22B: G - Unknown Bacterium

Learning Objectives

• Maintain a pure culture of the unknown organism provided
• Determine physical characteristics of the organism provided
• Inoculate various biochemical tests and be able to read and understand the significance of each test, whether positive or negative
• Use the information generated by testing, along with the given reference flow chart and identification texts, to deduce the Genus and Species designation of the unknown organism provided
• Hand in a report of the testing performed (Unknown identification sheet), a journal of how you arrived at the identification you indicated and a TSA plate containing the unknown organism streaked to demonstrate isolated colonies

It is virtually impossible to identify bacteria based on physical characteristics alone. This is due to the fact that there are only a few basic shapes and physical features commonly seen in the prokaryotic world. Instead, biochemical testing has been used to make bacterial identification down to the “species” level. These schemes are based on creating and matching biochemical profiles of the production of enzymes, acids and gases by isolated pure cultures of a given microorganism. Identification schemes and flow charts can be found in reference texts such as “Bergey’s Manual of Determinative Bacteriology” or “The Prokaryotes”.

Each group of students will receive a TSA slant or broth containing a pure culture of an unknown bacterium belonging to the Family Enterobacteriaceae. It is the responsibility of the group to maintain stock cultures of the organism provided. Working stock cultures will be used to inoculate the various biochemical test media over the next several weeks and should be fresh and free from contaminants. A reserve stock culture should be made and after incubation and comparison with the original slant, kept with the original slant in the refrigerator.

It is critically important that aseptic techniques are used during transfers and inoculations to prevent contamination of your cultures. If contamination is suspected, you will be able to fall back to your reserve stock. If you fail to maintain a reserve stock you will not be able to recover your organism if disaster strikes. The instructor will not provide a new culture for you to start with in the middle of the unknown exercises.

Student Responsibility

It is your responsibility to:

• keep your organisms alive and fresh to run tests
• check with us if you question purity of your organism or your test results
• appropriately select media to identifying unknowns
• ask for help
• keep your bacteria in pure culture
• check your results in a timely fashion (which may be CRITICAL for certain tests)
• ask for ID books when you cannot find them (the Bergey’s Manual chapter is in eCampus)
• put everything back where you found it
• ask for media and/or test materials
• come to lab prepared to run tests (your lab exercises, notebook, etc.)
• NOT write in the above ID books

Note

• Each student will hand in their own separate report even though you have performed the work together as a group.
• Remember it is import to keep your own journal and not to plagiarize other students in the group.

MATERIALS NEEDED

• TSA plates
• TSA slants
• TSB broths
• Clean glass slides
• Gram stain reagents
• Oxidase strips and reagent
• Various biochemical media (distributed in sets during subsequent lab periods)

THE PROCEDURES

Schematic of identification procedure

1^st session  ⇒ 2^nd session  ⇒ 3^rd session  ⇒ 4^th session  ⇒ 5^th session

Gram stain oxidase test Decarboxylase broths Casein hydrolysis optional tests

Streak plate catalase test Deaminase agar Lipid hydrolysis

TSA slant/TSB O-F glucose Gelatin hydrolysis starch hydrolysis

SIM O₂ tests

TTC motility Carbohydrates

IMViC tests nitrate test

Urea hydrolysis

1^st Session

1. Inoculate a TSA slant using an inoculum from the original culture you have been given.
2. Inoculate a TSB broth from the original culture.
3. Streak a TSA plate for isolated colonies using the 3 section method, using the original agar slant culture. You will check for purity of the culture with this plate as well as use it to characterize the colony structure.
4. Run tests listed in schematic above (see individual exercises for specifics on each test).
5. Incubate all cultures at 25º C or 37º C as directed by your instructor.
6. Gram stain your unknown organism using the TSB broth culture. Note the shape, arrangement and Gram reaction of your organism.
7. Place the original stock slant labeled with your group names and instructor in the refrigerator. Inoculate a new working culture each week to have a fresh culture to inoculate new media.

2^nd Session

1. Observe your new slants looking carefully for signs of contamination. Compare to the original slant noting the color (note pigmentation), texture, opacity and odor.
2. Describe the colony morphology displayed by isolated colonies observed on the streaked plate (refer back to the Colony Morphology experiment).
3. Observe the biochemical reactions and record the results on your unknown identification sheet. Make any additional notes in your journal.
4. Run tests listed in schematic above (see individual exercises for specifics on each test).
5. Incubate at temperature recommended by instructor.

3^rd Session

1. Observe the biochemical reactions and record the results on your unknown identification sheet. Make any additional notes in your journal.
2. Run tests listed in schematic above (see individual exercises for specifics on each test).
3. Incubate at temperature recommended by instructor.

4^th Session

1. Observe the biochemical reactions and record the results on your unknown identification sheet. Make any additional notes in your journal.
2. Run tests listed in schematic above (see individual exercises for specifics on each test).
3. Incubate at temperature recommended by instructor.

5^th Session

1. Observe the biochemical reactions and record the results on your unknown identification sheet. Make any additional notes in your journal.
2. Run tests listed in schematic above (see individual exercises for specifics on each test).
3. Incubate at temperature recommended by instructor.

6^th Session

1. Read and record final tests results according to each separate procedure in the laboratory manual. Review the resources available in the laboratory (Bergey’s Manual and other reference textbooks). If you are still unsure of the identity of your unknown, inoculate other tests as suggested in the reference texts for next class.

IT IS YOUR DECISION ABOUT WHAT OTHER TESTS SHOULD BE RUN FOR THE IDENTIFICATION OF YOUR BACTERIUM. This decision should be made with the identification tables in mind, identifying which tests might further identify your organism.

2. Streak a fresh TSA plate for isolated colonies. This plate will be handed in with the final identification report.

7^th Session

Hand in your:

1. Unknown identification sheet (per table)
2. Your journal (per table)
3. The streaked plate of the unknown (1 per group)

MEDIA LIST for unknown bacterial identifications

Ask for the medium. Most of this media is available presently. If out, fresh media can be made upon request.

Gelatin deep DNAse agar plate

MacConkey or EMB agar plate EMB agar plate

Methylene blue lipid agar plate Thioglycolate broth

MRVP broth Litmus milk

Nitrate broth Blood agar plate

Uni-OF glucose Malonate (discs)

ONPG discs Mannitol salt agar plate

pH media (3, 5, 10) Starch agar

Phenylalanine deaminase agar slant Skim milk agar plate

Decarboxylase (Moeller's) broths: SIM

arginine decarboxylase (ADC) broth Simmon citrate agar slant

lysine decarboxylase (LDC) broth TTC motility MRVP broth TSIA slant

ornithine decarboxylase (ODC) broth Urea broth

Sugars

ASK for a sugar ……

adonitol maltose xylose

arabinose mannitol cellibiose

dextrose/glucose mannose malonate

dulcitol melibiose lactose

esculin raffinose inulin

fructose/levulose rhamnose trehalose

galactose salicin inositol

sorbitol sucrose

BACTERIAL UNKNOWN SHEET

Note

• Tests that are italicized and in bold MUST be run, and reactions reported. All other tests performed must be reported as + or if run.
• Tests NOT RUN do not have to be reported.

GRAM REACTION ___________________ DNAse __________

Casein hydrolysis __________

MICROSCOPIC MORPHOLOGY AND Starch hydrolysis __________

ARRANGEMENT ___________________ Urea hydrolysis __________

Gelatin hydrolysis __________

Lipid hydrolysis __________

DESCRIPTION OF COLONY: ONPG __________

Color (clear, white, pigmented (if so, what color?) TSIA __________

Nitrate reduction __________

Distinctive colony characteristics: Phenol red sugars:

colony shape/ type of margin __________ Glucose (no gas)_________

elevation of colony __________ Glucose (with gas)________

other? __________ Lactose __________

Sucrose __________

Mannitol __________

Other sugars? __________

GROWTH CHARACTERISTICS: Circle one for each

O₂ needs: aerobe or facultative anaerobe? __________

Optimal growth T: 25^o C 30^o C 37º C __________

O-F glucose: oxidative, fermentative, or nonfermenter? __________

Other tests run (names + reactions)? ____________________

BIOCHEMICAL TESTS (+ or -?):

Oxidase __________

Catalase __________

Indole __________

Methyl red __________

Voges-Proskauer __________

Citrate __________

Motility __________

H2S __________

Phenylalanine deaminase __________

Decarboxylase tests:

Arginine __________

Ornithine __________

Lysine __________

THE BACTERIUM WAS IDENTIFIED AS : _________________ __________________

(genus) (species)

Contributors and Attributions

• Jackie Reynolds, Professor of Biology (Richland College)