2Date:Blood Agar

Date:Describe the colonies and the hemolysis for each if you can tell.

If you Gram stained, which colony type is G+? If you do tests for your G+ ID, would you use these colonies?

1/2Date:Chocolate Agar

Date:Describe the colonies.

If you Gram stained, which colony type is G+? If you do tests for your G+ ID, would you use these colonies?

2Date:Gram stain:

Gram(+): color, cell shape,

estimated cell size

Date:

2Date:Gram stain:

Gram(-): color, cell shape,

estimated cell size

Date:

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Final Unknown Project Data Collection Table (continued)

Day Media Inoculated / Test performed

From Where?

Results: Describe what all results looked like.

Interpretation: What do results indicate about organism’s size, shape, cell wall, metabolism

2/3

2/3

3/4

3/4

4/5

4/5

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Insert Photos of Results Here:

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Photos, continued:

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Bio 2192 Gram(+) Organisms and Expected ResultsMedia/Test Bacillus

megaterium (Bm) BSL 1

Bacillus subtilis (Bs)

BSL 1

Enterococcus faecalis (Ef)

BSL 2

Staphylococcus aureus (Sa)

BSL2

Staphylococcus saprophyticus

(Ss) BSL 1

Streptococcus mitis (Stmi)

BSL 2

Streptococcus pneumoniae (Spn) BSL 2

Streptococcus pyogenes (Spy)

BSL 2Gram Stain (+) purple (+) purple (+) purple (+) purple (+) purple (+) purple (+) purple (+) purple

Cell Shape bacillus bacillus cocci cocci cocci Cocci (weird on Choc. plate)

cocci cocci

O2

Catalase (+) bubbles (+) bubbles (-) (+) bubbles (+) bubbles (-) (-) (-)Oxidase (-) (-) (-) (-) (-) (-) (-) (-)

TSA with bloodgrowth, grayish gamma? or beta

growth, grayish gamma? or beta

growth, gamma

growth, beta growth, gamma growth, alpha growth, alpha growth, beta

Carb

ohyd

rate

Tes

ts

MacConkey’s no growth no growth no growth no growth no growth no growth no growth no growthLactose Durham (Acid,Gas)

-,- red, no gas -,- red, no gas -,- red, no gas AG= yellow, gas -,- red, no gas -,- red, no gas -,- red, no gas AG= yellow, gas

Sucrose Durham (Acid,Gas)

-,- red, no gas +,+ AG = yellow, gas

+,+ AG = yellow, gas

+,- A=yellow; sometimes

orange, no gas

+,- yellow with orange Durham,

no gas

+,- A= yellow -,- red, no gas +,+ AG= yellow, gas

MR/VP-/- -/+(red ring) +(red) /+(red

ring)+(red)/+(red

ring)+(red) /- +(red)/- no growth

-,-+(red) /-

TSI Slant/Butt A/K yellow/orange-red

A/A yellow/yellow

A/Ayellow/yellow

Results inconsistent/

do not use this test

Results inconsistent/

do not use this test

Results inconsistent/

do not use this test

Results inconsistent/

do not use this test

Results inconsistent/

do not use this test

TSI gas (-) (-) (-)TSI H2S (-) (-) (-)

Prot

ein

Test

s

Lysine (-) (-) (-) (-) (-) (-) (-) no growth (-)SIM sulfur (-) (-) (-) (-) (-) (-) (-) (-)SIM indole (-) (-) (-) (-) (-) (-) (-) (-)SIM motility (-) (-) (-) (-) (-) (-) (-) (-)Phenylalanine wk + pale

green(-) (-) (-) (-) (-) (-) no growth (-)

Urease wk + fuchsia slant (-) (-) wk + fuchsia slant (+) fuchsia (-) (-) (-)

Antib

iotic

s

Novobiocin (NB) > 16 mm = S

sensitive (20 mm)

sensitive (25 mm)

sensitive (16 mm)

sensitive (26 mm)

resistant sensitive (23 mm)

Sensitive(18-35 mm)

sensitive (16 mm)

Bacitracin (A) >10 mm = S

resistant resistant resistant resistant resistant resistant resistant sensitive (14 mm)

Optochin (P) > 14 mm = S

resistant resistant resistant resistant resistant resistant sensitive (22 mm)

resistant

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Bio 2192 Gram(-) Organisms and Expected ResultsMedia/Test Citrobacter

freundii (Cf)BSL 2

Escherichiacoli (Ec)

BSL 1

Klebsiella pneumoniae (Kp)

BSL 2

Proteusvulgaris (pv)

BSL 2

Pseudomonas aeruginosa (Pa)

BSL 2

Serratia marcescens

(Sema) BSL 1

Shigella flexneri (Sf) BSL 2

Gram Stain (-) pink (-) pink (-) pink (-) pink (-) pink (-) pink (-) pinkCell Shape bacillus bacillus bacillus bacillus bacillus bacillus bacillus

O2

Catalase (+) bubbles (+) bubbles (+) bubbles (+) bubbles (+) bubbles (+) bubbles (+) bubblesOxidase (-) (-) (-) (-) (+) purple (-) (-)

TSA with bloodgrowth, gamma growth, gamma

or betagrowth, gamma growth, grayish

may have beta edges

growth, grayish, may be iridescent silver may be beta;

growth, alpha may change to beta over

time

growth, gamma

Carb

ohyd

rate

Tes

ts

MacConkey’s growth (+ lac, fuchsia)

growth (+ lac, fuchsia)

growth (+ lac, fuchsia)

growth, (-lac)

growth, (-lac)

growth, (-lac)

growth, (-lac)

Lactose Durham acid, gas

+,+ AG= yellow, gas

+,+ AG= yellow, gas

+,+ AG= yellow, gas -,- red, no gas -,- red, no gas -,- red, no gas -,- red, no gas

Sucrose Durham acid/gas

AG= yellow, gas -,- red, no gas AG= yellow, gas A=yellow -,- red, no gas AG= yellow, gas -,- red, no gas

MR/VP + red/- + red/- + red/- + red/- -/- + red OR -/+red ring

+ red/-

TSI Slant/Butt A/A yellow/black=

yellow)

A/A (yellow/yellow)

A/A (yellow/yellow)

A/A (yellow/black=

yellow)

K/K no growth (red/red)

A/A or K/A(yellow/yellow)

(red/yellow)

K/A(red/yellow)

TSI gas (+) (+) (+) (+) (-) (-) (-)TSI H2S (+) black (-) (-) (+) black (-) (-) (-)

Prot

ein

Test

s

Lysine (-) (+) purple (+) purple (-) (-) (-) OR (+) (-)SIM sulfur (+) black (-) (-) (+) black (-) (-) (-)SIM indole (-) (+) red ring (-) (+) red ring (-) (-) (-)SIM motility (+) black (+) cloudy (-) (+) black (-) (-) (-)Phenylalanine (-) (-) (-) (+) green (-) (-) (-)Urease (-) (-) (+) fuchsia (+) fuchsia (-) (-) (-)

Antib

iotic

s

Novobiocin (NB) > 16 mm = S

resistant resistant (11 mm)

resistant resistant resistant resistant (10 mm)

resistant

Bacitracin (A) >10 mm = S

resistant resistant resistant resistant resistant resistant resistant

Optochin (P) > 14 mm = S

resistant resistant resistant resistant resistant resistant resistant

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Post Lab Questions1. What two things will a MacConkey plate tell you about your Unknown organism?

a. b.

2. What three things did you determine about your unknowns from your day two Gram Stain / microscope work?

a. b. c.

For the following questions use the Gram (+) and Gram (-) Tables on the previous pages.

3. If you could choose one media/ test to differentiate between E. coli, Citrobacter and Klebsiella, which one would you choose?

Why would you choose this particular test?

4. Which one media/ test would you use to differentiate between Proteus, Pseudomonas and Shigella?

5. Which antibiotic disc test would you use to differentiate between Streptococcus mitis and

Streptococcus pneumonia?

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1. Use the Gram(+) and Gram(-) tables to identify the following unknown organisms.

Test Results for Organism “A” Results for Organism “B”

MacConkey Pink colonies Clear colonies

TSI Yellow tube, bubbles in media Red slant, yellow butt

MR-VP MR red MR red

SIM No red ring, no black color no fuzziness

No red ring, no black color, no fuzziness

Oxidase No color No color

Urea Pink Yellowish

Phenylalanine Yellow after reagent added Yellow after reagent added

Sucrose Yellow, 25% gas in Durham tube Red

Correctly written

scientific name: A. B.

Results for Unknown “C” Results for Unknown “D”

Cell Shape Cocci Cocci

Catalase Bubbles No bubbles

A disc Growth up to disc Growth up to disc

P disc Growth up to disc 22 mm zone of inhibition

Novobiocin Growth up to disc 30 mm zone of inhibition

Hemolysis No color change in media Media is an olive green color

Correctly written

scientific name: C. D.

The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms, our associate dean Linda Martin for many kinds of aid, and our dean John Cornish.

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