Plate Pouring Protocol

00:08:50
https://www.youtube.com/watch?v=ey19jM6y7-c

摘要

TLDRThis video tutorial guides viewers on preparing LB agar plates, essential for culturing bacterial colonies or isolating populations from mixtures. The process begins by gathering materials including LB agar powder, sterile water, and antibiotics. Measurements are crucial, with specific grams of powder allocated per liter to achieve ideal agar concentration. The preparation involves mixing the powder with water, labeling with autoclave tape, and sterilizing in an autoclave at 121°C. Post-autoclave, the agar is cooled in a water bath to ensure antibiotics remain effective. Detailed instructions cover pouring techniques, ensuring sterility, and conducting antibiotic checks using known resistant strains. Emphasis is placed on maintaining hygiene and handling protocols before storing plates under optimal conditions.

心得

  • 🧫 LB agar plates are essential for microbial culture.
  • 🧪 Use premixed agar powder for efficiency.
  • 🌡️ Autoclaving is crucial for sterilization.
  • 🔍 Proper labeling prevents mix-ups.
  • 🧬 Antibiotics require careful dilution.
  • 🔥 A flame maintains a sterile environment during pouring.
  • 💧 Cool agar to 60°C before adding antibiotics.
  • 🧤 Use protective gloves when handling hot items.
  • 🕒 Allow plates to dry overnight for best results.
  • ❄️ Store finished plates at 4°C to prolong shelf life.

时间轴

  • 00:00:00 - 00:08:50

    This video provides a detailed guide on making LB agar plates, commonly used for culturing bacterial colonies. First, gather materials: LB agar powder (37g per liter), sterile water, antibiotics at 1000x concentration, glassware, disposable pipettes, and autoclave tape. Measure out 8.14g of powder for 220ml of gel mix. Mix powder with water in a bottle, ensuring air isn't sealed to avoid cracking during autoclaving.\n\nAutoclave the mixture at 121°C for 20 minutes to sterilize. Prepare the bench by cleaning with ethanol. Count and label plates, remove antibiotics from freezer. Set a 60°C water bath to cool the autoclaved mix, preventing the denaturation of antibiotics when added. Once sterilized and cooled, add antibiotic and pour plates, starting with a pipette to measure accurately. Allow plates to solidify at room temperature and dry overnight. Store at 4°C, check for contamination, and test antibiotic efficacy before use. Conclude by noting the importance of isolating individual colonies as isogenic populations for experiments.

思维导图

视频问答

  • What is the purpose of LB agar plates?

    LB agar plates are used to culture bacterial colonies and to select specific populations from complex mixtures.

  • What are the main ingredients in LB agar powder?

    The LB agar powder typically contains peptone, sodium chloride, and agar.

  • Why is it important to not seal the bottle airtight before autoclaving?

    Not sealing the bottle airtight prevents cracking during the sterilization process.

  • How do you test the antibiotic effectiveness on LB agar plates?

    Test using two strains: one resistant and one non-resistant to the antibiotic. Only the resistant strain should grow if the plates are prepared correctly.

  • What should you do if the LB agar solidifies before pouring all plates?

    If solidified, either remake the gel mix or re-liquefy using an autoclave or microwave, taking care not to overboil.

  • Why do you use autoclave tape on the bottle?

    Autoclave tape indicates that sterilization temperature has been achieved by darkening after 10 minutes at 121°C.

  • Why should plates be labeled on the bottom instead of the top?

    Labeling the bottom prevents misidentification if the lid is switched or upside down.

  • Why is it necessary to cool the agar to 60°C before adding antibiotics?

    60°C is cool enough to prevent antibiotic denaturation while keeping the agar liquid.

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  • 00:00:03
    [Music]
  • 00:00:05
    lb auger plates are commonly used to
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    culture individual bacterial colonies
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    from freezer stocks or to select
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    individual populations from complicated
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    bacterial mixtures in this video we'll
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    walk you through the steps for making lb
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    auger plates we'll show you all the
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    materials you'll need for the process
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    show you how to sterilize your plates
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    indicate best practices during plate
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    pouring and will demonstrate how to test
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    your plates by the end of this video you
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    should be able to prepare LV auger
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    plates with your antibiotic of
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    interest to begin the plate pouring
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    process we'll gather all of our
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    necessary materials first we have our lb
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    augur powder for every liter of molten
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    augur that we make we'll measure out 37
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    G of premixed lb AER powder consisting
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    of 5 G of peptone 10 G of peptone from
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    casin 10 G of sodium chloride and 12 G
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    of Ager AER these items can be bought
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    premixed or you can buy each separately
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    we prefer to buy them premix to save
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    time this composition will make 1.2%
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    augur plates next we have our sterile
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    water our goal is to make 30 gels in 60
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    mm x 15 mm plates Each of which can hold
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    roughly 5 to 7 Ms of lb augur on plates
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    this size you can easily distinguish
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    about 100 individual colonies if you're
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    going to be doing high volume screening
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    we recommend larger plates for the 30
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    plates we only need roughly 200 Ms of
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    water but just in case we spill a little
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    along the way we'll add 220 Ms to start
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    because we'll be using these plates for
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    selection we'll need antibiotic we keep
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    our antibiotics as stock Solutions at
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    1000x concentration and dilute them as
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    necessary next comes our glass wear and
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    disposables we will make the 220 Ms of
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    molten lb AER in a 500 mil bottle your
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    bottle should always have plenty of
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    extra volume so your lb AER doesn't boil
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    over in the autoclave we will use the
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    sterile pipets to Alo quat the gel mix
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    finally we'll use the autoclave tape to
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    label our
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    bottle now that we have everything ready
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    it's time to start making our plates the
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    first step is to measure out the elbag
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    powder it takes 37 G of powder to make 1
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    liter of gel mix since we only need 220
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    Ms of gel mix we will measure out 8.14 G
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    of elbag powder now we add the elbag
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    powder and the sterile water to the
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    bottle and swirl to form a colloid do
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    not make the seal airtight an airtight
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    seal can lead to cracking during the
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    sterilization process next we add a
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    piece of autoclave tape to the bottle
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    autoclave tape darkens when it has been
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    at least 10 minutes at 121° C in the
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    autoclave and lets us know our lb augur
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    has reached the appropriate
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    sterilization temperature we also add a
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    piece of lab tape to the bottle with our
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    initials the date and the bottle
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    contents this will clear up any
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    confusion should we accidentally leave
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    our bottle in the autoclave or if
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    somebody opens the autoclave before we
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    do autoclaves are used to bring
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    materials and media to high temperature
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    at high pressure the high temperature
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    steril izes our materials while the high
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    pressure keeps liquids from boiling over
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    some organisms and certain spores can
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    however survive in the harsh conditions
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    of the autoclave be sure to check the
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    literature for the appropriate
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    sterilization techniques if you're
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    working with any weird and wonderful
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    organisms to begin we place our lb aoid
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    in the autoclave and seal the door we
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    then set the autoclave to reach 121° C
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    under 20 psi for 20 minutes and start
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    the autoclave cycle do not use the
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    autoclave if you have not been prop L
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    [Music]
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    trained well the elbag sterilizing in
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    the autoclave we will prepare our bench
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    for plate pouring first we find or make
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    an empty section of bench with a working
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    flame then we spray down the bench with
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    70% ethanol and wipe it with a paper
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    towel once the bench is clean we count
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    out the appropriate number of plates and
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    leave them stacked in an easily
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    accessible location be careful not to
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    stack them too high lest we have an
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    architectural
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    disaster now we label our plates with
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    the medium and the antibiotic they will
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    contain we recommend using a batch
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    labeling system to speed up this process
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    for instance we simply label our bags
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    instead of our plates if you label your
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    plates in more detail be sure to write
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    on the bottom of the plates instead of
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    the
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    top next we take our antibiotic out of
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    the freezer we keep our antibiotics
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    stored as 1000x stock Solutions and
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    dilute these as necessary most
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    antibiotics can be stored in water but
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    some like Chlor fenicol must be stored
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    in other solvents like ethanol check
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    with the antibiotics manufacturer for
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    further
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    details we next set a water bath to 60°
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    C making sure that the water bath has
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    enough water to submerge about 75% of
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    our molten lb AER bottle we will cool
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    the lb AER mix in the water bath once
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    the autoclave cycle is complete 60° C is
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    a good temperature because it's warm
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    enough for the lb auger to remain in
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    liquid form yet cool enough that most
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    antibiotics will not
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    denat once the autocave cycle is
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    complete it's time to retrieve our
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    bottle of molten lb augur when handling
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    anything coming out of the hot autoclave
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    you should always wear thermally
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    protective gloves before we retrieve our
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    augur it's good practice to leave the
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    autoclave door open just a crack for
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    about 10 minutes this will allow any
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    excess steam to escape and will cool our
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    sample slightly after 10 minutes we
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    fully open the autoclave remove our
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    samples and leave the autoclave door
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    open just a crack as a courtesy to the
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    next user next we place the bottle with
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    the molten lb Aug mix into the 60° C
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    water bath submerging about 75% of the
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    bottle be careful not to let any of the
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    water bath water touch the lip or the
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    top of the bottle as this water is not
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    likely to be sterile after at least 5
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    minutes we remove our molten gel mix
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    from the water bath as a good rule of
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    thumb if you can hold the bottle with
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    lab gloves it's likely cool enough to
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    add antibiotic you can use a laser
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    thermometer for a more accurate
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    temperature
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    [Music]
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    measurement to begin the plate pouring
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    process we light the flame at our gel
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    pouring station and dilute our
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    antibiotic into the 60° C augur mix
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    we're adding 220 microl because we're
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    diluting our antibiotic 1 to 1,000 into
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    220 mL of augur once the antibiotic is
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    added it's time to start pouring our
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    plates it's usually faster easier and
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    more sterile to pour the molten gel mix
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    straight from the bottle however it's
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    good practice to measure out the first
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    couple of plates using a sterile pipet
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    until you get a good feel for the
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    appropriate volume don't worry if you
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    overfill or spill a little that's why we
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    made 220 mL of alagar instead of 200
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    you'll get the hang of it and before you
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    know it you'll be a plate pouring Pro as
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    you pour your plates be careful to recap
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    them quickly since that little flame
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    certainly won't kill everything that
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    could fall on them we stack our plates
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    in piles of about five as we pour make
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    sure you swirl each plate after you pour
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    it if your elb augur partially
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    solidifies you should stop pouring and
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    remake the gel mix if you're making
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    plates without any antibiotic you can
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    alternatively reify the lb augur by
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    running it through the autoclave again
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    or by microwaving if you microwave
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    beware of
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    overboiling it takes roughly 30 minutes
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    for our plates to solidify at room
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    temperature however we leave them out
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    overnight to allow them to dry
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    once dry we place the plates in a
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    plastic bag with an absorbent material
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    to reduce
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    condensation for long-term storage we
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    keep our plates in the cold room at 4°
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    C even when stored at this temperature
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    plates should always be checked for
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    contamination prior to use and should
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    never be stored for more than a
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    month once our plates have solidified
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    and dried we test them to make sure the
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    antibiotic functions properly to do so
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    we pull out two plates on the first
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    plate we we streak out a strain that we
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    know to be resistant to the antibiotic
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    on a second plate we streak out a strain
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    that's not resistant to the antibiotic
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    we then incubate both plates overnight
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    at the appropriate growth temperature if
  • 00:08:11
    we prepared our plates properly only the
  • 00:08:14
    resistance strain should
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    grow if only the resistance strain grows
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    our plates are ready for use or storage
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    at 4°
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    C isolating individual colonies on
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    plates is a crucial step in many
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    experiments because colonies arise from
  • 00:08:32
    single bacteria and are considered
  • 00:08:34
    isogenic thank you for watching our
  • 00:08:36
    plate pouring protocol video if you'd
  • 00:08:38
    like to watch more videos or browse more
  • 00:08:40
    written molecular biology protocols
  • 00:08:43
    please visit our website and blog agene
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标签
  • LB agar
  • bacterial culture
  • antibiotics
  • autoclave
  • sterilization
  • plate pouring
  • microbiology