Gram- staining technique lab
Hans Christain Gram is the Danish microbiologist who developed the Gram staining technique. This technique is one of the most significant techniques used in the field of microbiology. Gram staining provides a key for identifying and classifying bacteria- because bacteria are transparent and - therefore difficult to see under the compound microscope. Staining the specimens can increase visibility and help identify the microbes. Staining involves first heat –fixing the bacteria to a slide, then saturating the slide with a dye that reacts with various parts of the cell to stain. Heat fixing and staining may kill the bacteria, but the shape of the organism will be preserved. Methylene blue and crystal violet are commonly used stains. These dyes are positively charged and- therefore bind to the negatively charged polysaccharides and proteins on the cell wall surface, and on the inside of bacterial cells.
Gram staining can divide bacteria into two groups: Gram- positive and Gram-negative.
Objectives
Students will learn the Gram staining technique and distinguish between Gram-positive and Gram-negative bacteria.
Materials needed
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Bacterial culture
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Crystal violet –Ammonium Oxalate solution
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Gram's Iodine solution
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Counterstain -Safranin
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Decolorizing alcohol
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Apron, safety goggles, and gloves.
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Inoculating loops
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Distilled water
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Bunsen burner
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Slides
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Procedure
Students should review all safety procedures and the proper aseptic techniques before they start working. Students should wear safety goggles, aprons and gloves.
Smear preparation
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1.
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Sterilize the inoculating loop by holding it in the flame of the Bunsen burner for a few seconds, then allow it to cool of for ~ 15 seconds.
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2.
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Place a loopful of distilled water in the center of a clean slide
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3.
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Take a small amount of the bacterial culture by using the inoculating loop and place it in the center of the water drop.
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4.
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Mix the culture with the distilled water by using a circular motion and spread it in the center of the slide.
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5.
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Sterilize the inoculating loop again and allow the slide to air dry.
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6.
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Fix the slide by passing it 2-3 times over the Bunsen burner, and make sure that the smear side is up. Make sure that you do not expose the slide to excessive heat so that the slide will not break or be damaged.
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Staining the culture
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7.
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Place about 5 drops of the crystal violet stain to flood the culture smear by using a dropper, and allow it to stand for about one minute, then gently rinse the slide off with tap water for about 5 seconds and drain.
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8.
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Place 5 drops of the iodine solution onto the culture and allow it to work for about 1 minute, then gently rinse the slide and drain by shaking off the excess water.
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Decolorizing the stain
Alcohol should be used; this step is very important and the procedure is especially sensitie to it.
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Place 5 drops of 95% alcohol in a way that flows down the slide from one end and continue until the solvent alcohol is no longer colored, if the smear is too heavy, you might have to repeat the process one more time. If you decolorize too long, the crystal violet stain may be completely removed.
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Gently rinse the slide with water, and pat dry with lint-free paper, make sure that you pat it gently so that the smear will not rub off.
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Counter staining the smear
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Place about 5 drops of the safranin solution on the smear and allow it to stand for about 25 seconds
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12.
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Gently rinse the safranin away with water an dry it gently by using bibulous paper to remove excess water
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Observing the slide
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Examine the slide under an oil immersion (100x) objective lens.
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Gram -positive bacteria will appear blue to purple, and gram-negative bacteria
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will decolorize but retain the reddish or pinkish color of the counterstain.
Evaluation
Students will write a lab report including the procedure and results including an illustration for the shape, color and classification of the bacteria that have been observed.
