THE 2. Microscope Slide 3. Gram Staining

THE 2. Microscope Slide 3. Gram Staining

THE AMERICAN UNIVERSITY IN CAIROBIOLOGY DEPARTMENTSCIENCE 453 : BIOLOGY FOR ENGINEERS REPORT No.124/2/96Objective:To test a sample of laboratory prepared bacteria and categorise itaccording to Christians gram positive and gram negative classes and also byviewing it under a high powered microscope and oil immersions; classify itsshape and note any special characteristics.Introduction:Bacteria was categorised into two groups in 1884 by the DanishBacteriologist Christian, gram positive and gram negative by a stainingtechnique where the ability to avoid de-coloration of Crystal Violet solutionby alcohol would render the category of gram positive, and gram negative if thebacteria is de-coloured. This could be noted by the final colour of thebacteria: a violet colour where Gram positive and a pink colour of the Safraninadded pending the de-colouring process.Materials:1. Bacteria Sample 2.

Microscope Slide 3. Gram Staining Kit and Wash Bottlesa. Crystal Violet Solution b. Iodine Solution c.

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95% Ethyl Alcohol d.Safranin e. Distilled Water 4.

Bibulous Blotting Paper 5. Microscope 6. OilProcedure:A.

Preparation :1. Bacteria is cultivated on agar jelly in an incubator at 25C for 24 hours. 2.Obtain a microscope slide and with a toothpick, smear a thin coat of thebacteria sample onto the slide 3. Cover the smear with a drop Crystal Violetand leave standing for 20 seconds 4. Wash off the stain with distilled water;drain and blot off the excess with bibulous paper.

5. Apply Grams Iodine onthe smear and leave to stand for 1 minute. 6. Drain the excess iodine and apply95% Ethyl alcohol for 20 second duration or till the alcohol runs clearly fromthe slide. 7. The smear should rinsed for a few seconds with distilled water tostop the action of the alcohol.

8. Drain and blot off the excess with bibulous9. Introduce Safranin to the smear and leave standing for 20 seconds. 10. Washoff the stain with distilled water; drain and blot off the excess with bibulouspaper. 11. Leave the slide to air dry.

B. Examination:1. Place the slide under microscope on low powered lens. 2. Move the slideusing the apparatus until the sample can be seen as a blur under the microscope.3. Focus the lens to ensure that there is a sample directly under the lens.

4.Move to higher powered lens, repeat step 3. 5. Move to higher powered lens,repeat step 3 6.

Move microscope aside and add Oil immersion, leave for a fewseconds and re-examine the slide.Note Shape and colour and any other observations.Results and Observations:It was evident by visual examination that the alcohol was de-colouring or aleast partially de-colouring the bacteria.The sample appeared a dark pink or close to violet by the naked eye; amicroscope was needed to ensure results.Under the low powered microscope shades of pink were noted.

Under the medium power, the shades were more clear but no shape could be madeout.Under the high powered microscope clumps of pink rod (bacilli) shaped bacteriacells could be observed.Under Oil Immersion and high powered lens the cells could seen more spaced outand thus a clearer indication of the pink colour, bacilli shape and spores couldbe made out in the individual cells.Conclusion:The Shape was noted as Bacilli (Rod-like) shaped cells; a gram variableshape, distinct in either Gram Negative or Gram positive bacteria.The final colour of the cells were stained pink by the Safranin showingthe de-coloration of the crystal violet proving the bacteria is of the gramnegative class.Under oil immersion the cells became more sparse and under the highpowered lens of the microscope spores could be seen, as little bubbles, in thecells. This tells us that the bacteria was in its terminal state.

The presence of spores in the bacteria at its terminal state tells usthat the bacteria could be an old culture. Old bacteria cultures which are grampositive tend to de-colour, yet more slowly than gram negative bacteria. Thespeed of de-coloration was not inspected very clearly thus no further conclusioncould be reached, yet it is possible that this an old culture of Bacilli shapedGram Positive bacteria.Recommendation:It is recommended that the same sample be tested again for de-coloration; focusing on de-coloration speed. If the de-coloration is fast thenthe sample is definitely gram negative, slow de-coloration would tell us it isgram positive. For future samples it would be recommended to keep the bacteriasample for this specific test for only 16 hours as recommended to avoid thepresence of old cultures which are anomalous to this test.Category: Science

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