BACTERIA LAB

Purpose of Lab

The purpose of this lab is to define the effect of ultraviolet light on the amount of denatured bacteria in a sample. To find the best time and distance possible to most effectively use ultraviolet radiation to kill bacteria, our TSA engineering design team has run a lab to find the best dimensions, time under light, voltage, etc. for our prototype. The lab consisted of two tests: a test for distance of the source of ultraviolet rays from the bacteria sample (centimeters), and a test for how long (in minutes) the bacteria is subjected to ultraviolet radiation. The hypothesis for the first test was if the bacteria is closer to the source of UV light, more bacteria would be denatured. The second hypothesis was if the amount of time under UV light were to increase, then the amount of denatured bacteria cells would increase as well.

Outlined Procedure

Preparation:

Step 1: Prepare the petri dishes. Fill a container will 240 mL of hot water (1 cup). Mix in 60 mL of corn starch to make the solution for cell growth. Mix solution thoroughly. Pour 30 mL of corn starch solution into each petri dish.

Step 2: Prepare the human saliva. If using someone else’s saliva, wear plastic gloves and a mask. Fill a plastic cup with 60 mL of water. Take at least 5 cotton swabs and thoroughly wipe around mouth. Put the used ends of the cotton swabs inside the cup of water. Mix well and let the cotton swabs sit in the water for 10 minutes. Mix again after 10 minutes. Drop 10 mL of saliva solution into each corner of each petri dish (4 drops). Tape the petri dishes closed.

Step 3: Turn on the incubator. Fill a beaker with water and put the thermometer with it. Using the thermometer in water, make sure the incubator is at 95 degrees Fahrenheit (35 degrees Celsius). Leave the petri dishes inside the incubator for 24 hours.

Step 4: Find a material to suspend the UV light above where your petri dishes will be (our lab used the fischertechnik blocks to suspend the UV light, but other materials such as cardboard can be used). Tape back of the UV light to your material to suspend the UV light, and use the ruler to measure out the correct distances: 3 cm, 7.5 cm, and 12 cm.

Step 5: After 24 hours has passed, take the petri dishes out and wrap half the dish with tin foil. Take a sharpie marker and label each dish as follows: Time A (5 minutes), Time B (10 minutes), Time C (15 minutes). Do the same with the variable “Distance” with distances 3 cm, 7.5 cm, and 12 cm.

Lab:

Step 6: Put on the UV protection glasses, plastic gloves, and a mask. Put the “Time A” sample under the UV light 7.5 centimeters away from the bulb. Turn the UV light on for the time for the sample. For “Time A”s case, it would be 5 minutes.

Step 7: Turn off the UV light and remove the petri dish from under the bulb. Remove the foil and tape from the dish and use the sharpie marker to mark the side and bottom of the petri dish side that was covered in your foil. This foil side works as a control group.

Step 8: Get a plastic cup and fill it with a cup of water. Mix it together with 5 mL of Methylene blue microscope stain. This stain will make it easier to see the bacteria under the bacteria.

Step 9: Take a cotton swab and swipe it across the side of the petri dish that had foil over it. Wipe this cotton swab in a small circle on a microscope sample slide. Put a single drop of the stain solution onto the circle from the swab. Place a sample cover on the stain drop solution.

Step 10: Place this sample slide under a microscope. Take pictures of your sample under different magnifications (We used magnifications x10, x40, and x100). Taking pictures with the camera of a smart phone will suffice. Be sure to label pictures with their magnification, which petri dish they came from, and whether it was under foil or not.

Step 11: Repeat steps 9 and 10 for the side of “Time A” that was not covered in foil.

Step 12: Repeat steps 6 through 11 for samples “Time B” and “Time C” for their corresponding times under the UV light.

Step 13: Repeat steps 6 through 11 for the “Distance” samples and use a constant time of 5 minutes for each “Distance” sample.

Data Interpretation:

Step 14: Make observational notes of how the pictures of the bacteria look under foil vs. under direct ultraviolet radiation. Note the proportion of denatured cells including observations such as broken membranes, lack of DNA on insides, or bacteria being misshaped.

Step 15: Using a counting app, count the number of denatured and healthy bacteria for at least 1 picture from every petri dish sample. Compare the denatured and healthy bacteria numbers to find the proportion of these cells. Repeat for each petri dish sample.

Step 16: Make a stacked bar graph to compare to the proportion of healthy bacteria cells compared to those denatured. Also make a line graph using the denatured proportions to compare which distance and which time works the best, respectively. (Excel is an effective way make the graphs and visualize the data)

Microscope Images

Test Sample x40 Magnification Lens

Test Sample x100 Magnification Lens

The Effects of Time

It was found that the foil did its job at setting a control group for the lab. Since UV radiation only affects what it touches directly, the bacteria could not be denatured with a foil covering preventing light from touching it. The lowest proportion of healthy bacteria was “Time A” with proportion of 0.95. “Time A” also had the lowest proportion of denatured cells when put under direct UV light with a proportion of 0.56 denatured cells. “Time B” had a proportion of 0.83 denatured cells and “Time C” had a proportion of 0.845. The proportion of denatured cells increased by nearly 0.30, so the most significant change of denatured cell proportion was between 5 and 10 minutes. The change from 10 to 15 minutes (“Time B” to “Time C”) only had an increase of 0.015

It was found that 10 minutes would be the optimal time to hold air in an HVAC system under ultraviolet radiation. It had the most significant difference in time as the difference between 5 minutes and 10 minutes as the proportion of denatured bacteria had increased by 48%. “Time C” had a larger proportion of denatured bacteria cells than “Time B”, but the difference was less than 2% which isn’t enough of a difference to reasonably spend an extra 5 minutes powering our UV prototype. So, when building our prototype and implanting it into the current HVAC system, the contaminated air would only need to be exposed to UV lights for 10 minutes to kill off bacteria efficiently and effectively. The hypothesis that more time would denature more bacteria was correct.

The Effects of Distance

The relationship between distance and denatured cell proportion was not as easy to see. “Distance A” had a denatured bacteria proportion of 0.97 denatured bacteria and “Distance C” had denatured every bacteria cell. Distance B was between both distances but had only denatured 53% of total bacteria cells.

The nonlinear relationship between distance and denatured bacteria. As “Distance A” and “Distance C” have nearly the same proportion of denatured cells, it can possibly be assumed that distance at this scale does not make a difference in denatured cell count.

However, since “Distance B” drops to nearly half of the other distance, the relationship between distance and denatured bacteria proportion becomes hard to read.

The optimal distance would have a different process to determine said distance. The lab’s purpose was to find the relationship between distance and proportion of denatured bacteria and then the centimeters used in the lab would help determine the scale our prototype would need to be in feet. But as seen in the Analysis section, no relationship was found. If we were to go strictly off the data from “Distance C” and “Distance A”, the distance the bacteria is from the UV would not matter, as the prototype will have the bacteria in close quarters with the UV light. The hypothesis that a closer distance would make for more denatured bacteria was incorrect.