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4.The slides are allowed to dry in the air or by holding them high above a Bunsen flame.5. When the film is dry, the slide is passes, film side up, three times through the Bunsenflame.Staining with Basic Dyes1.The slides fixed in the first part of the procedure is placed on the wire staining screenover the waste jar.2.Each of the fixed smears is flood with approximately five drops of one of the dye andtime is allowed for it to act.3.The stained preparations are washed with water from the wash bottle.4.The slides are dried between blotting paper.
5.The stained preparations are examined under the oil-immersion objective of the microscopeand drawings of a good field are made.B. Negative or Indirect Staining1.From the broth cultures provided, 2 loopfuls are transferred to a slide and a small drop ofthe nigrosine solution is added. Mix thoroughly with your inoculating needle and spread ina thin film with the edge of a glass slide2.A tooth pick is used to scrape the surface and the crevices of your teeth to obtain tartar forstaining. The tartar is mixed on a slide with a small drop of water and stain with nigrosine.3.Air dry. Do not fix with heat.4.Examine under the oil-immersion objective and drawings is made on the notebook.C. Differential Staining Techniques – Gram Stain1.Smears of Bacillus cereus, Streptococcus faecalis, and one mixed smear of Escherichia coliand Micrococcus luteus are prepared. These preparations are fixed with heat.2.Stain with crystal violet for about 30 seconds.3.Rinse with water.4.The film is covered with Gram’s iodine and is allowed to act for about 30 seconds.5.Rinse with water.6.Decolorize with 95% alcohol.7.Rinse with water.8.Counterstain with safranin for 20 – 30 second.9.Rinse with water and blot dry.10.Examine under the oil immersion objective.D. Structural Stains – Endospore Stain1.Smear of B. cereus or C. sporogenes cultures is prepared, the smear is dried in air, and isfixed with heat.
2.The slides is placed on a staining rack above boiling water.3.The smear is covered with small pieces of paper towel, is kept saturated with malachitegreen (5% aqueous solution), and continue heating for 5 minutes.4.Wash gently with water.5.Counterstain with safranin for 30 seconds.6.Wash with water and blot dry.7.Examine under the oil immersion objective and make drawings.Results:A. Direct Staining with Basic DyesFigure 1 is theKlesiella pneumoniaestained withmethylene blue dye. It is a coccobacillus which isoval and similar to a coccus.Magnification: 100 x10X
Figure 2 is theBacillus cereusstained with crystalviolet dye. B.cereus is a rod, cylindrical shapedsingle bacillus.Magnification: 100 x 10XFigure 3 is theEnterococcus faecalisstained withmethylene blue dye. E.faecalis is in the form ofcoccus and diploccus and arranges in short chain.

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Term
Summer
Professor
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Tags
Bacteria, gram negative bacteria, basic dyes

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