Lab 8:   UV irradiation experiment

Mutations can change the sequence of bases in the DNA.  It can also harm the DNA in other ways.  Although, in some cases, these mutations may not have an effect, in many other situations, this change can be harmful to the bacteria.  There are a variety of chemicals, environmental factors and energy that can cause mutations.  In this lab, we will be looking at the effect of radiation on causing mutations in the DNA.

If radiation has high energy, then it will have a short wavelength.  If you look at the figure below, you will see that X-rays and Gamma Rays fall under this category.  This type of radiation is called ionizing radiation.  It is used to sterilize medical supplies and in food preservation.  In this laboratory, we will be studying non ionization radiation which has a longer wavelength but less energy than ionizing radiation.  UV radiation is an example of this second type of radiation.  It is much less penetrating than ionizing radiation and therefore, direct exposure of the organism to UV radiation is required. 

This picture is taken from Microbiology:  An introduction by Tortora, Funke and Case, 2007

The mechanism whereby UV radiation causes problems is by the creation of thymine dimers in the DNA.  This means that two thymine residues that are next to each will bind to each other.  This binding will inhibit DNA replication since the DNA polymerase will not be able to read this section of the DNA. 

In this experiment, each student will work individually.  Each student will be assigned a time and an organism (shown in the table below).  You will be going to the hoods in groups.  If you are assigned the shorter time, you will go to the hood first.

You will learn in this laboratory that UV radiation cannot pass through the lid of Petri dishes or even through an index card.  Therefore, unless you are assigned the last condition (15 minutes with the Petri dish lid on), you must REMOVE THE PETRI DISH LID or your experiment will not work

UV exposure Time

UV lab protocol

Each person will take one plate and their designated organism.

You then want to completely swab the organism over the entire plate.

When your instructor tells you that it is time to go to the hood, you will take your plate and bring it to the hood.  You will take the cover off (unless you were assigned to have the Petri dish lid on) when you have the plate under the hood as shown below.  You do not want to contaminate the plates.

Then you will put an index card over half of the plate. 

You will then leave the plate under the UV lamp for your assigned time.  Make sure that your plate is UNDER the UV lamp so that the organisms are exposed to the UV light. 

Make sure to mark which side of the plate was covered with the index card.

Important:  make sure that your plate is completely under the UV lamp.  Other students will have their plates under the UV lamp at the same time.  But, if there is not enough room for your plate, then you must wait.

You want your plate to be as close to the UV lamp as possible.

Safety precaution:  Do not look directly into the UV light as it can cause damage to your eyes.  Do not expose your direct skin to the UV either.  Wear gloves.

After your plate has been exposed to the UV light, discard the index card, cover the plate and incubate it at 37 degrees C for 24 hours.

In the next lab period, you will observe the plates.

Expected Results

Which side of this plate was covered with an index card?

SA

Most sensitive to UV

More organisms will die

BS

Most resistant to UV

More organisms will survive

Forms endospores

With increasing time, we should get less organisms surviving.