16: Germicides and UV Light

Learning Outcomes

By the end of this lab period you should be able to:

• Demonstrate familiarity with ways that microbial growth can be controlled in the environment.
• Articulate the limitations of different methods of microbial control.
• Describe features of organisms that are more resistant to control methods.

Introduction

One of the most important means of preventing the transfer of infectious organisms in healthcare (and at home, and in preparing food!) is the control of these organisms in the environment. This can be done by a variety of mechanisms some of which include the application of heat (pasteurization/incineration), chemicals (bleach, alcohol), radiation (UV light, gamma rays), and filtration. Today we are going to explore the effects of UV light as well as a variety of germicides on the growth of microorganisms.

UV Light

We are constantly being exposed to radiation - in fact, the light that you see, and the radiowaves that allow you to text your friends and family with your cell phone are all forms of radiation. This type of long-wavelength radiation is low frequency and low energy and cannot damage your cells (or microorganisms). However, some forms of radiation that have shorter wavelengths are higher energy and can be very damaging to cells. Ultraviolet (UV) light can cause serious burns, and lower wavelength radiation such as X-rays or gamma-rays can ionize molecules (ionizing radiation) resulting in massive cellular damage and death (Figure \(\PageIndex{1}\)).

UV light is frequently used as a way to control microbial growth in the environment. Researchers working with animal cells will often expose their lab benches/hoods to UV light before starting their experiments to reduce the number of bacteria in the environment and reduce the risk of contamination. In hospitals, UV light is being used to disinfect patient rooms and is being studied as a way to ensure safer indoor air as well.

Today we will expose E. coli and B. subtilis to UV light to see what effects it has on bacterial growth. How long do you need to expose either of these organisms to UV light in order to kill them? B. subtilis makes endospores, does this make a difference in the amount of exposure time required for disinfection?

Figure \(\PageIndex{1}\): The electromagnetic spectrum. Inductiveload, NASA, CC BY-SA 3.0, via Wikimedia Commons

Germicides

You encounter chemical germicides on a daily basis. Each time you wash your hands with soap, you are applying a low-level germicide to your skin. Do you use hand sanitizers? They contain alcohol, another chemical germicide. Do you add bleach to a load of clothes? Bleach is a strong disinfectant classified as a high-level germicide. Germicides are designed to reduce the levels of microorganisms on skin or surfaces to safe levels that will not cause disease. Some germicides are so strong that they can be used to sterilize surfaces or plasticware.

Germicides work by damaging microorganisms. They are frequently made from molecules that react with proteins and DNA, damaging them beyond repair and killing the cell. Some, like alcohol or soap, disrupt cellular plasma membranes. The effectiveness of a germicide is based on its mechanism of action, its concentration, and the time that the surface is exposed to the germicide. Other factors are also at play - including the number of microorganisms that are present, and the environment itself. It’s faster to disinfect a surface clean of visible debris than to disinfect a surface covered with vomit or feces (not to be too graphic!).

Today we will test four different germicides, each at three different concentrations against three different organisms. We will first coat small ceramic beads with a bacterial culture. We will then disinfect the beads by soaking them in the disinfectant solution. Then we will place the beads in fresh media to determine if any bacteria were still alive on the beads and able to grow (Figure \(\PageIndex{2}\)).

Figure \(\PageIndex{2}\): Schematic for setting up the germicides experiment.

Materials

UV light experiment

• UV safety glasses
• Short wavelength UV light source
• Cell spreaders and alcohol for flaming and sterile transfer pipettes
• Cardboard or black paper for shielding
• NA plates (two per lab partner pair)
• Bacillus subtilis 48-hour (or longer) broth culture
• E. coli 24-48 hour broth culture

Germicides experiment

• Bleach (0.01%, 1%, and 10%)
• Lysol® Brand II (0.01%, 1% and 10%)
• Hydrogen peroxide (0.01%, 1%, and 10%)
• Isopropyl (rubbing) alcohol (10%, 50%, 70%)
• Sterile ceramic beads
• Sterile 60 mm Petri dishes
• Sterile filter paper
• Sterile transfer pipettes
• Three tubes of nutrient broth per student pair
• Needle-nose forceps for manipulating the beads
• Alcohol wipes for sanitizing the forceps (or can be sanitized by dipping in alcohol and flaming)
• S. aureus 24-48 hour broth culture
• P. aeruginosa 24-48 hour broth culture
• E. coli 24-48 hour broth culture

Experiment

UV Light Day 1

1. Obtain two NA or TSA plates. Label the bottom of each with the name of the organism, your name, and the amount of time the organism will be exposed to UV light.
2. Use a sterile transfer pipette to place 2-3 drops of culture onto the plate and use the spread plate method to create a uniform inoculation. When incubated this should create a bacterial lawn.
3. Repeat with the second organism (E. coli).
4. Bring your plates to the UV light source and turn the source on using the SHORTEST wavelength option. Remove the lid (plastic absorbs UV light!) and cover ½ of the plate with black paper or cardboard. Expose the other half of the plate by leaving it directly under the light. Set the timer for the amount of exposure you were assigned.
5. When the timer goes off, remove the plate from the UV light, replace the lid, and incubate at 37^oC for 48 hours or 30^oC for 5 days.

UV light Day 2

1. Remove your plates from the incubator. Observe for growth. You should have confluent growth (a lawn) on the side NOT exposed to UV light).
2. On the other side, note the amount of growth. Your instructor may ask you to estimate the percentage of growth on the exposed side vs. the unexposed side. Alternatively, you could express the growth as “confluent growth”, “individual colonies” or “no growth”.
3. After recording your data, dispose of plates in the biohazard waste.

Germicides Day 1

1. Using sterile forceps (or forceps sanitized with alcohol wipes), place three sterile beads into a sterile microcentrifuge tube.
2. Use a sterile plastic transfer pipette to transfer 1 mL of a 24-48 hour culture of bacteria to the tube with the beads (your instructor will assign your group an organism to work with).
3. Allow the bacteria to thoroughly coat the beads (incubate for about 5 minutes).
4. Place a piece of sterile filter or bibulous paper into a sterile petri dish. Remove the beads using sanitized forceps or by decanting the tube. The filter paper should absorb excess bacteria.
5. Fill three new sterile microcentrifuge tubes with three different concentrations of disinfectant. You will be assigned ONE of the disinfectants to work with.

Table 1: Concentrations of Different Disinfectants for Testing

Disinfectant	Concentration 1	Concentration 2	Concentration 3
Bleach	0.1%	1%	10%
Lysol	0.1%	1%	10%
Hydrogen Peroxide	0.1%	1%	10%
Isopropyl (rubbing) alcohol	10%	50%	70%

6. Place one bead into each tube and allow the bead to be disinfected/sanitized for 5 minutes.
7. Remove the beads using forceps or by decanting onto a new, clean, and sterile piece of filter paper in a new, sterile petri dish.
8. Place each bead into a culture tube containing nutrient broth and incubate for 48 hours at 37oC.

Germicides Day 2

Compare each of your incubated tubes to an uninoculated control. Note where tubes are cloudy, indicating growth, and where they remain clear, indicating no growth. Record your results and share them with the class.

Data

UV Light

Compare your results with the class data. Your instructor may ask you to estimate the percentage of growth on the exposed side vs. the unexposed side. Alternatively, you could express the growth as “confluent growth”, “individual colonies” or “no growth”.

Table 2: Student Data Table for Collecting Class UV Light Results

	B. subtilis		E. coli
Exposure Time (minutes)	Unexposed	Exposed	Unexposed	Exposed
T=0
T=
T=
T=
T=
T=

Questions - UV Light

1. What was the purpose of the “unexposed” half of the plate?
2. This is not a quantitative exercise. Keeping this in mind, what is the general trend between bacterial death and UV exposure time?
3. Which organism survived the longest and why?
4. Why were you told to remove the plastic Petri plate lids before exposing the bacteria?

Germicides

Compare your results with the class data. Where there was growth, put a “+” or “G” symbol in the table. Where there was no growth, put a “-” or “NG” symbol in the table.

Table 3: Student Data Table for Collecting Class Germicides Data

	Bleach			Hydrogen Peroxide			Lysol			Isopropyl Alcohol
Organism	0.1%	1.0%	10%	0.1%	1.0%	10%	0.1%	1.0%	10%	10 %	50%	70%
P. aeruginosa
S. aureus
E. coli

Questions - Germicides

1. Which organism seemed to withstand disinfection best? Does this make sense based on what you know about the environment in which the organisms is typically found?
2. Which disinfectant performed the best overall?
3. Did any of the data not make sense? If so, what experiment would you design to better understand this data?