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Lab 7: Sterilization

  • Page ID
    24020
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    UV Radiation:

    What effect does ultraviolet light exposure have on bacterial growth?

    Ionizing radiation (e.g. x-rays, gamma radiation) carries enough energy to remove electrons from molecules in a cell. When electrons are removed from molecules, free radicals are formed that damage the cell leading to DNA damage, mutations, and cell death. Non-ionizing radiation, such as ultraviolet (UV) light, excites electrons in molecules. The excitation of electrons in DNA molecules often results in the formation of extra bonds between adjacent pyrimidines (specifically thymine) in DNA. When two pyrimidines are bound together in this way, it is called a pyrimidine dimer. These dimers often change the shape of the DNA in the cell and cause problems during replication ultimately leading to cell death. Both ionizing and non-ionizing radiation are used to control the growth of microorganisms in clinical settings, the food industry, and in laboratories.

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    WHAT IS THE EFFECT OF UV LIGHT EXPOSURE ON 3 DIFFERENT SPECIES OF BACTERIA?

    1. Each table will be given one of the following bacterial species/stages:

    • Staphylococcus aureus
    • Serratia marcescens
    • Bacillus cereus (vegetative)
    • Bacillus cereus (spores)

    2. Working as a table, use a cotton swab to make a lawn on the following 8 plates:

    • no UV exposure (control)
    • 15 seconds
    • 30 seconds
    • 1 min
    • 3 min
    • 5 min
    • 10 min
    • 10 min with the plate fully covered with a lid

    3. Put your plates (with the half lids) under UV light for the appropriate exposure time. Make sure to mark on the bottom of your plate which half of the plate was exposed to UV light and incubate them at 37°C.

    Note

    UV light can burn your skin and eyes. Take proper precautions to protect yourself.

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    Steam Demo:

    How does steam sterilization work?

    Of the various methods we have developed to sterilize materials, steam is the most effective, common, and economical. The device used most commonly for this purpose is an autoclave, also known as a steam sterilizer. Autoclaves are relatively safe and easy to operate and extremely effective at killing microbial vegetative cells and spores. Similar to home pressure cookers, which create pressure and high temperatures to shorten cooking times, autoclaves use super-heated steam under pressure to kill microbes. Sterilizing temperatures are usually 121-127°C (250-260°F) for at least 15 minutes.

    We can test the effectiveness of an autoclave by using commercially available ampules (sealed glass capsules of bacterial spores). Bacterial spores are used because they are the most difficult to kill and therefore the highest measure of antimicrobial killing efficiency.

    If the bacteria live after sterilization, they produce acid which turns the indicator yellow. If the bacteria are dead the indicator will remain purple.

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    TESTING THE EFFICIENCY OF STEAM STERILIZATION:

    4. Make sure you look at the tubes, know what they look like AND what they mean:

    • Negative Control = ampule autoclaved → not incubated = no growth = purple color
    • Positive Control = ampule not autoclaved → incubated = growth = acid produced = yellow color
    • Sterilization success = ampule autoclaved → incubated = no growth = purple color
    Vial Placement of Vial Results (yellow or purple) Did the Spores Survive?
    A (-) control = autoclaved but not incubated
    B (+) control = not autoclaved, incubated at 55°C
    C Autoclaved then incubated at 55°C
    D Placed inside multiple containers, autoclaved, then incubated at 55°C

    Chemical

    How effective are chemical germicides?

    Chemical germicides should reduce the number of pathogens on a surface, in a liquid, or on/in living tissues. Germicides designed for use on surfaces are called disinfectants. Germicides designed for use on living tissues are called antiseptics. To test the effectiveness of a chemical against pathogens, glass beads are coated with bacteria, exposed to different concentrations of germicides, and finally transferred to a growth medium. After incubation, the growth rates are determined. The more effective the germicide, the less bacterial growth there will be.

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    HOW EFFECTIVE ARE FOUR DIFFERENT COMMON GERMICIDES?

    5. Each table will be assigned one of the following disinfectants, which they will then need to dilute to the following concentrations:

    • Bleach (1%, 0.1%, and 0.01%)
    • Bench-top disinfectant (100%, 50%, 25%)
    • Hydrogen peroxide (3%, 0.3%, 0.03%)
    • Alcohol (50%, 30%, 10%)

    6. One side of the table will use Escherichia coli and the other side will use Staphylococcus epidermidis.

    7. Each side of a table will need 5 empty Petri dishes, 7 TSB broth tubes, and 5-6 beads.

    It may be helpful to view the summary figure next page before proceeding.

    8. For this experiment you will have four controls, you can ASEPTICALLY prepare the first set now.

    • Negative control A = broth, uninoculated → to make sure broth isn’t contaminated
    • Positive control A = broth + one loopful of bacteria from your culture → to make sure bacterial culture provided is healthy and growing

    9. ASEPTICALLY pipette 10 ml of sterile water into SMALL empty, pre-labeled Petri dishes. Then, pipette 10 ml of each of your 3 dilutions into empty Petri dishes. Below is an example of what your dishes would look like if you had been assigned bleach.

    Pipette water first. Then, go from your lowest to highest dilution.

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    10. With sterile forceps drop 6 beads into your Escherichia coli or Staphylococcus epidermidis broth tube and wait 1 minute.

    11. Using a glass Pasteur pipette, remove the broth and discard into the beaker of disinfectant provided.

    12. Pour out the beads and the remaining volume of broth onto filter paper. With sterile forceps separate the beads, place the lid back on and allow to air dry for 10 minutes.

    13. Place one bead in each Petri dish of disinfectant and one water Petri dish. Wait 10 minutes.

    14. Into the fifth plate, place a sterile and uncoated bead.

    15. With sterile forceps remove the beads and place each bead in a broth tube. Mix thoroughly and incubate them at 37°C.

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    exercise

    If your bacteria is susceptible to bleach, can you guess what the expected results will be for each condition?

    Use the following symbols:

    • + = growth
    • +/- = little growth
    • ng = no growth

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    Results:

    For your UV plates draw your results below:

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    For your broth plates complete the table below using +/- symbols. This is a relative way of measuring growth. You will have to determine for yourself what is high growth and what is low growth but your controls should help.

    • +++ = high growth, very turbid
    • ++ = average growth, turbid
    • + = low growth, slightly turbid
    • +/- = almost no growth
    • ng = no growth
    Bleach Bench-top Disinfectant H2O2 Alcohol
    0.01% 0.10% 1% 25% 50% 100% 0.03% 0.30% 3% 10% 30% 50%
    E. coli
    S. epi

    Lab 7: Sterilization is shared under a CC BY-SA license and was authored, remixed, and/or curated by Nazzy Pakpour & Sharon Horgan.

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