Essam Elsayed
Yusuf Figueroa
The identification of unknown bacterial species
BIO 265 W2L5
Prof. Bonaventura
Lab Report
Abstract
This study focused on identifying an unknown bacterial isolate using Gram staining, oxidase
testing, and the API 20E biochemical test strip. After conducting these tests, the results were
compared to the bioMérieux reference database, leading to the identification of the organism as
Klebsiella pneumoniae. This bacterium is commonly found in environmental sources but is also
known to act as an opportunistic pathogen under certain conditions. These findings underscore
the importance of the API 20E system in clinical microbiology for the rapid and accurate
identification of bacterial species.
Introduction
The gastrointestinal tract hosts a diverse community of bacteria that play crucial roles in digestion,
nutrient absorption, and maintaining overall health. However, some bacteria in this region can
cause infections or diseases. Notable examples include Escherichia coli and Salmonella, which
are well-known pathogens associated with gastrointestinal illnesses (Willey, Sherwood, &
Woolverton, 2017). Additionally, certain species, such as Klebsiella pneumoniae, are typically
environmental but can act as opportunistic pathogens under specific conditions, particularly in
immunocompromised individuals (Forbes, Sahm, & Weissfeld, 2014).
This experiment aimed to identify an unknown bacterial isolate using a combination of Gram
staining, oxidase testing, and the API 20E biochemical test strip. The API 20E strip is a widely
utilized tool in microbiology laboratories, facilitating the identification of enteric bacteria by
conducting a series of standardized biochemical tests (bioMérieux, n.d.). Its ability to streamline
bacterial identification makes it an invaluable resource in both clinical and research settings.
Materials and Methods
The identification of an unknown bacterial species begins with the application of Gram staining to
determine whether the bacteria are Gram-positive or Gram-negative. This procedure also provides
information about key characteristics, including morphology, shape, and motility by flagella.
The catalase test is a biochemical assay used to detect the presence of the enzyme catalase in
bacteria. Catalase breaks down hydrogen peroxide (H₂O₂) into water (H₂O) and oxygen (O₂), a
reaction that protects cells from oxidative damage caused by hydrogen peroxide, a byproduct of
metabolic processes (Cappuccino & Sherman, 2014).
The oxidase test is a biochemical assay used to determine the presence of cytochrome c oxidase;
an enzyme involved in the electron transport chain of aerobic respiration. This test identifies
bacteria capable of using oxygen as the terminal electron acceptor in respiration (Cappuccino &
Sherman, 2014).
The oxygen requirements of the bacteria are then assessed to determine whether it grows
exclusively in the presence of oxygen, only in its absence, or under both conditions.
All these tests are conducted to narrow down the
potential family of the unknown species.
To identify a bacterial species within the Enterobacteriaceae family, the API 20E strip test can be
utilized. This test consists of 20 individual biochemical tests designed to analyze various metabolic
activities and enzymatic properties of the bacteria. The results from these tests are then interpreted
collectively to determine the specific species (Becton, Dickinson &Company, 2014).
1. The ONPG test is a biochemical method used to detect the presence of the enzyme βgalactosidase in bacteria. This enzyme breaks down lactose into glucose and galactose.
Unlike lactose fermentation tests, which require the presence of both permease and βgalactosidase, the ONPG test specifically identifies bacteria that produce β-galactosidase,
even if they lack lactose permease (Willey, Sherwood, & Woolverton, 2017). The test is
considered positive when the color changes from colorless to yellow, indicating the
presence of β-galactosidase. If the solution remains colorless, the result is negative.
2. The ADH test is a biochemical test used to measure the decarboxylation activity of the
arginine decarboxylase enzyme on the amino acid arginine. The test determines the
ability of bacteria to decarboxylate arginine, producing ornithine and increasing the pH,
which is reflected by a color change in the medium (Baird-Parker, 1984). The test is
considered positive when the color changes from yellow to purple or orange-red, while a
negative result is indicated by the medium remaining yellow.
3. The LDC test is a biochemical test used to measure the activity of lysine decarboxylase,
an enzyme that decarboxylates the amino acid lysine to produce cadaverine and carbon
dioxide. This reaction increases the pH of the medium, which is typically indicated as
positive by a color change from yellow to purple. A negative result is reflected by the
medium remaining yellow, indicating the absence of alkaline conditions (Cappuccino &
Sherman, 2014).
4. The ODC Test is a biochemical test that measures the activity of ornithine decarboxylase,
an enzyme that decarboxylates the amino acid ornithine to produce putrescine and carbon
dioxide. This reaction leads to an increase in pH, making the medium more alkaline. A
positive result is indicated by a color change from yellow (acidic) to purple (alkaline),
while a negative result is reflected by the medium remaining yellow, indicating no
decarboxylation and no increase in PH (Cappuccino & Sherman, 2014).
5. The CIT test is used to determine the ability of a microorganism to use citrate as its sole
carbon source. Organisms that can utilize citrate as a carbon source produce the enzymes
citrase or citrate-permease to transport citrate into the cell. These organisms also convert
ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, creating an
alkaline environment in the medium (Cappuccino & Sherman, 2014). The citrate media
is green before inoculation and turns blue if the culture is positive for citrate utilization
(Tankeshwar, 2016).
6. The H2S production test detects the ability of bacteria to reduce sulfur-containing
compounds like thiosulfate to hydrogen sulfide (H2S) during metabolism. When H2S is
produced, it reacts with ferrous ammonium sulfate, resulting in a black precipitate, which
is a positive test for H2S production. If no black precipitate forms, the result is negative
(Cappuccino & Sherman, 2014).
7. The URE test is used to detect the ability of bacteria to produce the enzyme urease, which
hydrolyzes urea to ammonia and carbon dioxide. The ammonia produced raises the pH,
causing a color change in the medium, from yellow or orange to pink, indicating a positive
result (Cappuccino & Sherman, 2014).
8. The TDA test detects the presence of the enzyme tryptophan deaminase, which
deaminates tryptophan to produce indole and pyruvic acid. The addition of a reagent,
such as ferric chloride, results in a brown or red color if the enzyme is present, indicating
a positive result (Cappuccino & Sherman, 2014).
9. The IND test detects the ability of bacteria to produce indole from tryptophan via the
enzyme tryptophanase. After incubation, the addition of an indole reagent, such as
Kovac's reagent, will result in a red or pink color in the medium, indicating a positive
result (Cappuccino & Sherman, 2014).
10. The VP (Voges-Proskauer) test detects the production of Acetoin through the addition of
reagents A and B. A change in color from yellow to red indicates a positive result,
suggesting that the organism ferments glucose via the butanediol pathway (Cappuccino &
Sherman, 2014).
11. The GEL test detects the production of gelatinase, an enzyme that hydrolyzes gelatin into
amino acids and peptides. A positive result is indicated by a liquefied medium after
incubation and cooling, while a negative result is reflected by the solidification of the
medium (Cappuccino & Sherman, 2014).
12. Glu, Man, Ino, SOR, RHA, SAC, MEL, AMY, and ARA — These tests assess the ability
of bacteria to ferment various sugars and compounds, including glucose, mannitol,
inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, and arabinose. Positive
results typically result in color changes in the medium, indicating acid production
(Cappuccino & Sherman, 2014).
Glu (Glucose Test): This test determines the ability of the microorganism to ferment glucose.
Man (Mannitol Test): This test detects the ability of the organism to ferment mannitol, a sugar
alcohol. Ino (Inositol Test): This test assesses whether the organism can ferment inositol, a sugar
alcohol. SOR (Sorbitol Test): This test measures the organism's ability to ferment sorbitol, a
sugar alcohol. RHA (Rhamnose Test): This test checks for the fermentation of rhamnose, a sugar
found in many plant-derived substances. SAC (Sucrose Test): This test determines if the
microorganism can ferment sucrose, a disaccharide. MEL (Melibiose Test): This test detects the
fermentation of melibiose, a disaccharide. AMY (Amygdalin Test): This test evaluates the ability
of the microorganism to hydrolyze amygdalin, a compound found in seeds of certain fruits. ARA
(Arabinose Test): This test checks the ability of the microorganism to ferment arabinose, a fivecarbon sugar.
The API 20E strip consists of 20 small, dehydrated test tubes and cupules, each containing specific
reagents to test the biochemical properties of bacteria (Cappuccino & Sherman, 2014). To
perform the test, a bacterial suspension is first prepared and adjusted to match the McFarland
turbidity standard, ensuring the appropriate bacterial concentration. The suspension is then
inoculated into all the tubes of the API strip.
For tests that require an anaerobic environment, mineral oil is added to the cupules to create a
sealed, oxygen-free environment (Cappuccino & Sherman, 2014). The strip is incubated at 37°C
for 18 to 24 hours. After incubation, the tubes are observed for color changes to determine positive
or negative results for most of the tests.
However, certain tests require additional processing:

TDA test: Add 1 drop of 10% ferric chloride to the TDA tube and observe for a color
change within 2 minutes.

IND test: Add 1 drop of Kovac’s reagent to the IND tube and observe for a color change
within 2 minutes.

VP test: Add 1 drop of solution A (alpha-naphthol) and 1 drop of solution B (potassium
hydroxide) to the VP tube and observe for a color change within 10 minutes (Cappuccino
& Sherman, 2014).
After obtaining the results from the API 20E strip, each positive test result is assigned a numerical
value, while negative results are assigned a value of zero. The tests on the strip are grouped into
sets of three, with the values of the positive tests within each group summed to generate a singledigit code. These digits are then combined to form a 7-digit numerical profile, which represents
the metabolic and biochemical characteristics of the microorganism (Cappuccino & Sherman,
2014).
This 7-digit code is entered into the bioMérieux identification database, a comprehensive system
designed to identify microorganisms. The database matches the code with profiles of known
bacteria to provide a probable identification. This system is part of bioMérieux’s broader suite of
microbiological tools, widely used in clinical, food, pharmaceutical, and environmental
laboratories for accurate and reliable bacterial identification (Cappuccino & Sherman, 2014).
Results/Discussion
After performing a Gram stain and examining the sample under the microscope, the unknown
bacterial species were identified as Gram-negative bacilli, non-spore-forming, and non-motile. The
catalase test yielded a positive result, indicating the presence of the enzyme catalase, which breaks
down hydrogen peroxide (H₂O₂) into water (H₂O) and oxygen (O₂). In contrast, the oxidase test
produced a negative result, signifying the absence of cytochrome c oxidase in the bacterial cell
membrane.
After incubation in a tubular medium, bacterial growth was observed both at the top of the tube,
where oxygen is present, and at the bottom where oxygen is absent. This pattern indicates that the
bacterial species is facultatively anaerobic, capable of surviving in both aerobic and anaerobic
environments.
All these results suggest that the bacterial species belongs to the Enterobacteriaceae family. To
identify the specific species within this family, the API 20E strip test was performed. After
incubation at 37ºC for 24 hours, the results were observed as follows, with the corresponding
numerical values assigned to positive results:
1. ONPG: Positive (1)
8. TDA: Negative.
15. SOR: Positive. (4)
2. ADH: Negative.
9. IND: Negative.
16. RHA: Positive. (1)
10. VP: Positive (1)
17.SAC: Positive. (2)
4. ODC: Negative.
11. GEL: Negative.
18. MEL: Positive. (4)
5. CIT:
12. GLU: Positive. (4)
19.AMY: Positive (1)
13. MAN: Positive. (1)
20.ARA: Positive (2)
14. INO: Positive (2)
21. OX: Negative
3.
LDC: Positive. (4)
Positive. (2)
6. H2S: Negative.
7.
URE:
Positive. (1)
By adding the numerical values of the positive results in each group of three tests, a 7-digit
profile code was generated. This code, 5215773, was then entered into the bioMérieux
identification database, which matches biochemical profiles to specific bacterial species.
According to the bioMérieux database, the bacterial species was identified as Klebsiella
pneumoniae with an identification accuracy of 97.7%.
Conclusion
The API 20E strip is an invaluable tool for bacterial identification in clinical microbiology
laboratories. It enables rapid and efficient identification by combining multiple biochemical tests
into a single, streamlined system. In this study, the unknown organism was identified as Klebsiella
pneumoniae, showcasing the strip's versatility in detecting both opportunistic microbiota and
pathogenic bacterial species. Further tests, such as antimicrobial susceptibility testing, are critical
in clinical applications to guide effective treatment, as different Klebsiella strains may exhibit
varying resistance profiles.
References
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