3-5 Negative Stain - c Flame loop and use it to add 2-3...

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1 Experiment 3-5: Negative Staining (pgs. 84-85 ) Purpose : To learn the procedure for doing the negative stain Bacteria are transparent and difficult to see if doing a wet mount. Negative staining will stain the background of the slide and not the organism - dye used is Nigrosine (or India ink) - this is an acidic dye (chromophores have a negative charge), which cannot penetrate the organism whose surface is negatively charged - useful for determining cell morphology and size - no heat-fixing means no cell distortion/shrinkage Materials and Methods: TSB cultures of : Stain: Klebsiella pneumoniae Nigrosine Staphylococcus epidermidis Negative Staining: Feather-edge smear a. Put a drop of Nigrosine on clean slide b. Flame loop and use it to transfer a loopful of nigrosine to another clean slide
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Unformatted text preview: c. Flame loop and use it to add 2-3 loopfuls of bacteria to the drop of nigrosine d. Flame loop e. Use a 2nd slide and move towards the drop f. Let dye spread along the 2nd slide g. Then push the 2nd slide away from the region of the drop to create a smear thick on one end and thin on the other h. Air dry and examine under oil-immersion Notes on working with Nigrosine : 1. Try to work quickly because the nigrosine will tend to dry out 2. Cracks can be created upon drying (or things can be on the slide) which can be mistaken for organisms 3. Try to create a thinner area because you will not see anything if the stain is too thick Negative Stain -Klebsiella pneumonia Negative Stain -Staphylococcus epidermidis...
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