MICROBIOLOGY MB102b
LAB REPORT #4:
Gram staining of Staphylococcus aureus and Escherichia coli
Muhammed Sayed Hassan ID: 171741 GROUP (F)
Submitted to: L.A. Suzanne ali
Gram staining of Staphylococcus aureus and Escherichia coli
INTRODUCTION This lab covered smear preparation of Staphylococcus aureus and
Escherichia coli on the microscopic slide with the heat fixation method, Then gram staining both bacteria with a Primary stain, Mordant,
Decolorizing agent, and a counterstain. Then identifying its color under the light microscope. The gram staining method depends on the properties of the gram-negative and gram-positive peptidoglycan layers, Outer membrane and LPS content which all cause a different gram reactions. Aim of the lab was to differentiate between gram-negative bacteria and gram-positive bacteria with gram staining.
MATERIALS Chemical agents: 1. Immersion oil 2. Crystal violet 3. Gram's Iodine 4. Ethanol (95%) 5. Safranin 6. Distilled water 7. Ethanol (70%)
Biological agents: 1. Staphylococcus aureus 2. Escherichia coli
Equipments: 1. Light Microscope 2. Microscopic slide 3. Aluminium foil sheet 4. Inoculation loop 5. Bunsen burner
PROCEDURE a) Smearing of bacteria 1. 1 drops of water was put on the microscopic slide. 2. The inoculation loop was sterilized by bunsen burner. 3. 1 loopful was put on the microscopic slide. 4. The bacteria was smeared on the slide. b) Heat fixation of bacteria 5. The bacteria was heated with bunsen burner with indirect flame fixation. c) Gram staining of bacteria 6. The microscopic slide was put on the aluminium foil sheet. 7. 2 drop of Crystal violet were dropped on the smear for 1 minute. 8. Microscopic slide was rinsed gently with distilled water. 9. 2 drops of Iodine were dropped on the smear for 2 minutes. 10. Microscopic slide was rinsed gently with distilled water. 11. The slide was decolorized with ethanol for 30 Seconds. 12. Then microscopic slide was rinsed gently with distilled water. 13. 2 drops of Safranin were dropped on the smear for 1 minute. 14. Microscopic slide was rinsed gently with distilled water. 15. Then the slide was drought for about 10 minutes before examination. d) Microscopic examination of the slide 16. 1 drop of immersion oil was dropped on the slide. 17. The slide was put in the microscopic clip. 18. Knob was zoomed until the lens touched the oil concave surface.
19. Then the slide was examined with the Oil lens. NOTE: Repeat the same procedures for the two bacteria: Staphylococcus aureus and Escherichia coli.
RESULTS
Fig(1): Gram positive stain of Staphylococcus
aureus bacteria shown under a light microscope.
F ig(2): Gram negative stain of E.coli bacteria shown under a light microscope.
DISCUSSION Above fig(1) shows gram positive stain Staphylococcus aureus bacteria gave (Violet) color under light microscope using oil-immersion lens. Fig(2) shows gram negative stain Escherichia coli bacteria which gave (red-pink) color using the same lens for fig(1).
CONCLUSION The aim of the lap was successfully achieved which is differentiating between gram positive and gram negative bacteria through gram staining.