Stain Protocols-BIOL 2420 Gram Stain 1.Perform a bacterial smear,as discussed in Figure 3-52 on page 150 of your lab manual. 2.Saturate the smear with crystal violet for 1 minute 3.Rinse the slide gently with water. 4.Saturate the smear with iodine for I minute. 5.Rinse the slide gently with water. 6.Decolorize with Gram decolorizer(acetone/alcohol)for 3-5 seconds ONLY;if you leave the decolorizer on too long,it will bleach the crystal violet out of a gram positive cell! 7.Rinse the slide gently with water. 8.Counterstain with safranin for 1 minute 9.Rinse the slide gently with water 10.Carefully blot the slide dry with bibulous paper. 11.Observe the slide under the microscope,using proper microscope technique. With proper staining technique.. Gram positive bacteria will stain purple. Gram negative bacteria will stain red/pink TIP:When making a smear of an unknown organism to Gram stain,place three nmon an pole yor staining technique,it will be reflected in the known organisms,and you'll know you need to do the stain again to get accurate esults. Smith-2010 Page2of4 Stain Protocols – BIOL 2420 Smith – 2010 Page 2 of 4 Gram Stain 1. Perform a bacterial smear, as discussed in Figure 3-52 on page 150 of your lab manual. 2. Saturate the smear with crystal violet for 1 minute. 3. Rinse the slide gently with water. 4. Saturate the smear with iodine for 1 minute. 5. Rinse the slide gently with water. 6. Decolorize with Gram decolorizer (acetone/alcohol) for 3-5 seconds ONLY; if you leave the decolorizer on too long, it will bleach the crystal violet out of a gram positive cell! 7. Rinse the slide gently with water. 8. Counterstain with safranin for 1 minute. 9. Rinse the slide gently with water. 10. Carefully blot the slide dry with bibulous paper. 11. Observe the slide under the microscope, using proper microscope technique. With proper staining technique… Gram positive bacteria will stain purple. Gram negative bacteria will stain red/pink. TIP: When making a smear of an unknown organism to Gram stain, place three organisms on your slide: a known gram positive, a known gram negative, and your unknown (and LABEL which is which!). That way, if there’s a problem with your staining technique, it will be reflected in the known organisms, and you’ll know you need to do the stain again to get accurate results