Preparing a Gram Stain

Now that we have obtained a pure culture we are ready to prepare a gram stain so that we could see the shape, size and arrangement of the bacteria. Also, we could determine whether the bacteria are gram-negative or gram-positive.

First we take a sample from our pure culture and heat fix the sample onto a slide. Then we placed the slide on a dry rack over a sink at our lab bench. We then covered the smear with a Crystal violet stain and left it on the slide for 20 seconds.

Afterwards we rinsed off the crystal violet with distilled water.

We put the slide back on the dry rack and then covered the smear with Gram’s iodine for 1 minute.

We rinsed off the Gram’s iodine with distilled water. Then we held the slide at a 45-degree angle and added the decolorizing reagent (EtOH) drop by drop until the color stopped running.

We immediately rinsed off the decolorizing reagent and covered the smear with a Safranin stain for 1 minute.

After 1 minute we rinsed the slide with distilled water to remove the excess Safranin.

Once all the Safranin excess was gone we blot water from the slide with pieces of bibulous paper.

Now we are ready to examine the stained smear under the microscope using the oil immersion lens and look what we saw!

Looking closely at the slide we realized that we did not in fact obtain a pure culture. As you can see we have at least two different types of bacteria one that is gram-positive and seems to have spores and another that is gram-negative. Therefore, Dr. Pathakamuri helped us obtain a more pure culture and at the next lab we will gram stain the new sample.