Today, we took a gram-stain of our bacteria 'L' in order to figure out if it was gram-negitive, gram-positive or gram-variable. Unlike the stain we did before (a simple stain) we used not just Crystal Violet but had to use Iodine (Gram's Iodine to be exact) and Safranin. However before we could use all the fun colorful chemicals above we had to recreate a simple stain. As mentioned previously (earlier blog post) we smeared, air dried and than heat-fixed the bacteria onto a slide. We than stained the bacteria using the Crystal Violet by covering the stain for 20 to 30 seconds over a slide drying rack. Then rinsing the slide with distilled water, we used Gram's Iodine and covered the smear for 1 minute. Then using 95% EtOH we held the size at a 45 degree angle and ran the color off the slide (decolored the sample). After this we rinsed again with distilled water and used Safranin to coverthe slide for 1 minute. Once finished we blotted the slide dry wth Bibulous paper and viewed it under a microscope on 400x using immersion oil. Our bacteria turned out to be Gram-negitive! This is because it did take up the Safranin! The peptidoglycan layer in Gram-Negitive Bacteria is so small that it cannot keep hold of the Crystal Violet once EtOH is applied. We know that because of this thin layer of peptidoglycan, the new color Safranin can hold (which is why the results below show a pink/red bacteria smear! (If it were Gram-Positive it would've been purple!)
this was the results under 400x
Rinsing Off The Slide With Distilled Water
The results at a lower magnification (100x)
Hope You Enjoied!
Annie B
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