Stufflebean 0

Identification of an Unknown Microorganism with
Laboratory-Based Microbiological Testing
Andrew M. Stufflebean
November 6, 2009
Dr. Ben Neely – BIOL 310 L91
Department of Biology, College of Charleston, Charleston, SC

Unknown Culture: (#12)

I can identify the unknown bacteria as belonging to the family Enterobacteriaceae. which categorize bacteria according to species-specific characteristics and traits. specifically the species Enterobacter aerogenes. morphological classification. and cultural growth on various media. aerogenes species and those of the unknown: Gram-negative bacilli. Such test included: differential staining. and cross-contamination of the culture is absent from the samples used. which ranged from morphological differentiation to biochemical analysis and comparison. Upon completion of the two-month diagnostic laboratory testing. The positive identification of the unknown bacteria was attributed to the correlation amongst the known results of the E. testing shifted to more internal physiological requirements of the bacteria. lactose fermenter. cell shape. The experiments range from a wide variety of diagnostic criteria. From these results and observations. Charleston. As the weeks progressed. Experimentation focused on . I was able to construct a dichotomous key. Introduction The main goal of the unknown microorganism identification is to utilize the various experiments performed in the microbiology laboratory. oxidase negative. cell arrangement. Experiments consisted primarily of laboratory testing. Experiments ranged from differential staining. and biochemical intracellular/extracellular enzymatic activity. mainly structurally characteristics of the organism. 2009 Abstract The classification and identification of an unknown microorganism was completed through a series of microbiological test. College of Charleston. eliminating bacterial species whose results contradicted those of the unknown organism.Stufflebean 1 Identification of an Unknown Microorganism with Laboratory-Based Microbiological Testing Andrew M. SC November 6. and citrate positive. indole/methyl-red negative. a battery of testing is completed with emphasis on interpreting the results of the “known” bacteria used and comparing the results with those of the unknown. The classification of the unknown bacteria is based upon the assumption that the original culture is pure. which compared the results of known bacterial species with those of the unknown bacteria. (L-53) Early testing of the unknown organism centered primarily on general. Each week. Stufflebean Unknown Organism #12 Department of Biology.

an isolated colony from the streak plate was selected and used to inoculate both a working and reserve agar slant. As such. oxygen requirements. the successful identification of E. allowing for visual taxonomic classification amongst the unknown bacteria and the “known” bacterial species used throughout experimentation. The following bacterial strains were used throughout the entire experimentation and identification period. The slants were then incubated at 37ºC for 48 hours and then moved to the refrigerator for later use. the later experiments specifically allow for the differentiation amongst species at a family and genus level.1. and then placed in the refrigerator for storage prior to selection by students. Next. incubated at 37ºC for 48 hours. The cultures were evaluated through visual observation on a variety of characteristics. aerogenes as the unknown bacteria is conclusive with careful cross-referencing and comparison of my results from the experiments. with those of the species listed in Table 33. pg. 1) Bacterial Test/Control Strains. Tests were performed on the control strains.) . Materials and Methods Unknown Bacterial Strains. All unknown bacterial strains were selected at random by lab students. a streak plate was prepared for the culture and incubated at 37ºC for 48 hours.Stufflebean 2 metabolism.) Bacillus subtilis B. 1) is constructed. providing for the elimination of irrelevant bacterial possibilities of our unknown culture. specific enzymatic activity. (Table 4. a dichotomous key (Fig. alongside the unknown strain. The following bacterial species were used: A. From these results. After the selection of the unknown bacteria labeled (#12). and differentiation amongst principal groups within a bacterial family. The identity of the unknowns remains with the lab instructors only. (L-53 A) Cultural characteristics of the unknown bacteria were evaluated on both nutrient agar plates and nutrient agar slants. Results build on one another as the week’s progress. As the early experiments identified broad characteristics of bacteria. The Microbiology lab instructor provided the unknown bacterial strains in agar deep slants that had been previously inoculated with a specific microorganism.

) Proteus vulgaris F.) Escherichia coli E. Three types of media were utilized in determining the nutritional and physical requirements of different bacteria in accordance with standard protocol and procedures for each specific test. The stain was viewed under a microscope at 100x magnification with oil-immersion. and Counterstain. The differential staining test results were recorded in Table 2. test controls (A/D/G) were used alongside the unknown strain. Two differential staining tests were performed according to standard procedures and protocol. and Counterstain. Proper staining procedures were followed with the appropriate application of the Primary Stain. test controls (A/B/D/F) were used alongside the unknown strain. The stains were viewed under a microscope at 100X magnification with oil-immersion. Unknown Results were recorded in Table 1. results were gathered based on distribution of growth in comparison to relative position of bacteria in the agar tubes. One test was used to determine the oxygen requirements for the unknown organism. Mordant. test control (A) was used with the unknown strain that was used. (1) For the Gram Stain. dispersing the bacteria. Cultures were then incubated at 37ºC for 48 hours. For the Spore Stain. The agar tubes were placed between the palms of the hands and rotated. followed by inoculation with test controls and the unknown strain.) Staphylococcus aureus H. Sterile brain-heart infusion agar was liquefied at 100ºC then cooled to 45ºc.) Pseudomonas aeruginosa G.Stufflebean 3 Enterobacter aerogenes C. The test was performed according to standard procedures and protocol.) Streptococcus pyogenes. Two plates of the media were used . For the MacConkey agar. test controls (C/D/G) were used along with the unknown strain. as well as the control. (1) For the Oxygen requirement. Microorganism Cultivation on Various Media. (1) For the Phenylethyl alcohol agar. (1) Differential Staining. After incubation. then placed in an iced water bath. Atmospheric O2 Requirements. test controls (A/D/F) were used along with the unknown strain. Decolorizing agent. Decolorizing agent. Proper gram staining procedures were followed with the appropriate application of the Primary stain.) Enterococcus faecalis D.

The final extracellular enzyme activity test was the Gelatin Hydrolysis. Results for the milk agar plate were determined based on appearance of the medium surrounding growth. Following their incubation. in determining both extracellular and intracellular enzymatic activity. the tubes were put in the refrigerator for 30 minutes to allow for solidification. All tests were performed in accordance with standard protocol and procedures for experimentation. Tubes that remained liquid were . Test control results for the various biochemical tests can be found in Table 33. Plates were incubated at 37ºC for 24 to 48 hours. For the Starch hydrolysis. (1) For the extracellular enzymatic activity experiments. For the CLED agar. the plate was flooded with Gram’s iodine for 30 seconds. The results for the unknown were recorded in Table 1. with the unknown results recorded in Table 3. All media were divided into 4 equal sections and inoculated with the test strains in a straight line across the media. test controls (A/D/F) were all used alongside the unknown strain in the three separate experiments.Stufflebean 4 with test controls (A/D) and the unknown in both plates. Results were recorded based on amount of growth. and the results for the unknown were recorded in Table 3. milk agar plates were used. The tubes were incubated for 48 hours at 37ºC. Gelatin deep tubes were inoculated with the test controls and the unknown strain. Following incubation of the starch agar. Various tests were utilized in the experimentation of both test controls and the unknown strain. and appearance of the medium surrounding the growth. Both plates were divided into four equal sections and inoculated with the three test controls and the unknown. Biochemical Activities of microorganisms. The second plate used test controls (A/D/E) and the unknown strain. The tubes were observed based on the state of the medium. appearance of growth. Appearance of the medium around the bacterial growth was observed.1. test controls (B/F/G) were used alongside the unknown strain in one plate. starch agar plates were used. Inoculated plates were incubated at 37ºC for 48-72 hours in the inverted position. For the Casein Hydrolysis.

For the oxidase experiment. Phenol red lactose.Stufflebean 5 incubated for an additional seven days. identifying oxidase activity of the bacteria. Triple sugar-iron agar slants were . and sucrose broths with Durham tubes were used. For the carbohydrate fermentation. a small amount of zinc was added to the tubes. which was divided into four sections and incubated. as well as standard incubation at 37ºC for 24-48 hours. Color of the colonies after addition of the reagent were observed and recorded. The medium used was the Trypticase soy agar plate. test controls (B/D/E/F) were used along with the unknown bacterial strain and a control tube. the unknown bacterial strain was tested for the presence or absence of bubbles following addition of hydrogen peroxide to a sample of the culture. For the TSI agar. Following incubation. Results for the unknown were recorded in Table 3. test controls (C/D/F) were used alongside the unknown strain and an un-inoculated control. about two to three drops of the reagent (p-aminodimethylaniline) were added to surface of the plate with a sterile swab. the 15 tubes were observed on the basis of the color of the medium and gas production within the Durham tube. with results for the unknown bacterial strain listed accordingly. If red coloration did not appear. Following incubation. Red coloration after Solution A and B were added and the color change after the addition zinc to the non-red tubes in beginning were recorded in determining whether or not Nitrate was reduced. Following incubation. dextrose. For the Nitrate reduction. For the intracellular enzymatic activity experiments. test controls (A/D/F) were used along with the unknown bacterial strain. Five tubes per broth media were inoculated with test strains. For the Catalase test. a variety of tests were performed in accordance of standard protocol and procedures. test controls (B/D/E/F) were used alongside the unknown strain. five drops of both (sulfanilic acid) and (-naphthylamine) were added to the broth cultures. Trypticase nitrate broth was inoculated with the strains and incubated. (1) The complete list of performed test is listed in Table 3.

Methyl-Red. The color of the medium was observed and recorded in determining the fermentation of Glucose. five drops of a methyl red indicator were added to the tubes. . the most important tests for this particular bacterial strain were conducted. Differential staining methods were used to observe the morphological characteristics of the unknown bacteria. in the Citrate utilization test. The color of the reagent layer was observed and recorded in determining Tryptophan hydrolysis of the bacteria. Following incubation. (1) Finally. 2. including the Indole. and Citrate tests. Table 33. Following bacterial growth. and 3.1 list the various results for the control organisms used throughout experimentation in the laboratory. as well as numerical listing of experiments and their results in Table’s 1. For all three experiments. (1) Results Completed experiments and results can be found on pages 1-3 of Table 4. Simmons citrate agar slants were inoculated with the test strains and incubated. MR-VP broth was inoculated with each test strain and incubated. along with the color of the medium that indicated whether or not the bacteria utilized citrate. which was also seen in experiment 26 with the SIM agar deep tubes. Differential Staining. as well as the presence of blackening within the medium. The result from the gram stain was indicative of a gram-negative microbe with rod shaped (bacilli) cells and variable arrangement. For the Methyl-Red experiment. In the Indole test. After incubation. Blackening within the medium indicated the production of Hydrogen sulfide. Lastly. the presence or absence of growth was noted.Stufflebean 6 inoculated with the test strains and incubated for 24 hours. tubes were examined for Butt and Slant color. test controls (B/D/E) were used along with the unknown strain and a control for each test. SIM agar deep tubes were inoculated with the test strains and incubated. 10 drops of Kovac’s reagent were added to the tubes. Following incubation. All procedures and protocol for each experiment were careful followed according to the Microbiology Lab Manual.

and was the step in narrowing down the identity of the unknown.Stufflebean 7 as seen under microscopic examination following the stain. The appearance of the yellow medium in the lactose. Casein. The results from these are explained in the table and serve only to classify similarities between the unknown . (Table 2) Microorganism Cultivation on Various Media. (Table 3) Biochemical Activities of microorganisms. The unknown was also a non-spore forming bacteria which further narrowed down the potential identity of the organism. and Gelatin Hydrolysis test were recorded in Table 3. and the yellow appearance of the medium are attributed as positive test for lactose fermenters. further separating the gram-negative bacilli into two groups as seen in Figure 1. dextrose. (Table 1) Atmospheric O2 Requirements/Cellular Respiration. as seen in the Carbohydrate fermentation. (Table 1) The unknown’s ability to use alternative compounds as electron acceptors in cellular respiration was also be seen in the Nitrate test. (Table 3) This characteristic also affects the fermentation pathways. The results from the gram stain were reinforced following the completion of both the Phenylethyl alcohol agar test and the MacConkey agar test. Completion of the CLED agar test confirmed the positive lactose fermentation of the unknown. Also. and sucrose broth cultures were indicative of a positive reaction of acidic by-products that changed the methyl-red indicator within the tubes. with the reduction of Nitrate to Nitrite. The gram stain divides bacteria in two specific groups of microorganisms. Extracellular enzymatic activity seen in the Starch. the color of the colonies. the production of a bubble in the Durham tube indicated positive fermentation and production of a CO2 by-product. The scattered growth throughout the brain-heart infusion agar deep tube was indicative of a Facultative Anaerobe. (Table 1) Both media are inhibitory to either gram-positive or gramnegative bacteria. indicated by the absence of growth on the inhibitory PEA agar and the presence of growth on the MacConkey agar. The abundant growth on the CLED agar.

the IMViC test. Fig. The three test performed in this experiment are characteristic of the bacterial family. 1) Regarding the Methyl-red test. the negative result was attributed to the lack of red coloration after the addition of the reagent. which is absent in members of the Enterobacteriaceae family.Stufflebean 8 microorganisms and the control strains. As such. 2) The unknown bacteria also showed a positive result for motility in the SIM agar. (Table 3) Discussion . the indole negative organisms due not hydrolyze tryptophan. (1) The negative result from the Oxidase test indicated the absence of the production of cytochrome oxidase. were indicative of negative results for the production of Hydrogen Sulfide in both experiments. which allows for taxonomic differentiation of the unknown organism with members of the same family. indicating the use of Citrate as an alternative source of Carbon to be utilized in the production of energy. Hydrogen sulfide test. (1. Lastly. Enterobacteriaceae. 26) and the TSI agar (Exp. (Table 3) Further fermentation results are examined in the Triple Sugar-Iron agar test. (1) The positive reaction is observed with abundant growth on the slant and a blue coloration of the medium. the yellow color of the broth after the addition of the reagents indicates a negative test for the fermentation of glucose with production oh acidic by-products. Table 1) The citrate test resulted in a positive reaction for my unknown strain. the most important results were seen in Experiment 25. 2) In the Indole test. (Table 3) Such a result classifies the unknown as fermenting either Lactose and/or Sucrose. (1. The Catalase test showed a positive result for the production of Catalase after the addition of Hydrogen peroxide to a sample of the unknown culture. 24). (Table 3) The presence of bubbling indicated the facultative anaerobe has utilized Catalase to break apart the Hydrogen peroxide into Oxygen and Water. (1. (Table 3) The absence of blackening in both the SIM deep agar (Exp. indication an acidic slant and an acidic butt. (1.

and Citrate tests differentiated the common similarities in results between E. Throughout the duration of laboratory experimentation. 1) The final identification of the bacteria hinged upon the results recorded from the three most critical tests performed. (Fig. all of the unknown results listed in Tables 1-3. separating the test strains used in lab from the unknown strain. The Indole. aerogenes and not E. I am able to classify my unknown microorganism as the species E. Methyl-Red. 2) The results were organized into a Dichotomous key. coli. (2) The results gathered from the various experiments confirm the unknown strain of bacteria was indeed E. (1) Specifically. (Table 33. coli. and comparison of my results with those of the test control species used in lab. match those of the known bacteria E. the negative result of the unknown to this test identified the microbe as the species E.1 and Bergey’s . coli and E. aerogenes in Table 33. aerogenes and not E.1) Prior to the IMViC test. (Table 33. aerogenes. In referencing the results for the unknown listed in Tables 1. Following the Indole test.1) Lastly. aerogenes listed in Table 33. the Methyl-Red test was used to differentiate between enteric bacteria that ferment glucose to acidic by-products. 2. characteristic of E. The Indole test results confirmed the identity of the unknown as being enteric (Table 3).1. Early experimental results for the unknown strain clearly identify the bacteria as being Gram-negative lactose-fermenting bacilli. aerogenes. (Tables 1-3) From this point on. the identity of our unknown organism was limited to the family of microorganisms with shared results. (1. we can classify the unknown as an enteric bacteria belonging to the class of microbes known as Gammaproteobacteria. aerogenes was reinforced with the positive result in the citrate test. the identity of the unknown as E. aerogenes. thorough analysis of observations. specifically the family Enterobacteriaceae.Stufflebean 9 After weeks of diagnostic laboratory testing. and 3 with those listed for the control strain of E. it can be concluded that all experiment occurs matched those of the known bacterial strain.

Inc. By completing experimentation from a broad to a more specific standpoint. Pearson Education. Lastly. 2008. Acknowledgements I would like to acknowledge the assistance and guidance of both Tracy Hirsch and Dr. G. Bergey’s Manual of Determinative Bacteriology. I want to thank both Dr. Microbiology. J. Nutritional/Physical Requirements for the cultivation of an unknown microorganism . The identification of an unknown serves as a capstone research project for Microbiology students. fostering the information and concepts being taught throughout the semester.. Cappuccino. California 2.. utilized in our everyday life. Literature Cited 1. 9th ed. 8th ed. Holt. for her assistance in running the various experiments and interpreting the results of each test appropriately. Maryland TABLE 1. I would like to thank my lab partner Ashley Clune. Ben Neely. Lippincott Williams & Wilkins. Also. we are able to build the skills essential for deductive reasoning. 1994. J. Morrison and the lab manager for providing all of the necessary supplies and equipment to run the test. (2) The work of identifying an unknown bacterial culture is crucial to building a foundation of skills that reinforced the information covered in lecture.Stufflebean 10 Manual of Determinative Bacteriology. a Laboratory Manual.

Electrolyte Deficient medium (Lactose fermenters will lower pH of medium. # Experiment Type: 11 Gram Stain Result(s) of Unknown: (Gram Negative) Cells appear red/pink due to application of Safranin (Negative. TABLE 2. with red color attributed to pH indicator in medium with presence of Coliform bacilli) *Red indicated acidic by-products resulting from lactose fermentation of medium. CLED: Cysteine Lactose 15 Abundant growth of yellow colonies on medium. 15 Phenylethyl alcohol agar (Medium inhibitory to growth of Gram negative bacteria) Moderate to abundant growth with red colonies forming and red coloration of medium. Atmospheric O2 Requirments (Facultative Anaerobe) Scant growth on medium with clear colonies and no change in color of medium. 15 MacConkey Agar (Non-inhibitory effect on Gram negative bacteria. changing from blue to yellow. with bubbles and breaks in medium.Stufflebean 11 Exp. No Spore formation) 13:A Spore Stain Cells appear red/pink due to application of Safranin . # 18 Experiment Type: Result(s) of Unknown: Distribution of growth throughout. Differential Staining Methods for the classification of an unknown microorganism Exp.

No Zone of Hydrolysis Casein Hydrolysis (-) Negative: No change in medium. # Experiment Type: Result(s) of Unknown: (Extracellular Enzymatic Activity) Starch Hydrolysis (-) Negative: Black medium. medium appears blue Citrate Test 26 (Intracellular Enzymatic Activity) Hydrogen Sulfide Test (-) Negative: Yellow/clear medium. No production of Gelatinase 22 (Intracellular Enzymatic Activity) 30 Catalase Test (+) Positive: Presence of bubbling after addition of Hydrogen Peroxide to unknown (Intracellular Enzymatic Activity) 29 Nitrate Reduction (+) Positive: Unknown remains red after addition of Solution A and B 31 (Intracellular Enzymatic Activity) (-) Negative: No color change of colonies Oxidase Test 23 (Intracellular Enzymatic Activity) Carbohydrate Fermentation 24 25 Lactose Yellow medium with bubble (A/G) Dextrose Yellow medium with bubble (A/G) Sucrose Yellow medium with small bubble (A/G). No zone of Proteolysis Gelatin Hydrolysis (-) Negative: Gelatin remains solid. indicates Fermentation of Lactose and/or Sucrose H2S Production (-) Negative: Absence of blackening within medium (Intracellular Enzymatic Activity) (-) Negative: No color formation after addition of Kovac’s reagent Indole Test (-) Negative: Medium remains yellow after addition of Methyl-Red Methyl-Red Test (+) Positive: Growth present. Biochemical Activities of an unknown microorganism Exp. absence of blackening Motility (SIM agar) (+) Positive: Culture growth beyond line of inoculation .Stufflebean 12 TABLE 3. variable (Intracellular Enzymatic Activity) Triple Sugar-Iron Agar Test (+) Positive: Yellow Butt/Yellow slant.

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