Lab Blog Week 1

Introduction:

For the first week in a lab as grandiose as this, a solid introduction that covers what is expected of the student and outlines the structure for the semester is the first step to starting on track in the S-STEM train program. This past week, we learned common protocol and procedure in the lab bootcamp, overviewing what is to come for the semester. I was taught the common procedure for completing a gram stain, and how to make sense of the results. The procedure for creating media was also outlined to me along with the inoculation process. I also shadowed a project that tested the survival rates of e coli under direct UV radiation.

Method:

On the first day of training camp, as a class we discussed how to be prepared before entering the lab. It is important to always come in prepared with goggles, a notebook, and a pen. Make sure everything that can be done at home, such as research, is done at home. It is important to utilize all the lab time with things that can only be done in the lab. Email the lab technicians what supplies you will be needing 2-3 days before planning to use it, to give ample time for preparation. For biological disposal, pour into the large glass jar, and place dish in the bin, ensuring all glass flasks are labelled correctly with initial and date. It’s important to label everything with what it is, its date, and initials, especially for chemical disposal.

Pipettes are used in the lab for incredibly small measurements and measures in microliters. The blue pipette is the largest, with a range of 200-1000 microliters. The pipette should always be kept upright. The first ‘stop’ is the indicated volume the pipette is set to. It is important to go slow when lifting samples, remain in a stable position when collecting, and to avoid touching the sides of the container with the pipette. Always ensure the tip is sterile, paying attention to any air bubbles.

On the next day of lab boot camp, we went over lab blogs, and how they should be structured. Included should be an introduction of the week, that gives background information as well as the papers being used, if any. It also includes a method section, results section, and a discussion section, which can be used for possible flaws in experimental design, as well as what the plan is for the continuing week. We discussed the process of experimental design, and how it all begins with research. By finding a gap in knowledge, we can form a question. When forming a design, one must think of all controls and how to keep them constant. It’s important to keep track of all the independent and dependent variables, to make sure anything that could disrupt data is being held constant.

Make sure every single move or step is recorded, so that they can be replicated over and over.

A gram stain is the process of dying the bacteria with crystal violet, to see it and determine certain properties. To begin, clean the area that is about to be used. Prepare one stain plate and turn on Bunsen burner. Run inoculation loop through flame until it glows red across, to ensure sterilization. Wait for it to cool then collect sample. Place onto the stain plate within the circle. Take the stain plate and pass over flames gently and patiently, to slowly have the liquid sample engrain onto the plate without having the flame kill the life. Once dried on, move to rack on the sink, and marinate entire sample with crystal violet for one minute. Use DI water to rinse off dye, keeping stream off to the side, not direct onto sample. Saturate sample with grams iodine for one minute and repeat DI rinse process. Saturate sample with ethanol, to allow the dye to permeate the cell walls. Wait ten seconds before rinsing with DI water. Finally, saturate sample with safranin for 45 seconds. After rinsing with DI water, use blotting paper to remove any access moisture.

The making of broth is referring to making liquid media, which is used to grow bacteria. To make liquid media, select a flask that isn’t too large, yet can contain all of the desired volume, bearing in mind that any sample should not go past half the flask’s volume. Once a flash has been selected, fill it with some DI water initially. Media is made with TGY: a 3-1-3 ratio of tryptone(3g), glucose (1g), and yeast(3g), per liter. To find desired values of TGY, divide the gram-ratio values by whatever value was used to split up the one liter, into the desired value amount of liquid media. After calculations have been made, carefully measure out each ingredient to the calculated increment using the scale. Add into previous graduated cylinder, then once entire ratio is added, take another graduated cylinder filled with DI water, and use to pour carefully up to volume. Label with initial, date, volume, and what it is.

I also got to work on the Activated Methyl Cycle project, which aims at pinpointing how durable E. coli is when exposed to UV radiation at different durations, and microjoules. We planned on testing seven different combinations of exposure rates and durations. We started by filling up seven Eppendorf tubes with 900 microliters of LB broth. We labeled seven pieces of paper and placed 100 microliters of the E. coli sample, after it had been Nano-dropped and gram-stained, onto its surface. Using the Ultraviolet Crosslinker, we subjected each sample the radiation as so: Treatment 1 was exposed to 70 microjoules/cm2 for 1 minute, Treatment 2 was exposed to 70 microjoules/cm2 for 2 minutes, Treatment 3 was exposed to 60 microjoules/cm2 for 1 minute, Treatment 4 was exposed to 60 microjoules/cm2 for 2 minutes, Treatment 5 was exposed to 50 microjoules/cm2 for 1 minute, Treatment 6 was exposed to 50 microjoules/cm2 for 2

minutes, and Control was exposed to 90 microjoules/cm2 at 2 minutes (This is what is documented to be the strength and duration of ultraviolet radiation to kill E. Coli). After the timer beeped for each sample, we used the pipette to pick back up each sample and place it back into their respected Eppendorf tube. After distributing each treatment into their tube of LB broth, we used a pipette to collect 100 mL of a solution and dropped it into the middle of an Agar plate. We repeated this procedure with all solutions. We then used inoculation procedures to sterilize a tool, putting it over the Bunsen burner for 30 seconds. Before using the tool to swirl the solution in the agar plate, we test to see if the tool sizzles first in the corner of the plate, to prevent killing the sample. Swirl tool around dish, and repeat sterilization between each agar plate.

Results:

(Figure 1) Gram-staining helps to determine if bacteria is gram-positive or gram-negative, based on how it holds the crystal violet dye. It allows one to be able to see the shape of the bacteria. Figure shows the different shapes: bacillus, cocci, and tetrad.

I have not yet had a chance to look at the results of the different exposure and duration rates of ultraviolet radiation on the E. coli samples.

Discussion:

This past week included a lot of information on getting to know the lab. We outlined common procedures regarding disposal, labels, pipettes and microscopes. It is important to really understand these basic procedures of the lab, since we must encounter them every day. It also allows for the most success to happen, since the procedure prevents any unpreparedness or common mistakes from occurring. Some common mistakes made in lab can easily be prevented by keeping the workstation organized, and tidy. I also got to learn the procedure for completing a gram stain. I need to be sure to act with caution when

 

applying the different dyes at the end of the process, since the amount of time it sits on the sample is crucial to being able to view the sample with accuracy. For example, if the ethanol doesn’t stay on long enough, its possible the results can appear different since it didn’t fully coat the sample and thus couldn’t carry out its function of helping the dye permeate the cell wall. For creating media, ensure the calculations are always accurate when doing the conversions of the TGY ratio. A common way results can be tainted, is if each ingredient isn’t measured up accurately, which can result in a ratio of ingredients that is different from what is called for. I had a chance to see some of these procedures applied, when working on the activated methyl cycle project. Looking back on what was done, a place of possible error might have been during the inoculation process. It is important to let the tool cool down enough before swirling the agar plate, as to not kill off any more life, especially in an experiment like this, where their durability to live through certain conditions is what is being measured. In the future, I will test if my tool sizzled more gently, or wait longer for the tool to cool, to give more confidence that all the damage seen on sample is only from the ultraviolet radiation, and not the hot tool. Next week, my plan is to learn the function of many of the machines in the lab and learn of more common procedures that take place. I also plan on seeing the results of the e. coli and how they ended up doing under the different rates and exposure lengths of ultraviolet radiation.