Table of Contents
1.0 INTRODUCTION & OBJECTIVES ........................................................................ 2
1.1 Introduction & Background of the Experiment .................................... 2
1.2 Objectives ................................................................................................ 3
2.0 PROCEDURE ........................................................................................................................ 3
3.0 RESULTS & DISCUSSION ........................................................................................... 5
3.1 Results...................................................................................................... 5
3.2 Discussion ................................................................................................ 7
4.0 CONCLUSION ..................................................................................................................... 9
5.0 REFERENCES .................................................................................................................... 11
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1.0 INTRODUCTION & OBJECTIVES
1.1 Introduction & Background of the Experiment
Bacterial identification is a fundamental component of microbiological analysis and
plays a crucial role in diverse fields such as medical diagnostics, food safety, environmental
microbiology, and biotechnology. Accurate identification of bacteria is essential for
understanding microbial diversity, determining pathogenicity, and developing appropriate
treatment strategies. While microscopic techniques such as Gram staining can offer basic
insights into bacterial morphology and structural features (e.g., capsules, endospores), they are
insufficient for identifying bacteria at the genus or species level (Tortora et al., 2020).
To achieve precise bacterial identification, biochemical testing serves as a vital
methodology. These tests leverage the principle that each bacterial species possesses a unique
DNA sequence, which in turn encodes specific enzymes responsible for catalysing distinct
metabolic reactions (Madigan et al., 2021). By inoculating bacteria into differential media and
assessing the presence of metabolic end products, microbiologists can create a biochemical
"fingerprint" that aids in the identification of unknown bacterial isolates.
In this experiment, Staphylococcus and Escherichia coli were selected as representative
Gram-positive and Gram-negative bacteria, respectively. Several biochemical tests were
employed, including observations of colony morphology on nutrient agar, growth patterns on
MacConkey agar (a selective and differential medium), and a series of differential tests such as
starch hydrolysis, methyl red, Voges-Proskauer, and citrate utilization. Each of these tests
targets a specific metabolic trait: for example, the starch hydrolysis test indicates the production
of amylase, while the methyl red and Voges-Proskauer tests assess the fermentation end-
products of glucose metabolism. The results of these tests collectively contribute to the
identification process by comparing observed biochemical traits against known profiles of
reference organisms. The practical experience gained through such exercises not only
strengthens laboratory skills but also reinforces theoretical knowledge about microbial
metabolism, enzymatic function, and diagnostic microbiology.
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1.2 Objectives
• To observe the colony morphology of Staphylococcus and E. coli on nutrient agar.
• To test bacterial growth on selective and differential MacConkey agar.
• To detect amylase activity using the starch hydrolysis test.
• To determine glucose metabolism via Methyl Red and Voges-Proskauer tests.
• To assess citrate utilization as a carbon source.
• To identify bacteria based on biochemical test results.
2.0 PROCEDURE
Nutrient agar
Staphylococcus and The samples were
E. coli were streaked incubated at 37oC
on Nutrient agar for 24 hours
The growth were
observed and
documented
McConkey agar
Staphylococcus and The samples were
E. coli were streaked incubated at 37oC
on McConkey agar for 24 hours
The growth were
observed and
documented
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Starch Hydrolysis Test
Staphylococcus and The samples were
E. coli were streaked incubated at 37oC
on Starch agar for 24 hours
The presence of
Iodine was poured
bacteria were
over the surface of
observed and
starch agar
documented
Methyl Red Test
The colonies were
The samples were
transferred into the
incubated at 37oC
glucose phosphate
for 24 hours
broth
The test tubes were 2 drops of methyl
shaken, and colour red solution were
changes were dropped into the
observed culture
Voges Proskauer Test
The colonies were The samples were
transferred into the incubated at 37oC for 24
glucose phosphate broth hours
2 drops of creatine
The test tubes were solution, and 1 ml of 4%
shaken, and colour potassium hydroxide
changes were observed solution were dropped
into the culture
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Citrate Utilisation Test
The colonies were
The samples were
streaked onto the
incubated at 37oC
slant of Simmon-
for 24 hours
citrate medium
Colour changes, and
bacterial growth
were observed
3.0 RESULTS & DISCUSSION
3.1 Results
The experiment involved a series of tests to examine the morphological and
biochemical characteristics of Staphylococcus and Escherichia coli (E. coli). Observations
were made based on their growth patterns on various media and their reactions in differential
biochemical tests. The outcomes include colony morphology on nutrient and selective agar,
starch hydrolysis activity, and responses to the Simmons-Citrate test. Some results aligned with
theoretical expectations, while others showed deviations that suggest possible contamination,
technique errors, or medium-related limitations. The following table summarizes the results
obtained:
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Staphylococcus
Nutrient agar MacConkey agar Starch hydrolysis Simmon-citrate agar
Growth (+)
or (-) (+) (+) (+) (-)
Distribution
of growth
Table 1: Distribution of Growth of Staphylococcus on Different Agar Medium
Figure 1: Methyl Red test Figure 2: Voges Proskauer test
E. coli
Nutrient agar MacConkey agar Starch hydrolysis Simmon-citrate agar
Growth (+)
or (-) (+) (-) (+) (-)
Distribution
of growth
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Table 2: Distribution of Growth of E. coli on Different Agar Medium
3.2 Discussion
The experiment was done to examine bacterial morphology, Gram reaction, and
presence of such structures as capsules and endospores. The sample tested were Staphylococcus
and E. coli. These samples’ properties are tested on nutrient agar, MacConkey agar, starch agar,
Methyl Red test, Voges Proskauer test, and Simmon-citrate agar. The identification of these
samples is done based on their formation shapes, their response towards different Gram stain
agar (positive or negative).
For the first experiment on nutrient agar, the morphology of both Staphylococcus and
E. coli are observed. From the results documented in Table 1, the structure of Staphylococcus
is shown, it can be observed that Staphylococcus colonies forms in an irregular shape. Same
goes to the formation of E. coli on nutrient agar, they form colonies that shapes in an irregular
form. The formation of colonies of Staphylococcus on nutrient agar in the experiment aligns
well with the theory that Staphylococcus on nutrient agar form colonies that made up a round
to irregular in shape, and they also tend to form swarming growth throughout the plate
(Sapkota, 2022). On the other hand, the observation of shape of E. coli of the experimental
result, is slightly different from the theory. Theoretically, the colonies of E. coli form as a
somewhat circular shape on a nutrient agar plate (Basavaraju & Gunashree, 2022), meanwhile
the experimental results indicate a formation of an irregular shape. This is believed due to the
lack of neatness of streaking of sample on the nutrient agar plate, which possibly could have
resulted in ununiform formation of colonies where the areas are mixed up with one another.
Moving on to the McConkey agar, this part focuses on determining whether a bacterium
is Gram-positive or Gram-negative. McConkey agar is a selective medium that inhibits the
growth of Gram-positive bacteria (Carolina Knowledge Center, 2024). Hence, a theory can be
made prior to the experiment that E. coli colonies should be observable on the McConkey agar
plate as E. coli is a Gram-negative bacterium, meanwhile colonies of Staphylococcus should
not be able to form on the plate as it is a Gram-positive bacterium. Despite the theories made,
the experimental results obtained goes completely against the theories made, this is because
Staphylococcus showed positive growth on the agar, meanwhile absolutely no colonies of E.
coli can be observed on the plate. From these unexpected findings, several assumptions can be
made from the results. Starting from the Staphylococcus, the presence of colonies on the
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McConkey agar is possibly just a contamination of external bacteria, which means the observed
colonies are not actually the Staphylococcus colonies, instead just another Gram-positive
bacterium from external contamination. This is probably due to unproper aseptic technique or
sanitising of workplace which may have caused the agar to be contaminated. On the other hand,
the absence of E. coli colonies is possibly due to the culture possibly contaminated with another
organism that is inhibiting E. coli growth or outcompeting it for nutrients, which have caused
the cells to be stressed and died. This assumption is backed by a study that McConkey agar
have shown to poorly perform in supporting growth of E. coli colonies (Mccarthy, Holbrook,
& Stephens, 1998).
In the next part, starch hydrolysis test, the samples are tested on their capabilities to
break down starch into glucose. From this experiment, the samples are streaked in respective
starch agar plates, and incubated, after that iodine is added onto the plate, clear halos
surrounding bacterial growth indicates that the bacterial species was able to hydrolyse the
starch resulting in the clear zone without starch, and if the growth does not exhibit clear zones
surrounding it, the starch is intact throughout the plate which means the bacteria was unable to
hydrolyse the starch (Hartline, 2023). Based on the observations of the bacteria activity on the
starch, the clear zones can be seen surrounding the Staphylococcus colonies, meanwhile the E.
coli sample is completely hidden by the black iodine solution. This indicates that
Staphylococcus is a bacterium that has the ability to hydrolyse starch into glucose. Contrary to
this, E. coli is proven to be starch hydrolysis negative as no clear zone can be observed from
its plate. Despite the prosper growth of E. coli on the agar, the absence of clear zones indicate
that it does not produce amylase, which is an enzyme that breaks down starch into glucose.
In the Methyl Red (MR) test, the goal is to determine whether a bacterium performs
mixed acid fermentation of glucose, resulting in stable acidic end products. After incubation,
the methyl red indicator is added; a red colour indicates a positive result, showing that the
bacteria produce and maintain a low pH environment. Theoretically, E. coli is expected to give
a positive MR result due to its ability to carry out mixed acid fermentation, which leads to the
production of sufficient acid to lower the pH and cause a red colour change (Tankeshwar, n.d.).
In contrast, Staphylococcus is generally expected to yield a negative result, as it does not
usually perform this type of fermentation and therefore does not produce enough acid to trigger
a colour change with the methyl red indicator.
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As for the Voges Proskauer (VP) test, it is designed to detect acetoin, a neutral
fermentation product formed via the butylene glycol pathway (Dahal, 2025). In this test, the
addition of Barritt’s reagents will result in a red colour if acetoin is present, indicating a positive
result. Theoretically, Staphylococcus is expected to be VP-positive, as it is known to produce
acetoin during glucose fermentation. On the other hand, E. coli is generally VP-negative, as it
does not utilize the butylene glycol fermentation pathway and therefore does not produce
acetoin.
Finally, the Simmons-Citrate test, the objective is to determine whether bacteria are
capable of using citrate as their sole carbon source. A positive result is indicated by a change
in colour of the medium from green to blue due to the alkaline by-products produced during
citrate metabolism (Aryal, 2022). Theoretically, Staphylococcus is expected to be citrate-
positive, capable of utilizing citrate and causing the colour change. Meanwhile, E. coli is
usually considered citrate-negative, as it typically lacks the ability to transport and metabolize
citrate under the test conditions. However, the experimental results obtained for this test
showed that neither Staphylococcus nor E. coli demonstrated any colour change in the
Simmons-Citrate medium. The agar remained green for both samples, indicating no detectable
citrate utilization by either bacterium, which contradicts the theoretical expectations,
particularly for Staphylococcus. Several possible explanations can be made for this unexpected
outcome. First, it is possible that the inoculation was too light, which may have resulted in
insufficient bacterial growth to observe a colour change, even if citrate metabolism did occur.
Also, (Aryal, 2022) also mentioned that some organisms are capable of growth but they do not
cause colour changes, perhaps that is the case for Staphylococcus here.
In conclusion, while some experimental outcomes supported the expected biochemical
characteristics of Staphylococcus and E. coli, several deviations highlighted the importance of
strict technique, accurate sample handling, and environmental control in microbiological
testing. These discrepancies emphasise that while theory provides a guideline, practical
application may yield variations that require interpretation and further verification.
4.0 CONCLUSION
The identification and differentiation of bacterial species through morphological
observation and biochemical testing remain vital tools in microbiology. This experiment
focused on two commonly studied bacteria Staphylococcus and Escherichia coli using a variety
of diagnostic tests including nutrient agar morphology observation, growth on MacConkey
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agar, starch hydrolysis, Methyl Red (MR), Voges-Proskauer (VP), and Simmons-Citrate
utilization. The findings largely aligned with established microbiological principles but also
revealed notable discrepancies that underscore the complexities and potential challenges in
experimental microbiology.
The morphological analysis on nutrient agar confirmed that both Staphylococcus and
E. coli formed irregular colonies, though this was inconsistent with the typical circular
appearance reported for E. coli (Basavaraju & Gunashree, 2022). Such deviations are likely
due to technical limitations during streaking. The growth on MacConkey agar presented an
unexpected result, where Staphylococcus a Gram-positive organism demonstrated growth,
while E. coli, a known Gram-negative bacterium, did not. This anomaly suggests possible
contamination or stress-induced inhibition of E. coli growth, highlighting the importance of
maintaining strict aseptic techniques and optimal culture conditions (Carolina Knowledge
Center, 2024).
Biochemical tests provided further insights. The starch hydrolysis test accurately
indicated Staphylococcus as amylase-positive and E. coli as amylase-negative, aligning with
known enzymatic profiles (Hartline, 2023). Similarly, the MR and VP tests reflected expected
results: E. coli tested positive in the MR test, confirming its mixed acid fermentation pathway,
while Staphylococcus tested positive in the VP test due to its production of acetoin via the
butylene glycol pathway (Tankeshwar, n.d.). The Simmons-Citrate test, however, yielded
unexpected results for both organisms, showing no colour change. This may be due to
insufficient inoculation or the limitations of the medium, which may not support growth or
colour shift for certain strains even if citrate is utilized (Aryal, 2022).
In summary, the experiment reinforced theoretical concepts while also highlighting
discrepancies that occur in practice. Factors such as contamination, improper technique, and
suboptimal growth conditions can significantly affect experimental outcomes. Therefore,
consistent methodology, proper environmental control, and critical evaluation of anomalies are
essential in microbial identification. The integration of multiple tests rather than reliance on a
single diagnostic criterion ensures a more accurate and robust identification process, critical in
clinical and environmental microbiology.
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5.0 REFERENCES
Aryal, S. (10 August, 2022). MicrobiologyInfo.com. Retrieved from Simmons Citrate Agar-
Composition, Principle, Uses, Preparation and Result Interpretation:
https://microbiologyinfo.com/simmons-citrate-agar-composition-principle-uses-
preparation-and-result-interpretation/
Basavaraju, M., & Gunashree, B. (11 November, 2022). IntechOpen. Retrieved from
Escherichia coli: An Overview of Main Characteristics:
https://www.intechopen.com/chapters/84764
Carolina Knowledge Center. (16 April, 2024). Retrieved from Bacterial Growth on MacConkey
Agar: https://knowledge.carolina.com/professional-growth/activities/biology-
activities/bacterial-growth-on-macconkey-agar/
Dahal, P. (8 April, 2025). Microbe Notes. Retrieved from Voges-Proskauer (VP) Test: Principle,
Procedure & Results: https://microbenotes.com/voges-proskauer-vp-test/
Hartline, R. (18 February, 2023). LibreTexts Biology. Retrieved from 1.17: Starch Hydrolysis:
https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Man
ual_(Hartline)/01%3A_Labs/1.17%3A_Starch_Hydrolysis
Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2021). Brock
biology of microorganisms (16th ed.). Pearson.
Mccarthy, J., Holbrook, R., & Stephens, P. J. (1 September, 1998). ScienceDirect. Retrieved
from An Improved Direct Plate Method for the Enumeration of Stressed Escherichia
coli O157:H7 from Food:
https://www.sciencedirect.com/science/article/pii/S0362028X22016726
Tankeshwar, A. (n.d.). Microbe Online. Retrieved from Methyl Red (MR) Test: Principle,
Procedure, and Results: https://microbeonline.com/methyl-red-mr-test-principle-
procedure-results/
Tortora, G. J., Funke, B. R., & Case, C. L. (2020). Microbiology: An introduction (13th ed.).
Pearson.
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