In this video I'm going to show you how to make a bacterial smear for acid fast or zeal neolusine staining. Let's put on our goggles before we start. Howdy. Okay, first we're going to prepare a microscope slide. I'm going to get ready for differential staining by creating a slide with three circles on it. In that first circle on the left I'm going to put my positive control, my acid fast, known organism. In that third circle, the one on the right I'm going to put my non-assaged fast negative control. The unknown bacterial sample will go in the middle. It's a good idea to flip that slide over so the wax is on the bottom. That way the wax will not mix with my bacterial sample and it's an easy way to tell which side of the slide is the top side. Whichever side you can scratch the wax off on as the bottom. It can be helpful to label a paper towel with the names of the samples you'll be using for this slide. I'm going to use my co-bacterium smegmatis on the left as my acid fast control. Staphylococcus epidermitis on the right is my non-assaged fast control and my unknown will go in the middle. I'm going to take a dropper bottle of water and put a small drop of water in each of those circles. This is what I'm going to mix my bacterial samples into. Now my slide is ready for the bacterial samples. I'm going to start by transferring my co-bacterium smegmatis, my acid fast positive control into the circle of water on the left of my slide. First I need to sterilize my inoculation loop or wand. I put it into that micro-incinerator hob for 5 to 10 seconds. Before I can obtain my bacterial sample I will need to also let that loop cool another 5 to 10 seconds. Then I can take my cooled sterolube and gently run it across the median to obtain a colony of my co-bacterium. My co-bacterium is waxy so it's going to be difficult to mix that into the water sample. You might get some chunks so just take a little extra time to try and break that sample off and spread it evenly in that water droplet. Then I need to sterilize my loop. I do that between each stop. Next I will obtain my non-asset fast, my negative control and here I'm using Staflococus at pydermittis. I take my cooled sterolube and I just gently grab a colony off the surface of the media. I'm going to mix that sample into the water droplet in the circle on the right and again that's my negative control. My bacteria that does not have a waxy coating. I sterilize my loop. Let it cool and then I can obtain my unknown bacterial sample. I gently move my loop across a colony in that unknown sample and mix that into the water droplet in the center of the slide. Then I'm going to sterilize my loop one last time. It's now time to heat fix my bacterial smear. Now that my slide is all prepared I am going to put it on the platform on top of the micro incinerator. That will deliver heat that will kill the bacteria and stick it to the slide. When my slide is heat fixed and ready for staining those droplets of water will be completely evaporated usually about 5 minutes.