Microbiology Unknown Lab Report

Unknown D+ and Q-

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 Unknown D+

Introduction

Identifying unknown microorganisms is a critical skill in microbiology that

combines observation, biochemical testing, and deductive reasoning. Correct

identification influences medical treatment, food safety, and public health decisions. The

goal of this experiment was to correctly identify the assigned unknown organism " D+"

using a systematic approach, including differential staining and biochemical testing.

Initial Gram staining categorized the organism, and subsequent biochemical tests

confirmed the organism’s identity. Through careful observation, analysis, and

comparison to known characteristics, the identity of the unknown bacterium was

determined with confidence.

Materials and Methods

Materials:

- Unknown culture labeled " D+"

- Nutrient Agar (NA) plates

- Gram staining reagents

- Catalase test materials (3% hydrogen peroxide)

- Mannitol Salt Agar (MSA)

- Blood Agar plates

- Coagulase test kit

- Microscope

- Incubator at 37°C
 Procedure:

1. Gram Stain: A loopful of the unknown was smeared onto a slide, heat-fixed, and

subjected to Gram staining using crystal violet, iodine, alcohol decolorizer, and safranin.

Observations were made under oil immersion.

2. Catalase Test: A colony sample was placed on a clean slide, and hydrogen peroxide

was added. Bubble formation indicated a positive result.

3. Mannitol Salt Agar (MSA): The organism was streaked on MSA and incubated at

37°C for 24-48 hours. Growth and color change were recorded.

4. Blood Agar Test: The organism was streaked onto Blood Agar and incubated.

Hemolysis patterns were observed.

5. Coagulase Test: A coagulase test was performed by mixing bacteria with coagulase

plasma and observing clot formation.

Results

Observations:

Test Result Interpretation

Gram-positive cocci in Possible Staphylococcus

Gram Stain
clusters species

Confirms Staphylococcus
Catalase Test Positive (bubbling)
genus

Mannitol Salt Agar Growth with yellow Ferments mannitol → points

(MSA) colour change to S. aureus

Blood Agar Test

Beta-hemolysis (clear Indicates pathogenic
 zones) strain like S. aureus

Coagulase Test Positive (clot formation) Confirms Staphylococcus aureus

Gram Stain:

Appearance: Gram-positive cocci in clusters

Catalase Test:

Positive (Immediate bubbling observed)

Mannitol Salt Agar (MSA):

Growth observed; medium turned yellow, indicating mannitol fermentation

Blood Agar Test:

Beta-hemolysis (complete clearing around colonies)

Coagulase Test:

-Positive (Clot formation observed)

Flow Chart for Unknown D+

Gram Stain (Positive Result)

Positive (Purple)

- Staphylococcus aureus

- Enterococcus faecalis

- Bacillus cereus

- Bacillus subtilis

- Bacillus megaterium

- Lactococcus lactis
 - Staphylococcus epidermidis

- Micrococcus luteus

- Corynebacterium diphtheriae

Catalase Test Performed (Positive Result)

- Positive (Produced bubbles)

- Staphylococcus aureus

- Staphylococcus epidermidis

- Micrococcus luteus

Mannitol Salt Agar Test

- Positive (Growth with yellow fermentation)

- Staphylococcus aureus

Coagulase Test

- Positive (Clot formation)

- Confirms Staphylococcus aureus

Final Identification:

- Unknown " D+" = Staphylococcus aureus

Discussion

The identification of unknown " D+" was achieved through a series of methodical

tests that confirmed its traits. The Gram stain revealed Gram-positive cocci arranged in

clusters, immediately suggesting Staphylococcus or Micrococcus genera. The catalase

test was positive, indicating the Staphylococcus genus. Growth and fermentation on

Mannitol Salt Agar (yellow coloration) strongly indicated Staphylococcus aureus, known

for its ability to ferment mannitol. Beta-hemolysis observed on Blood Agar suggested a
pathogenic strain, and a positive coagulase test definitively confirmed the identity as

Staphylococcus aureus. Alternative organisms such as Staphylococcus epidermidis and

Micrococcus luteus were ruled out because they do not typically ferment mannitol and/or

do not cause beta-hemolysis. The final confirmatory coagulase test solidified the

identification. Possible sources of error could include improper sample handling during

Gram staining or cross-contamination during subculturing. However, consistent positive

results across multiple differential tests provided strong confidence in the outcome.

Conclusion

Based on Gram staining, catalase activity, mannitol fermentation, hemolysis

pattern, and coagulase production, the unknown bacterium "D+" was confidently

identified as Staphylococcus aureus.

 References

Leboffe, M. J., & Pierce, B. E. (2015). Microbiology: Laboratory Theory and Application

(4th ed.). Morton Publishing Company.

Chess, B. (2011). Laboratory applications in microbiology. McGraw-Hill Publishing.

Parker, N., Schneegurt, M., Tu, A. H. T., Lister, P., & Forster, B. M.

(2016). Microbiology.
 Unknown Q-

Introduction

Identifying unknown microorganisms is a fundamental practice in microbiology,

crucial for clinical diagnosis, treatment, public health, and scientific understanding. Using

a combination of morphological observations, Gram staining, and biochemical testing,

microbiologists systematically identify bacterial species. In this lab, the objective was to

identify the unknown microorganism labelled " Q-" using appropriate microbiological

methods. Initial tests provided broad classifications, and further biochemical analysis

pinpointed the species.

Materials and Methods

 Unknown culture labelled "Q-"

 Nutrient Agar (NA) plates

 Gram staining reagents

 Catalase test materials

 Triple Sugar Iron (TSI) Agar

 Microscope

 Incubator at 37°C

Procedure
 1. Gram Stain: A bacterial smear was prepared, heat-fixed, and Gram-stained. The

slide was examined under oil immersion to determine Gram reaction and

morphology.

2. Catalase Test: A sample of the unknown organism was placed on a slide, and

hydrogen peroxide was added. Bubbling indicated catalase positivity.

3. TSI Slant Test: The organism was inoculated into a TSI slant and incubated. The

slant and butt colours, along with gas production, were recorded to assess sugar

fermentation and gas formation.

Results

Test Result Interpretation

Eliminated Gram-positive
Gram Stain Gram-negative rods
organisms

Lactose and/or sucrose

Yellow slant/Yellow butt
TSI Slant fermentation with gas
with cracks
production

Narrowed to catalase-

Catalase Test Positive positive Gram-negative

rods

Flowchart for Unknown " Q-"

Gram Stain (Negative result)

Positive (Purple) | Negative (Pink)

Staphylococcus aureus

Enterococcus faecalis

Bacillus cereus

Bacillus subtilis

Bacillus megaterium

Lactococcus lactis

Staphylococcus epidermidis

Corynebacterium diphtheriae

Micrococcus luteus |

Alcaligenes faecalis

Klebsiella pneumoniae

Pseudomonas aeruginosa

Escherichia coli

Shigella flexneri

Salmonella typhimurium

Serratia marcescens
Proteus mirabilis

Providencia alcalifaciens

Catalase Test Performed (Positive result)

Positive (Produced bubbles) | Negative (No bubbles produced)

Alcaligenes faecalis

Pseudomonas aeruginosa

Escherichia coli

Shigella flexneri

Serratia marcescens

Klebsiella pneumoniae

Salmonella typhimurium

Proteus mirabilis

Providencia alcalifaciens

TSI Slant Test

 Yellow slant/Yellow butt, Cracks present → 8. Escherichia coli

 Red slant/Yellow butt, No cracks → 9. Shigella flexneri

 Yellow slant/Yellow butt, No cracks → 12. Serratia marcescens

  Final Identification: Escherichia coli

Discussion

The identification of Unknown “ Q-" proceeded systematically. The Gram stain

result immediately indicated a Gram-negative bacillus, allowing the elimination of all

Gram-positive organisms. The catalase test further refined the possibilities to catalase-

positive Gram-negative bacilli. Finally, the Triple Sugar Iron (TSI) test was essential in

distinguishing among the remaining organisms. The observed yellow slant/yellow butt

along with gas (cracks in the agar) is a classic reaction for Escherichia coli, which

ferments glucose, lactose, and/or sucrose with gas production.

Alternative organisms like Shigella flexneri and Serratia marcescens were

considered. However, Shigella typically shows a red slant (no lactose or sucrose

fermentation) and no gas production. Serratia shows a yellow slant/yellow butt but rarely

produces significant gas. The observed cracking and complete colour change confirmed

Escherichia coli. Possible sources of error include contamination, misinterpretation of gas

production, or variation in incubation times affecting colour intensity. Multiple

confirmations and clear results increased the reliability of the identification.

Conclusion

Based on Gram staining, catalase activity, and TSI reactions, Unknown " Q-" was

identified as Escherichia coli.

 References

Leboffe, M. J., & Pierce, B. E. (2015). Microbiology: laboratory theory and application.

Morton Publishing Company.

Chess, B. (2011). Laboratory applications in microbiology. McGraw-Hill Publishing.

McMeekin, T., Olley, J., Ratkowsky, D., Corkrey, R., & Ross, T. (2013). Predictive

microbiology theory and application: Is it all about rates?. Food Control, 29(2),

290-299.