Transcription
[Music] In this video, we are going to show you how to prepare a heat-fix bacterial film and stain it using Gram's method. For this method, you will need a bacterial culture, some distilled water (which can be found in the box by the sink), an inoculating loop (with the wire straightened by running between your thumb and forefinger), and a box of microscope slides.
Before we make a film, we need to remove any grease from the microscope slide. We will remove a slide from the box and make sure you have a single slide. You will note in the video that I have two slides stuck together; this is very common, so please make sure you check you have just the one. Engage the blue flame of your Bunsen burner and pass your slide through the flame several times to remove any grease.
Next, take your inoculating loop and sterilize it. Take the lid off the distilled water using the little finger of your dominant hand. Flame the neck of the water bottle and place three loopfuls of water onto your microscope slide. Put the lid back on the water bottle and make sure you sterilize your loop. You next pick up the plates containing your bacterial culture and, holding the inoculum loop like a pencil, pick off a small quantity of bacteria from a single colony. Please note: you do not need to take a lot of bacteria from your plate. Mix your sample of bacteria into the water on your microscope slide and mix in concentric circles out to about the size of two postage stamps. Sterilize your inoculating loop and allow the water to evaporate from your slide. This should take approximately two to five minutes.
So now you can see that all of the water has evaporated from my slide. So now I need to heat fix my film. This causes the bacteria to stick to the glass and prevents them from being washed away in the Gram staining process. So, as you've just seen on the video, you take your slide and you run it through the Bunsen flame three to four times. You're now ready to start Gram staining.
From the box by the sink, take out the crystal violet, Lugol's iodine, acetone, and some safranin. You also need to collect some blotting paper. Before doing anything else, turn on the cold tap and leave it running throughout the staining process. Place your slide on the staining rack and flood it with the crystal violet. You should then wait approximately 30 seconds to one minute before washing it off. Note: in this video, I've sped up the process. Next, flood the slide with Lugol's iodine. You should wait another 30 to 60 seconds before washing it off the slide.
Next is the critical part of the Gram staining process. Take your acetone and pick up your slide. Run the acetone down the slide before immediately rinsing it off. Do not let the acetone sit on the slide, as this will lead to a loss of color with Gram-positive organisms. Finally, add safranin to your slide. Leave this on the slide for one to two minutes before washing it off. You at this point remove excess water from your slide and get a piece of blotting paper. You can find these in the boxes at the end of the bench by the window. Place your slide on a piece of blotting paper and fold over the blotting paper and use it to blot dry the slide. Don't attempt to rub the water off; this will also remove bacteria. Once all the water has been blotted off, you are ready to view your stained film under the microscope.