Gram Staining

In the past three weeks of my internship, I’ve learned more about biology by doing different experiments and doing some researches. Last week, which was the last week of fall semester, I was taught how to do Gram-staining. It was easy and super interesting for me. With this method (Gram Stain) you can categorizes bacteria based on the physical and chemical structure of their cell wall, also it distinguishes between Gram positive and Gram negative groups.

After making a slide of your sample, in my case was the bacteria that I growth before, Gram staining involves four processes: first add the crystal violet to the sample on the slide and leave it for 30 seconds, and then rinse it with distilled water. Then add the Gram iodine for 1 minute, and rinse the slide with water again. Next we need to use a Gram decolorize for almost 5 seconds and rinse it with water after 5 seconds. It decolorizes the sample if it’s a Gram negative and it will remove the crystal violet. For the last part, add the Safranin to the slide and after 30 seconds rinse the slide with distilled water for almost 5 seconds. If it’s a Gram positive bacteria, it keeps the crystal violet and it looks purple under microscope.

The slide that I got from Gram staining it shows on the picture below.

So when I viewed my slide under a microscope I got this picture, which it shows that it’s Gram positive and bacilli.

Tryptic Soy Agar (TSA)

I needed to make a media for my bioluminescent bacteria to grow. The best media for growing it, is Tryptic Soy Agar (TSA).

Tryptic Soy Agar (TSA) is a general purpose medium that contains enough nutrients for a wide variety of microorganisms to grow.

First, I weighed two 20 grams of TSA with scalar. Then, I measured two 500 mL of distilled water with the graduated cylinder and poured 500 mL of distilled water from the graduated cylinder into the Erlenmeyer flask. I added magnetic stir bar to the flasks and placed flasks on the hot stir plates. At the same time, I pour 20 grams TSA into each flask and turned on the stir plate selection for mixing Agar and heat selection for boiling solution. I continued heating and string for dissolving Agar. It took approximately 15 minutes to all Agar was dissolved.

I covered each flask with a piece of foil and cut off two strips of autoclave tape and place the tape around flasks neck. The tape should hold the foil and flask together.

I wore the gloves and placed my flasks into the metal autoclave plate. Then, I put the flasks in the chamber and close the autoclave door. For autoclaving the TSA, I chose the media and then I hit the start. It took around 45-50 minutes to autoclave TSA. When autoclave finished, I took out my flasks and placed them into a water bath at 60 ºC for cooling down about 30 minutes. After that, I set up the biological safety cabinet to pure the solution into petri plates. At the end I placed my plates into incubator to use them later.

Isolation of luminescent Bacteria from Squid

My first project in the lab was isolation of luminescent bacteria from squid, I’m going to share my experiment and observation. But before doing the experiment I had a question that “what is Bioluminescent Bacteria?” I found out they are the bacteria that produce light and they are found in seawater, in marine animal’s guts and on the surface of decomposing fish, and in the specialized organs of squid fish that provide bioluminescent bacteria as a safe place to live and a source of food.

In order to isolate the bacteria from squid I used two different sources. The first method required a 3.0% NaCl solution, by mixing 30 gr NaCl into 1 Liter distilled water. Then I placed the squid in a container and add enough 3.0% NaCl solution to the container to almost cover the squid. i then place the container in a refrigerator 16 degrees C for 24 hours. After 24 hours I checked the squid for luminescent bacteria by gently agitating the water in a dark room.

The second method was the dissect the squid and collect the ink directly from the squid. I needed to collect the ink from behind the eyes and from the sack in the body. First I cut the gut without cutting the ink sack inside the body, and separate the sack carefully from the body and collected the ink on the palate. Then I cut the tentacles away from the eyes and harvest the ink from behind the eyes. Then after collecting all the ink just added enough 3.0% NaCl solution to a container and incubated it at 18-20 degrees C.

Here is the link that shows clearly how to harvest the squid ink: