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I have done an examination on the unknown waterfront Site 1 A sample. The gram stain procedure was performed , along with many various tube tests like Nitrate test,citrate test,SIM test,Lactose test ,glucose and Mannitol PR test. (A) Gram Stain Procedure CULTURE: Waterfront Site 1 A sample REAGENTS: Crystal violet,Gram’s Iodine,ethyl alcohol and safranin. EQUIPMENTS: Bunsen burner, inoculating loop, glass slides, bibulous paper, lens paper, emersion oil and microscope. METHOD AND PROCEDURE: 1. Stained the glass slide with WF site 1 A 2. Smeared the crystal violet on the stained slide – left for 1 minute. 3. Gentle washed the slide with tap water. 4. Used mordant Gram’s Iodine – left for 1 minute. 5. Gentle washed the slide with tap water. 6. Used alcohol for the decolorization drop by drop until it run clear – for 15 seconds. 7. Gentle washed the slide with tap water. 8. Counterstained with Safranin – left for 1 minute. 9. Gentle washed the slide with tap water. 10. Used bibulous paper to blot dry. Observed under 100X on microscope with the use of emersion oil. Result of Gram stain:- After following the gram stain procedure I have observed a gram negative Pink-rod shape mono bacilli under 100X microscope. (B) Other various tube tests REAGENTS: Indole, Nitrate A & B, Methyl Red EQUIPMENTS: Bunsen burner,inoculating loop METHOD AND PROCEDURE: Sterilize loop vertically in the blue flame of bunsen burner. From the rack,take the test tube containing WF Site 1 A sample culture. Remove the cap from tube ,heat the neck of the tube. Using aseptic technique take a loop full of the organism from WF Site 1 A. Again flame the neck of the tube and replace the cap and place the tube back in rack. Remove the cap of the media and heat the neck and inoculate it . Follow the same procedure to inoculate all the medias. RESULTS : Lactose – Negative SIM ( Indole) – Negative Mannitol PR – Negative Nitrate ( Nitrate A&B ) – Positive / color turned red Citrate – Positive / Blue Slant Glucose ( Methyl Red ) – Negative CONCLUSION : The gram stain was performed in order to identify whether the microorganism was gram positive or gram negative. I have observed a gram negative pink-rod shaped bacteria under the 100x microscope power. Various tube tests were examined and by putting all the results together,I have concluded that microorganism I have worked on was CITROBACTER FREUNDII . Citrobacter Fruendii is an anaerobe gram negative bacteria which can be found in soil,water,sewage,food and in intestinal tract of human and animal. It belongs to Enterobacteriaceae family.

I have done an examination on the unknown waterfront Site 1 A sample. The gram stain procedure was performed , along with many various tube tests like Nitrate test,citrate test,SIM test,Lactose test ,glucose and Mannitol PR test. (A) Gram Stain Procedure CULTURE: Waterfront Site 1 A sample REAGENTS: Crystal violet,Gram’s Iodine,ethyl alcohol and safranin. EQUIPMENTS: Bunsen burner, inoculating loop, glass slides, bibulous paper, lens paper, emersion oil and microscope. METHOD AND PROCEDURE: 1. Stained the glass slide with WF site 1 A 2. Smeared the crystal violet on the stained slide – left for 1 minute. 3. Gentle washed the slide with tap water. 4. Used mordant Gram’s Iodine – left for 1 minute. 5. Gentle washed the slide with tap water. 6. Used alcohol for the decolorization drop by drop until it run clear – for 15 seconds. 7. Gentle washed the slide with tap water. 8. Counterstained with Safranin – left for 1 minute. 9. Gentle washed the slide with tap water. 10. Used bibulous paper to blot dry. Observed under 100X on microscope with the use of emersion oil. Result of Gram stain:- After following the gram stain procedure I have observed a gram negative Pink-rod shape mono bacilli under 100X microscope. (B) Other various tube tests REAGENTS: Indole, Nitrate A & B, Methyl Red EQUIPMENTS: Bunsen burner,inoculating loop METHOD AND PROCEDURE: Sterilize loop vertically in the blue flame of bunsen burner. From the rack,take the test tube containing WF Site 1 A sample culture. Remove the cap from tube ,heat the neck of the tube. Using aseptic technique take a loop full of the organism from WF Site 1 A. Again flame the neck of the tube and replace the cap and place the tube back in rack. Remove the cap of the media and heat the neck and inoculate it . Follow the same procedure to inoculate all the medias. RESULTS : Lactose – Negative SIM ( Indole) – Negative Mannitol PR – Negative Nitrate ( Nitrate A&B ) – Positive / color turned red Citrate – Positive / Blue Slant Glucose ( Methyl Red ) – Negative CONCLUSION : The gram stain was performed in order to identify whether the microorganism was gram positive or gram negative. I have observed a gram negative pink-rod shaped bacteria under the 100x microscope power. Various tube tests were examined and by putting all the results together,I have concluded that microorganism I have worked on was CITROBACTER FREUNDII . Citrobacter Fruendii is an anaerobe gram negative bacteria which can be found in soil,water,sewage,food and in intestinal tract of human and animal. It belongs to Enterobacteriaceae family.