Antibiotics in Action

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    Biology Activity
    Resistance in Bacteria

    Introduction

    Natural selection is a rather simple idea. In a group of organisms, let's a say a litter of newborn wolf cubs, there will be some variation from cub to cub even though all the cubs have the same mother and father. The differences are caused by random mutations of the wolves' DNA. Some of the changes may make the wolf cubs better hunters and better survivors, and some of the changes may make them poorer hunters and poorer survivors.
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    Introduction
    General Safety Guidelines
    Microbiology Safety Guidelines
    Procedure
    Data Analysis

    For example, one cub may be born with larger teeth. This wolf may be a good hunter and survive to pass the genes for larger teeth on to the next generation of wolves. Another wolf cub may have smaller teeth. This wolf may be a poorer hunter, and may not live long enough to reproduce. Then the genes for smaller teeth will not be passed on to the next generation of wolves. This means that in time, most wolves will have larger teeth. In fact, if you look in a wolf's mouth, you will see its teeth are very large, but we don't recommend you get too close to the mouth of any wild carnivore!

    Charles Darwin helped develop this idea in the 1800s. He studied ecosystems around the world and was intrigued by the way animals and plants always had just the right features to live in their own environments. In the Galapagos Islands off the western coast of South America, he found lots of different species of finches—small seed-eating birds. On every island he found the finches had different kinds of beaks. The finches of each island had beaks suited to breaking open the particular seeds that were found there. The variation of beak type clearly indicated an adaptation to different types of food available on the different islands.

    Natural selection doesn't just affect big animals like birds and wolves. It also affects microbes, and this is not always a good thing. Our use of antibiotics can sometimes cause the evolution of bacteria that resist antibiotics. This is how it works: There are many many bacteria in a person who suffers from a bacterial disease. Some of them will be more resistant to antibiotics than others, because of random mutations. If the patient is treated with antibiotics, the antibiotics may not kill all the bacteria, but only those bacteria that are most susceptible to the antibiotic. The most resistant bacteria are left behind. When the surviving bacteria reproduce, the genes for resistance will be passed on to the entire next generation of bacteria. In short, sometimes antibiotics only kill the weak bacteria, leaving the stronger nastier ones behind.

    This happens when a patient stops taking antibiotics before the doctor says to do so. This also happens when antibiotics are given for diseases that aren't caused by bacteria, such as colds and flu. For more about how bacteria become resistant to antibiotics, please read Bugs Fighting Back: Basics of Bacterial Resistance.

    The lab that follows helps to illustrate the operation of natural selection with a particular strain of bacteria, Escherichia coli (E. coli), that is exposed to ultraviolet radiation. Most E. coli are sensitive to ultraviolet (UV) light and die. UV light has a particular wavelength that can be absorbed by the genetic material, the DNA molecules, causing physical damage and inactivation of the various cellular (chemical) activities controlled by the DNA. However, some bacteria can survive the exposure to UV, depending on exposure time, because of mutations already present in their DNA. These resistant strains can pass along the genetic survival mechanism when they reproduce, by fission. You will be able to identify the surviving bacteria and transfer them to new growth environments. The progeny from the survivors will also be tested to see which bacteria inherited the ability to flourish when exposed to UV light. Reproduction in bacteria can occur in 20 minutes or so, and resistant colonies can be apparent over a 24-hour period.

    Materials and Apparatus

    • Stock culture of Escherichia coli from biological supply company
    • Nutrient broth medium (sterile): peptone/beef
    • Nutrient agar medium (sterile): peptone/beef
    • Agar plates with lids
    • Sterile cotton
    • UV source (15 watt, in fluorescent desk lamp)
    • UV goggles(or sunglasses approved for blocking UV)
    • Transfer loops
    • Flame source: Bunsen burner or alcohol lamp
    • Incubator, set to 38ºC
    • Stop watch
    • Marking pencils

    Procedure

    Please read Microbiology Safety Guidelines before attempting this activity. Following accepted procedures for sterile transfer of bacteria cultures of E. coli and inoculation of agar plates and broth tubes, prepare to set up a series of plates for growing E. coli that will be exposed to UV light. As with any experiment, it is important to have controls built into the exercise. If we have two types of controls—an agar plate that is NOT inoculated as well as a plate that is inoculated but NOT exposed to UV light—what is the purpose of each arrangement?

    Day 1 Procedure

    1. Mass 1.0 grams of a soil sample to be used as a source of microbial decomposers. Several different sources of soil should be used including forest soil and soil with a high clay content.

    2. Label one agar plate on the BOTTOM with your initials and the date (why the bottom and not the lid?). Inoculate the agar with a sample from the stock E. coli culture.

    3. Wearing approved safety goggles for pretection against ultraviolet radiation, place the inoculated plate under the UV light at a vertical distance of 15 cm from the light source and uncover the plate. Turn on the light for 60 seconds. (Note to teachers: There are a number of variables here including the distance of the plate from the UV source, the intensity of the light source, the exposure time, and the particular wavelength of the UV light. The experiment could be expanded to take into account these variables, particularly if a certain combination of factors eliminates all bacterial growth.)

    4. Turn off the light, replace the cover on the agar plate.

    5. Incubate the plate for 24 hours at 38ºC.

    Day 2 Procedure

    1. After 24 hours, remove the plate from the incubator and examine for signs of bacterial growth.

    2. Count and record the number of bacterial colonies.

    3. Obtain a tube of sterile nutrient broth that will be used to transfer samples of any colonies on the agar plate.

    4. Using sterile technique, transfer a sample from ONE colony on the plate to the tube containing sterile nutrient broth. After replacing the sterile cotton plug, LABEL your tube. Then gently roll the tube between your hands to mix the contents.

    5. Incubate the tube for 24 hours at 38ºC.

    Day 3 Procedure

    1. For this part of the experiment, you will determine if you have produced bacteria that are resistant to the effects of UV light. Your culture tube of broth from Day 2 will be the source of bacteria selected out by the exposure to UV light on Day 1.

    2. Obtain 5 nutrient agar plates. Label the bottom of each plate with your initials and the date. Then label 1 of the 5 plates with GMC that represents the growth medium control. This tube is NOT inoculated but will be incubated.

    3. For the remaining 4 plates, inoculate each from the test tube culture that you incubated on Day 2.

    4. Of the 4 inoculated plates, select 1 and label on the bottom, IC for inoculation control. This plate will NOT be exposed to UV light.

    5. Take the 3 remaining inoculated plates and label each 1 min, 2 min, or 3 min for the different UV exposure times. With your individual plates at 15 cm from the UV light source, remove the lid of each plate when ready to expose the agar to the UV for the specified time.

    6. Cover and incubate all 5 plates at 38ºC for 24 hours.

    Day 4 Procedure

    1. After 24 hours of incubation, examine the 5 plates for any signs of colony growth. Record the number of colonies on each plate.

    2. Collect class data and compare results.

    Data Analysis

    1. What do the results of the two controls, GMC (growth medium control), and IC (inoculation control), tell you? How do the results compare with what you would EXPECT ? Explain.

    2. If no colonies grew on the plates that were exposed to UV light but did grow on the IC plate, what would that mean?

    3. In this experiment, what is the natural selection force?

    4. For bacteria that survive, what factors would determine the rate of growth?

     

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