Observing Bacteria Under A Microscope
I had the opportunity to work in a laboratory to stain tap water and observe the bacteria present in it under a microscope. Recently, I had been participating in an research experiment of my own where I investigated the effect of high oxidative stress on bacteria.
I came across the use of electrolyzed salt water or saline solution as a disinfectant for sterilizing surgical equipment. Usually people use alcohol sanitizers or acid based sanitizers to kill microbes. However, this was the first I heard of someone using electrocuted water as a means of disinfection. Studies show that this method is actually based on the product’s (electrolyzed salt water) ability to act as a strong oxidizing agent. Fun fact : bleach, an industrially used disinfectant for swimming pools and bathrooms, actually uses this concept too! However, unlike bleach, electrolyzed saline is not toxic and is completely chemical-free! I wondered why this was a commonly used method already, and hence, I started to research deeper into this concept.
For the first step of my multiple-part-study, I wanted to test different solutions with different oxidation strengths (henceforth referred to as ORP — Oxidation and Reduction Potential) to determine which would be the most effective. This included electrolyzed saline with different ORPs, which was achieved by varying the parameters (voltage, electrode size etcs.) of the electrolyzing equipment. The efficacy was tested by treating samples of tap water (tap water in India is undrinkable to the contamination of bacteria) with these different ORP solutions to see which had the greatest bacterial reduction.
There are multiple commonly used methods for counting organisms under a microscope, but for this experiment, I stained the samples of treated tap water with chemical dyes that can highlight bacteria under a microscope. First, the bacterial slides were stained with Safranin solution, a type of dye used to detect gram negative bacteria — bacteria that have a thin cell membrane and an outer lipid layer. Next, the bacterial slides were stained with Crystal Violet solution, a dye used to highlight gram positive bacteria — bacteria that have a thick cell membrane and no outer lipid layer. Gram negative bacteria does not retain Crystal Violet and hence can be differentiated from gram positive bacteria. The slides were further dyed with a solution called Gram’s Iodine, which further highlights gram positive bacteria to give it a violet color. Finally, when observed under a microscope, all the bacteria were highlighted irrespective of their grap type.
Next, all I had to do was manually count the number of bacteria. There are numerous ways to do this, this for the sake of simplicity. Images of the bacterial slides were taken and cropped into a 1225 x 1225 pixel square. A 3x3 grid was drawn on the cropped images (as seen in image 2) and three random squares were selected using a random number generator. The bacteria in each square were counted and the average was taken to generalize for the slide.
