19. Identification of Unknown Bacterial Cultures Introduction Special Safety Precautions: For this lab, precautions must be strictly enforced because we are dealing with unknown bacteria. All bacterial cultures are potential pathogens. Discard all biohazard material appropriately: Slides go in the metal tray. All spills must be reported immediately. Scrub your hands after the lab. Identification of unknown bacteria will allow the student to review major microbiological techniques learned during the course. Each student group will identify two assigned unknown bacteria: The methodology for bacterial identification will follow the approach used by medical microbiology laboratories. Use the identification table and the flow charts appended to the lab instructions to decide the next tests.
Describe colony and cell morphology for each organism
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.
Biochemical diagnostic tests
In a medical microbiology laboratory, the Gram stain and cellular morphology determine the choice of selective and differential media as well as the enzymatic assays that will be adopted. This approach saves both time and resources. In the case of the identification of an infectious agent, time is the essence. Appropriate and successful treatment depends on the correct identification and the patient’s life is at stake. Relationship to Class Instruction: BIOL&260
Lab report due on: _______________________________________
Goal of experiment: Identify unknown bacteria. Review microbiology core technical competencies.
Material and Methods Additional material available for download on ANGEL
Identification tables for Gram positive and Gram negative bacteria
Micrographs of bacterial morphology
Diagram of cell arrangement
Flow Chart of Tests for Gram positive and Gram negative bacteria.
Cultures are grown on BHI medium or TSA. Not all cultures will be available every quarter.
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Gram +
Gram -
Bacillus cereus
Citrobacter freundii
Bacillus subtilis
Escherichia coli lac+
Enterococcus faecalis
Klebsiella pneumoniae
Lactobacillus sp.
Neisseria sicca
Micrococcus luteus
Pantoea agglomerans
Staphylococcus aureus
Proteus mirabilis
Staphylococcus epidermidis.
Proteus vulgaris
Streptococcus salivarius
Pseudomonas aeruginosa Salmonella typhimurium
Culture media Mannitol Salt Agar (MSA)
Mac Conkey Agar (MAC) plates
Motility Butts
Eosin Methylene Blue Agar (EMB)plates
Brain Heart Infusion (BHI) Agar Plates
Motility Indole Ornithine (MIO) tubes
Triple Sugar Iron Slants
Urea broth
Polysaccharide medium
6.5% NaCl medium
Simmons citrate medium
Blood Agar
Procedure Gram Stain
Lab 5
Endospore Stain, capsule stain
Lab 17: Special Stains
Differential Media and Biochemical Tests for Gram negative Rods
Lab 8 and 9
Enzymatic Assays
Lab 10
Miscellaneous Inoculating loops
Gram stain reagents
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Clothespins (optional)
3% KOH
Slides
3% Hydrogen Peroxide
Hot plates
Malachite Green
Autoclaved toothpicks
Nigrosin
Cytochrome Oxidase strips
Methylene Bue
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. The flow chart determines the biochemical test and media you will use for your identification. 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.
Session 1: Colony Morphology, Gram Stain and First Enzyme Assays Record results on page 19-10 Record drawings and results of this section on the worksheet provided: page with original sketches and instructor’s initials must be stapled to lab report for credit.
1. Observe the colonies on the agar plate.
6. Rinse with water as described in step 3
Describe shape, color and aspect (mucoid, dry, etc…). Enter your notes in the space provided at the end of the instructions.
above.
7. Add a few drops of Gram’s decolorizer. Let it trickles down the slide. Rinse off with water after about 5 seconds or when the decolorizer is no longer colored as it flows over the slide. Decolorizing for too long will allow decolorization of gram-positive as well as gram-negative organisms and will defeat the purpose of the stain.
2. Prepare one bacterial smear for each sample. Label each slide carefully. Place slides on paper towel, smear side up.
3. Add 2-3 drops of crystal violet stain directly on the smear. Stain for 1 minute.
4. Rinse the slide by washing the stain off
8. Counterstain with Gram’s Safranin solution
with a gentle stream of water from a wash bottle (at this stage all bacteria will be stained purple by the crystal violet). Drain off the rinse water.
(about 1 drop) for 30 seconds.
9. Wash with distilled water. 10. Air-dry the slide, or blot (not rub) carefully
5. Add 2-3 drops of Gram's iodine solution.
with the corner of a paper towel or a Kimwipe. Do not rub off the material when you blot. The slide should be completely
Let the slide stand for 1 minute.
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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 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.
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
Fast Enzymatic Assays All results from this section will be presented in the form of a table. Decide which tests you need to run in order to identify your unknown cultures based on the results of this lab.
Catalase (Gram +) Catalase assay is used for both Gram + and Gram – bacteria; however, all Gram – bacteria in the list are catalase positive. the release of oxygen as a result of hydrogen peroxide breakdown. This appears as foaming.
1. Add a few drops of 3% hydrogen peroxide to a slide.
2. Pick some culture with sterile toothpick and
3. Record your results. mix with hydrogen peroxide and look for Write up a chart of experiments to be performed and be prepared to justify your choice.
Oxidase Reaction (Gram -) Determine the presence of cytochrome oxidase enzymes.
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1. Obtain a Pathotec reagent strip.
4. If the organism is oxidase positive, a purple color will develop within 30 seconds.
2. Lay the strip on a paper towel.
5. After one minute, the test is concluded and
3. Withdraw a sample of culture from the
any color development is ignored.
plate with an inoculating loop and rub on the strip. The strips are wide enough to allow for more than one test.
Session 2: Inoculate media Shared Assays for Gram + and Gram - Bacteria Motility 2. If the needle is not pulled straight out, it
1. A needle is simply jabbed straight into the
may make a second track with growth extending from the first track to the second.
agar to the 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.
3. Incubate until next session at room temperature.
Assays for Gram Positive Bacteria Only MSA for Catalase Positive 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 and Micrococcus luteus grow on MSA,but do not ferment mannitol. S aureus does ferment mannitol. appropriate organism on the corresponding half of the plate. It is OK to pick up from a streak if there are no single colonies.
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
2. Incubate at 35째C overnight. 19-5
instructions for lab 10.
3. Next session observe both growth and color of colonies. Interpret results according to
Endospore Stain for Catalase Positive 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.
5. Wash off safranin with a gentle stream of water.
2. Flood the paper with the malachite green
6. , Airdry slide or blot carefully and observe
stain and steam for 5 min. Add stain if it begins to dry. (Review procedure lab 8).
under oil immersion.
3. Discard paper towel in the regular paper
7. Draw your observations.
waste and gently wash the slide with water.
4. Flood the slide with the counter-stain safranin to stain the vegetative cells for 30 seconds.
Salt Tolerance Test Streptococci and Enterococci can be differentiated according to their tolerance to salt and fermentation of mannitol. If the results from the Gram stain and the catalase test indicate that the unknown is a Gram positive coccus which does not exhibit catalase activity, Streak a mannitol, 6.5% NaCl slant. Incubate for 24-48 hours at 35째C. If the unknown bacteria are Enterococci, the slant will turn yellow. Streptococci do not grow in the presence of 6.5% NaCl and do not ferment mannitol.
Assays for Gram Negative Bacteria (Oxidase Positive) Polysaccharide Medium (Cocci only) 1. Streak a polysaccharide medium plate and
3. Neisseria sicca produces starch. The
incubate for 48 hours at 35째C.
medium will display a dark blue color. Moraxella catarrhalis does not give a colored reaction. Agar shows a yellow color which is the color of the iodine solution.
2. Add a drop of Lugol iodine (from Gram stain kit) and record observation.
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Assays for Gram Negative Bacteria (Oxidase Negative) Motility Indole Ornithine 1. Inoculate with your unknown culture only
6. Glucose fermentation produces acid. The
if you identified it as a Gram (-) rod.
pH indicator changes from purple to yellow. If there is no ornithine decarboxylation, the medium will appear yellow.
2. A needle is s inserted straight into the agar to ¼ ″ of the 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. Non-motile bacteria are stuck along the original track.
7. Decarboxylation of ornithine causes an increase in pH. The deep appears a turbid purple to a faded yellow-gray color.
8. Add four (4) to five (5) drops of Kovacs’ reagent to the surface of the medium and shake gently. If there is no indole present, the Kovacs reagent stays bright yellow. If indole is present (positive reaction), the medium turns pink.
3. If the needle is not pulled straight out, it may make a second track with growth extending from the first track to the second.
4. Incubate overnight at 35°C. 5. Record the color of the deep before adding Kovacs reagent.
Urease Reaction 9. Inoculate tube of urea broth with one
10. Incubate overnight at 35°C.
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.
11. Observe color of medium. Bright pink indicates the presence of urease. If the medium appears slightly peach, incubate further to confirm urease presence.
Selective Media: MAC 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 and lactose fermentation are two separate observations. MAC and XLD are all streaked according to the following procedure. the corresponding section of the plate. It is OK to pick up from a streak if there are no single colonies.
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.
3. Incubate 48 hours at 35°C.
2. Working aseptically, pick one colony of the
4. Next session observe both growth and color
plate with an inoculating loop and streak a single line with the appropriate organism on
of colonies.
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Triple Sugar Iron (TSI) and Simmons Citrate Triple Sugar Iron measures the ability of an organism to ferment glucose, lactose, and sucrose and produce hydrogen sulfide. It used to differentiation Salmonella from other enteric bacteria. Simmons citrate will differentiate enteric bacteria according to their ability to ferment citrate.
Procedure 1. Use a needle to inoculate TSI.
4. Repeat for each type of culture.
2. Transfer aseptically culture on the needle.
5. Incubate 48 hours at 35째C.
3. Stab the butt. Withdraw the needle and
6. Read and interpret the results according to
swipe the slant in a zigzag motion. The cap must be loose to maintain aerobic conditions on the slant.
lab 9 instructions.
Simmons Citrate 1. Streak a Simmons citrate slant with the
3. Blue slants are positive and green slants are
culture to be identified.
negative for citrate utilization.
2. Incubate for 48 hours at 35째C for 24-48 hours.
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Lab Report (Grade doubled) The Identification of the Unknown Bacteria report is the last report for the quarter. Write it carefully and label appropriately all sections, figures, and tables. Proof read the text for correct content, spelling and grammar. Use appropriate APA style citations. The Lab Report Check List Introduction
Goal of lab: methods used to identify bacteria and practical applications
Experimental procedure
Include number of assigned unknown culture Mention briefly the materials and methods actually used. There is no need to repeat the detailed procedures, only note what was done differently.
Results
Colony morphology on agar Shape, cell arrangement and Gram stain Include worksheet on Gram stain with instructorâ&#x20AC;&#x2122;s initials See page 10. Biochemical tests and special stains as applicable Table summarize results
Discussion
Provide explanation for choice tests performed. Record observations in table format as much as possible. Flow chart of identification, either hand drawn or computer assisted. Do not use the flow chart provided on line.
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. Provide additional information on the biology of your unknown culture. Is it medically relevant? Is it normal human flora? Is it mainly an environmental microbe? Does it colonize mostly water or soil?
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Staple this page to your final report Description of Colonies on Agar Plate: use the descriptive terminology provided in 7 .Microorganisms in the environment II. Assigned Unknown #
Assigned Unknown #
Microscopic observations (Draw as carefully as possible a few cells; cell shape and arrangements must be clear). Include total magnification and label all visible structures Assigned Unknown #
Bacterial Sample
Assigned Unknown #
Gram Stain: Color of Smear
Degree of Stringiness
Assigned unknown #
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Initials