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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)

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Identification of an Unknown Microorganism with Laboratory-Based

Microbiological Testing
Andrew M. Stufflebean
Unknown Organism #12
Department of Biology, College of Charleston, Charleston, SC
November 6, 2009

Abstract
The classification and identification of an unknown microorganism was completed through a series
of microbiological test, which ranged from morphological differentiation to biochemical analysis and
comparison. Experiments consisted primarily of laboratory testing, which compared the results of known
bacterial species with those of the unknown bacteria. Experiments ranged from differential staining,
morphological classification, and biochemical intracellular/extracellular enzymatic activity. From these
results and observations, I was able to construct a dichotomous key, eliminating bacterial species whose
results contradicted those of the unknown organism. Upon completion of the two-month diagnostic
laboratory testing, I can identify the unknown bacteria as belonging to the family Enterobacteriaceae,
specifically the species Enterobacter aerogenes. The positive identification of the unknown bacteria was
attributed to the correlation amongst the known results of the E. aerogenes species and those of the unknown:
Gram-negative bacilli, lactose fermenter, oxidase negative, indole/methyl-red negative, and citrate positive.

Introduction
The main goal of the unknown microorganism identification is to utilize the various
experiments performed in the microbiology laboratory. Each week, 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 experiments range from a wide variety of diagnostic
criteria, which categorize bacteria according to species-specific characteristics and traits. The
classification of the unknown bacteria is based upon the assumption that the original culture is
pure, and cross-contamination of the culture is absent from the samples used. (L-53) Early
testing of the unknown organism centered primarily on general, mainly structurally
characteristics of the organism. Such test included: differential staining, cell shape, cell
arrangement, and cultural growth on various media. As the weeks progressed, testing shifted to
more internal physiological requirements of the bacteria. Experimentation focused on

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metabolism, specific enzymatic activity, oxygen requirements, and differentiation amongst
principal groups within a bacterial family. As the early experiments identified broad
characteristics of bacteria, the later experiments specifically allow for the differentiation amongst
species at a family and genus level. Results build on one another as the weeks progress,
providing for the elimination of irrelevant bacterial possibilities of our unknown culture. From
these results, a dichotomous key (Fig. 1) is constructed, allowing for visual taxonomic
classification amongst the unknown bacteria and the known bacterial species used throughout
experimentation. As such, the successful identification of E. 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 33.1.
Materials and Methods
Unknown Bacterial Strains. All unknown bacterial strains were selected at random by lab
students. The Microbiology lab instructor provided the unknown bacterial strains in agar deep
slants that had been previously inoculated with a specific microorganism, incubated at 37C for
48 hours, and then placed in the refrigerator for storage prior to selection by students. The
identity of the unknowns remains with the lab instructors only. After the selection of the
unknown bacteria labeled (#12), a streak plate was prepared for the culture and incubated at 37C
for 48 hours. Next, an isolated colony from the streak plate was selected and used to inoculate
both a working and reserve agar slant. The slants were then incubated at 37C for 48 hours and
then moved to the refrigerator for later use. (L-53 A) Cultural characteristics of the unknown
bacteria were evaluated on both nutrient agar plates and nutrient agar slants. The cultures were
evaluated through visual observation on a variety of characteristics. (Table 4, pg. 1)
Bacterial Test/Control Strains. The following bacterial strains were used throughout the entire
experimentation and identification period. Tests were performed on the control strains, alongside
the unknown strain. The following bacterial species were used: A.) Bacillus subtilis B.)

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

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

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

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inoculated with the test strains and incubated for 24 hours. Following bacterial growth, tubes
were examined for Butt and Slant color, as well as the presence of blackening within the
medium. Blackening within the medium indicated the production of Hydrogen sulfide, which
was also seen in experiment 26 with the SIM agar deep tubes. (1) Finally, the most important
tests for this particular bacterial strain were conducted, including the Indole, Methyl-Red, and
Citrate tests. For all three experiments, test controls (B/D/E) were used along with the unknown
strain and a control for each test. In the Indole test, SIM agar deep tubes were inoculated with the
test strains and incubated. Following incubation, 10 drops of Kovacs reagent were added to the
tubes. The color of the reagent layer was observed and recorded in determining Tryptophan
hydrolysis of the bacteria. For the Methyl-Red experiment, MR-VP broth was inoculated with
each test strain and incubated. Following incubation, five drops of a methyl red indicator were
added to the tubes. The color of the medium was observed and recorded in determining the
fermentation of Glucose. Lastly, in the Citrate utilization test, Simmons citrate agar slants were
inoculated with the test strains and incubated. After incubation, the presence or absence of
growth was noted, along with the color of the medium that indicated whether or not the bacteria
utilized citrate. All procedures and protocol for each experiment were careful followed according
to the Microbiology Lab Manual. (1)
Results
Completed experiments and results can be found on pages 1-3 of Table 4, as well as
numerical listing of experiments and their results in Tables 1, 2, and 3. Table 33.1 list the
various results for the control organisms used throughout experimentation in the laboratory.
Differential Staining. Differential staining methods were used to observe the
morphological characteristics of the unknown bacteria. The result from the gram stain was
indicative of a gram-negative microbe with rod shaped (bacilli) cells and variable arrangement,

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

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

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After weeks of diagnostic laboratory testing, thorough analysis of observations, and
comparison of my results with those of the test control species used in lab, I am able to classify
my unknown microorganism as the species E. aerogenes. (2) The results gathered from the
various experiments confirm the unknown strain of bacteria was indeed E. aerogenes. In
referencing the results for the unknown listed in Tables 1, 2, and 3 with those listed for the
control strain of E. aerogenes in Table 33.1, it can be concluded that all experiment occurs
matched those of the known bacterial strain. Early experimental results for the unknown strain
clearly identify the bacteria as being Gram-negative lactose-fermenting bacilli. (Tables 1-3)
From this point on, we can classify the unknown as an enteric bacteria belonging to the class of
microbes known as Gammaproteobacteria, specifically the family Enterobacteriaceae. (1, 2) The
results were organized into a Dichotomous key, separating the test strains used in lab from the
unknown strain. (Fig. 1) The final identification of the bacteria hinged upon the results recorded
from the three most critical tests performed. The Indole, Methyl-Red, and Citrate tests
differentiated the common similarities in results between E. coli and E. aerogenes. (Table 33.1)
Prior to the IMViC test, the identity of our unknown organism was limited to the family of
microorganisms with shared results. The Indole test results confirmed the identity of the
unknown as being enteric (Table 3). Following the Indole test, the Methyl-Red test was used to
differentiate between enteric bacteria that ferment glucose to acidic by-products. (1) Specifically,
the negative result of the unknown to this test identified the microbe as the species E. aerogenes
and not E. coli. (Table 33.1) Lastly, the identity of the unknown as E. aerogenes was reinforced
with the positive result in the citrate test, characteristic of E. aerogenes and not E. coli.
Throughout the duration of laboratory experimentation, all of the unknown results listed in
Tables 1-3, match those of the known bacteria E. aerogenes listed in Table 33.1 and Bergeys

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

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Exp. #

18

Experiment Type:

Result(s) of Unknown:
Distribution of growth throughout, with bubbles and breaks in medium.

Atmospheric O2
Requirments

(Facultative Anaerobe)
Scant growth on medium with clear colonies and no change in color of 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.

15

MacConkey Agar

(Non-inhibitory effect on Gram negative bacteria, 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.

Electrolyte Deficient
medium

(Lactose fermenters will lower pH of medium, changing from blue to yellow.

TABLE 2. Differential Staining Methods for the classification of an unknown microorganism

Exp. #

Experiment Type:

11

Gram Stain

Result(s) of Unknown:
(Gram Negative)
Cells appear red/pink due to application of Safranin
(Negative, No Spore formation)

13:A

Spore Stain
Cells appear red/pink due to application of Safranin

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TABLE 3. Biochemical Activities of an unknown microorganism
Exp. #

Experiment Type:

Result(s) of Unknown:

(Extracellular Enzymatic Activity)

Starch Hydrolysis

(-) Negative: Black medium, No Zone of Hydrolysis

Casein Hydrolysis

(-) Negative: No change in medium, No zone of Proteolysis

Gelatin Hydrolysis

(-) Negative: Gelatin remains solid, 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), variable

(Intracellular Enzymatic Activity)

Triple Sugar-Iron Agar Test

(+) Positive: Yellow Butt/Yellow slant, 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 Kovacs reagent
Indole Test
(-) Negative: Medium remains yellow after addition of Methyl-Red
Methyl-Red Test
(+) Positive: Growth present, medium appears blue
Citrate Test

26

(Intracellular Enzymatic Activity)

Hydrogen Sulfide Test

(-) Negative: Yellow/clear medium, absence of blackening

Motility (SIM agar)

(+) Positive: Culture growth beyond line of inoculation

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