Microbiology & lab The American University of
Semester spring 2025 Kurdistan
Date: 4\3\2025
Lab report
Bacterial (Gram) staining
Prepared by Supervised by
Khdher Ali
Jihan Khorsheed Assist. Prof. Dr. Rezheen
Fatah Abdulrahman, PhD
Sarmad Omar
Avan Khaleel
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Objectives:
• understand the purpose and significance of Gram staining in microbiology.
• differentiate between Gram-positive and Gram-negative bacteria based on their cell wall
composition.
• observe bacterial morphology, arrangement, and color under a microscope after staining.
Equipments
• Gram staining reagents:
• Crystal violet
• Iodine solution
• Absolute ethanol
• Safranin
• Distilled water
• Filter paper
• Microscope
• Immersion oil
Procedure
A pre-prepared bacterial slide which was prepared by Dr. Rzheen was used for the Gram staining
procedure. The process began with the application of crystal violet, which was left on the slide
for one minute to act as the primary stain. After rinsing with distilled water, iodine solution was
added and left for one minute , helping to fix the dye within the bacterial cells. Following
another rinse, absolute ethanol was applied for one to five seconds to decolorize the sample,
removing the crystal violet from Gram-negative bacteria while Gram-positive bacteria retained
the stain. The slide was then washed again with distilled water before safranin was applied as a
counterstain for one minute, allowing the Gram-negative bacteria to absorb the red dye. After a
final rinse with distilled water, the slide was gently dried using filter paper. A drop of immersion
oil was placed on the slide, and it was examined under a 100x lens using a light microscope.
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Results
Under microscopic examination, the bacteria were seen as rod-shaped (bacilli) and arranged
singly. The color of the stained bacteria appeared reddish-pink, indicating that they were Gram-
negative. This result confirms that the bacterial cells did not retain the crystal violet stain after
decolorization with ethanol, instead taking up the counterstain, safranin.
Conclusion
Gram staining is a differential staining method used to classify bacteria based on their cell wall
composition weather is gram-positive or gram-negative. In this experiment, the bacteria were
identified as Gram-negative due to their inability to retain the crystal violet-iodine complex after
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ethanol decolorization. Instead, they took up the safranin counterstain, appearing reddish-pink
under the microscope. This technique is essential for bacterial identification in microbiology and
medical diagnostics.
References
1. Assist. Prof. Dr. Rezheen Fatah Abdulrahman, PhD
2. The practical lab section
3. https://www.youtube.com/watc h?v=FJoEMPPr2Ho
4. https://www.youtube.com/watc h?v=MiGp8buyzXg
5. https://www.youtube.com/watc h?v=HxjN2mBLGCE
Sarmad > notes and materials
Jihan > procedure
Khdher > measurements and objectives
Avan > figures and conclusion
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