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17. Identification of Unknown Bacteria Identify unknown bacteria. Review microbiology core technical competencies.

Introduction Identification of unknown bacteria will allow the student to review major microbiological techniques learned during the course. Each student will attempt to identify two unknown bacteria: Number 1 is an unknown isolated from the environment Number 2 is assigned by the instructor and identified by a number The first step in any identification exercise is to describe colony and cell morphology for each organism. Next, a Gram stain test is performed to place the species in a broad taxonomic group. Gram stain results are confirmed with a KOH test. The observations from the morphology and Gram stain determine the next steps; therefore it is worth your time and effort to repeat a Gram stain in order to get a clear answer. A series of biochemical diagnostic tests is performed to identify further the organisms. Use the identification table appended to the lab instructions to decide what the next tests should be. Relationship to Class Instruction: BIOL&260 Lab report due on: _______________________________________

Material and Methods Cultures on BHI medium or TSA Gram + Bacillus cereus

Gram Serratia marcescens

Bacillus subtilis

Proteus vulgaris

Lactobacillus acidophilus

Enterobacter aerogenes

Staphylococcus epidermidis.

Escherichia coli lac+

Staphylococcus aureus

Pseudomonas aeruginosa

Streptococcus salivarius

Neisseria sicca

Micrococcus luteus

Klebsiella pneumoniae Culture media

Mannitol Salt Agar (MSA) Motility Butts Brain Heart Infusion (BHI) Agar Plates Blood Agar

Mac Conkey Agar (MAC) plates Eosin Methylene Blue Agar (EMB)plates Motility Indole Ornithine (MIO) tubes Urea broth 17-1




Gram Stain Isolation of Cultures Endospore Stain Differential Media and Enzymatic Assays Enzymatic Assays

Lab Instructions

Lab 5 Lab 3 Lab 8: Special Stains Lab 9 and 10 Lab 10

Miscellaneous Inoculating loops

Gram stain reagents

Clothespins (optional)

3% KOH


3% Hydrogen Peroxide

Hot plates

Malachite Green

Autoclaved toothpicks

Procedure Maintain a log of experiments and prepare an identification flow chart. The flow chart provided in the appendix includes all the unknown organisms supplied by the instructor. Students will prepare a flow chart for their own unknown cultures. DON’T INCLUDE TESTS THAT ARE NOT RELEVANT TO YOUR EXPERIMENTS. A. Morphology and Gram Stain 1. Observe the colonies on the agar plate. will be stained purple by the crystal Describe shape, color and aspect violet). Drain off the rinse water. (mucoid, dry, etc…). Enter your notes 5. Add 2-3 drops of Gram's iodine in the space provided at the end of the solution. Let the slide stand for 1 instructions. minute. 2. Prepare one bacterial smear for each 6. Rinse with water as described in step 3 sample. Label each slide carefully. above. Place slides on paper towel, smear side 7. Add a few drops of Gram’s decolorizer. up. Let it trickles down the slide. Rinse off 3. Add 2-3 drops of crystal violet stain with water after about 5 seconds or directly on the smear. Stain for 1 when the decolorizer is no longer minute. colored as it flows over the slide. 4. Rinse the slide by washing the stain off Decolorizing for too long will allow with a gentle stream of water from a decolorization of gram-positive as well wash bottle (at this stage all bacteria as gram-negative organisms and will defeat the purpose of the stain. 17-2

8. Counterstain with Gram’s Safranin solution (about 1 drop) for 30 seconds.

clusters, or regular packets of four or eight). Compare the Gram negative organism(s) with the Gram positive and note the difference in color. If not clearly evident, check your procedure with the instructor.

9. Wash with distilled water. 10. Air-dry the slide, or blot (not rub) carefully with the corner of a paper towel or a Kimwipe. Do not rub off the material when you blot. The slide should be completely dry before adding oil for microscopic examination.

NOTE: A thick stain takes longer to decolorize than a thin one, so the exact time cannot be specified. It is usually not more than 20 seconds. Too little or too much decolorization can affect your results. At this stage the Gram positive organisms will remain purple. If the smear is thick, they may appear so visually, but a thin smear from a broth culture may not be visible at all. Gram negative bacteria will be colorless at this stage.

11. Examine the stained slides with low power objectives and finally use the oil immersion lens. For this you will need maximum light by opening the diaphragm and adjusting the light source intensity. 12. Examine the bacteria and observe the size, shape (rod, spherical or curved), Gram staining (positive: purple; negative: pink), and arrangement (singly, in pairs, in chains, irregular

13. Sketch a few of the organisms. Do not try to sketch the entire field. Show the instructor the slide for initials on records.

KOH Test determine the stringing effect. If there is stringing (increased viscosity) within 15 seconds, the KOH test is positive, while the bacteria are considered to be Gram negative. Do your results from this test agree with those from the Gram stain?

1. Place one drop of 3% potassium hydroxide (KOH) solution on a clean slide. Transfer a good amount of bacteria from the culture medium with a sterile toothpick to the drop. 2. Mix the bacteria into the solution rapidly and in a circular motion.

4. Record your observations.

3. After 5-8 seconds, raise and lower the toothpick just off the slide surface to Special Safety Precautions Discard all biohazard material appropriately: Slides go in the metal tray.


Staple this page to your final report Description of Colonies on Agar Plate: Your unknown

Assigned Unknown #

Description of Cells Morphology microscope observation: Your unknown

Bacterial Sample

Assigned Unknown #

Gram Reaction Color

Your unknown Assigned unknown #


Degree of Stringiness


B. Selective Media and Enzymatic Assays Decide which tests you need to run in order to identify your unknown cultures based on the results of this lab. Write up a chart of experiments to be performed and be prepared to justify your choice. Running an unnecessary test wastes time and resources. For instance, don’t streak Gram (-) cultures on mannitol salt agar plates or a Gram (+) coccus on a Mac Conkey plate! We will share the media plates. Divide the plates into 2 sections and streak only one side with your culture. Let another student use the other half for his or her culture. Assays for Gram + and Gram - Bacteria Motility 2. 1. A needle is simply jabbed straight into the agar to the bottom and pulled straight back out. There is sufficient liquid so that motile bacteria usually maintain their flagella and can swim 3. through the medium, spreading away from the site of the inoculum. Nonmotile bacteria are stuck along the original track.

If the needle is not pulled straight out, it may make a second track with growth extending from the first track to the second. Incubate until next session at room temperature.

Assays for Gram + Bacteria MSA (Cocci) Mannitol Salt Agar (MSA) contains 7.5% NaCl, inhibitory to the growth of most bacteria other than staphylococci (Why?). It also contains mannitol as the carbohydrate source and a pH indicator, phenol red, for detecting acid produced by mannitol fermenting staphylococci. Phenol red is pink at pH>7 and yellow at pH<7. S. epidermidis grows on MSA but does not ferment mannitol, S aureus does ferment mannitol. 1. Using a sharpie, draw a line on the bottom of a MSA agar plate so as to divide the plate in sections. Working aseptically, pick one colony of the plate with an inoculating loop and make a single streak line with the appropriate organism on the

corresponding half of the plate. It is OK to pick up from a streak if there are no single colonies. 2. Incubate at 37°C overnight. 3. Next session observe both growth and color of colonies.


Catalase Catalase assay is used for both Gram + and Gram â&#x20AC;&#x201C; bacteria; however, all the Gram â&#x20AC;&#x201C; bacteria in the list are catalase positive. 1. Add a few drops of 3% hydrogen look for the release of oxygen as a peroxide to a slide. result of hydrogen peroxide breakdown. This appears as foaming. 2. Pick some culture with sterile toothpick and mix with hydrogen peroxide and

3. Record your results.

Endospore Stain (Bacilli) Bacteria from the genus Bacillus form endospores which are stained with malachite green. Gram positive rods should be tested for endospore formation. Use your original slant which is more likely to have developed spores. If you suspect you have a Bacillus culture, perform an endospore test. 1. Prepare smear from the organism tested.

4. Flood the slide with the counter-stain safranin to stain the vegetative cells for 30 seconds..

2. Flood the paper with the malachite green stain and steam for 5 min. Add stain if it begins to dry. (Review procedure lab 8).

5. Wash off safranin with a gentle stream of water. 6. , Airdry slide or blot carefully and observe under oil immersion.

3. Discard paper towel in the regular paper waste and gently wash the slide with water.

7. Draw your observations.

Assays for Gram - Bacteria Oxidase Reaction Determine the presence of cytochrome oxidase enzymes. 1. Obtain a Pathotec reagent strip. 2. Lay the strip on a paper towel.

4. If the organism is oxidase positive, a purple color will develop within 30 seconds.

3. Withdraw a sample of culture from the plate with an inoculating loop and rub on the strip. The strips are wide enough to allow for more than one test.

5. After one minute, the test is concluded and any color development is ignored.


Motility Indole Ornithine 1. A needle is simply jabbed straight into the agar to ¼ ″ bottom and pulled straight back out. There is sufficient liquid so that motile bacteria usually maintain their flagella and can swim through the medium, spreading away from the site of the inoculum. Nonmotile bacteria are stuck along the original track.

Ornithine decarboxylation is promoted by anaerobic conditions. 6. Decarboxylation of ornithine is indicated by the development of a turbid purple to a faded yellow-gray color. 7. Bright yellow color indicates both lack of ornithine decarboxylation and glucose fermentation. These observations must be recorded before Kovac’s reagent is added.

2. If the needle is not pulled straight out, it may make a second track with growth extending from the first track to the second.

8. Indole production is indicated by the formation of a pink to red color after the addition of three or four drops of Kovacs’ reagent to the surface of the medium and gentle shaking. If there is no indole present, the Kovac’s reagent stays bright yellow.

3. Inoculate with your unknown bacteria only if you identified them as Gram (-) rods. 4. Incubate overnight at 37°C. 5. The instructor will overlay all cultures with a few drops of sterile mineral oil.

Urease Reaction 1. Inoculate tube of urea broth with one colony of unknown bacteria, or, if there are no single colonies, with a small aliquot of bacterial culture if you identified them as Gram (-) rods.

2. Incubate overnight at 37°C. 3. Observe color of medium.

Selective Media: MAC and EMB Mac Conkey Agar (MAC) is used for the growth and differentiation of enteric bacteria (Gram (-), cytochrome oxidase negative). The differential property of MAC results from the inclusion of lactose and a pH indicator, methyl red, in the medium. Bacteria that can grow on MAC and ferment lactose produce acid, which changes the bacterial colonies and sometimes the surrounding agar medium to a pink color. Bacteria that can grow on MAC but do not ferment lactose produce tan colonies. Remember that growth on MAC and lactose fermentation are two separate observations. EMB plates can be used instead of MAC plates.


1. Using a sharpie, draw a line on the bottom of a MAC agar plate so as to divide the plate in 2 sections. Label sections with ID of bacteria you will streak.

corresponding section of the plate. It is OK to pick up from a streak if there are no single colonies. 3. Incubate 48 hours at 37째C. 4. Next session observe both growth and color of colonies.

2. Working aseptically, pick one colony of the plate with an inoculating loop and streak a single line with the appropriate organism on the

Lab Report Introduction: Goal of lab. Methods used to identify bacteria and practical applications Material and Method: Mention briefly the materials and methods actually used. There is no need to repeat the detailed procedures, only mentioned what was done differently. Explain the reasons you chose the tests you performed. Results: Record observations in table format as much as possible. Discussion and Conclusion: Which tests worked and which did not work. According to your results, what are your unknown bacteria and how did you come to that conclusion? This is an example of a table to log in your results. As long as you use a table, the format is up to you. Media

Organism # 376 Gram - Rod Observation Results

Motility MAC MIO

Diffuse red color Tan colonies Gray purple, Kovacs yellow



+ for motility Lactose negative Ornithine decarboxylase positive Indole negative Results

Cytochrome Oxidase Urease Other

No change in color


No change in color


Growth at Room Temperature

Red colonies

The organism is Serratia marcescens.


Identification of Unknown Bacteria