Growing Bacteria

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Steps to grow bacteria safely in a classroom laboratory.

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  • If bacteria dry out, they may die or just stop reproducing. If they get too crowded in a culture, or on a plate, they stop reproducing and start to die. Bacteria do not usually die when they are cold, but they do reproduce much more slowly. If you want to keep a bacterial culture for a long time, they can be preserved by being frozen. Too much heat will kill them, though. In fact, we use heat to sterilize equipment. The energy source and nutrients vary by the type of bacteria you are trying to grow. Glucose is the sugar most bacteria can eat and is usually the one used.
  • There are different types of nutrient agar, the type you choose will depend on the kind of bacteria you are growing. Many pathogenic (or disease causing) bacteria are grown on blood agar. Cultures from many genetically modified strains are resistant to weak antibiotics, so they are grown on plates with these antibiotics, to keep unwanted bacteria from contaminating the plates. I have grown yogurt bacteria on plates that only had agar, sterile water and skim milk power in the plate – so only bacteria who could live on lactose sugar would grow.
  • The incubator is needed to keep the bacteria warm and in the dark. The nutrient agar can be ordered already poured into plates, or you can buy agar that must be melted and poured into plates, or you can make your own from dried ingredients. Just remember to keep everything as sterile as possible as you work, using sterile technique. If you make your own agar, it may not be as sterile as store-bought, unless you have the ability to heat it in an autoclave.
  • To sterilize equipment, you must heat it with a flame, or autoclave it. An autoclave heats equipment to a high temperature and pressure for 20 minutes using steam. This kills the vast majority of organisms that might contaminate your sample.
  • Growing Bacteria

    1. 1. Growing Bacteria Goal 11.04
    2. 2. What do bacteria need to grow? • • • • Moisture Space A comfortable temperature (warm) An energy source and nutrients – – sugars (glucose and others), or – sunlight (for some types) – chemicals (for the Archaebacteria)
    3. 3. How do we provide what they need to grow? • Incubator – similar to an oven, can be kept at a nice warm temperature, around 32 C. • Nutrient Agar or Broth – contains energy source – other nutrients – moisture – certain antibiotics sometimes
    4. 4. Equipment needed: Incubator Nutrient Agar or Broth Image from Shelly Scientific www.shellyscientific.com - GI6 SHEL LAB Digital Laboratory Incubator Images from Connecticut Valley Biological www.connecticutvalleybiological.com Tryptic Soy Agar Plates, pkg of 10 Nutrient Broth 150 mL
    5. 5. How do we get the bacteria? • Environmental sampling: Take a very small part of the environment and streak it onto a plate or blend a sample and dilute it, and add the diluted sample to the plate. • Mother culture: Growing bacteria of a certain type. Bacteria of certain types can be ordered from science supply companies.
    6. 6. Dilution Start with the item in question: Liquefy it: This is your sample:
    7. 7. Dilution From your sample: Fill a sterile tube with 50% sample and 50% sterile broth: Repeat: And keep repeating until you reach the dilution you want: This is necessary because the original sample may contain billions of bacteria and each of these can become a colony. If you want to be able to tell what kind of bacteria are present, then you need to have only a few colonies.
    8. 8. Sterile Technique Bacteria are everywhere and can fall into containers from the air and from you. 1. Sterilize surfaces with 70% alcohol. 2. Wear gloves. Don’t touch your face or surfaces. 3. Keep everything closed until you need to use it. 4. Tilt open containers, and keep lids off of the counter. Hold them , without touching the inside. 5. Use sterile equipment, or run the loop, or glass spreader through a flame or dip in alcohol between uses.
    9. 9. Plating the bacteria Streaking a plate: 1. Take the swab or loop containing your sample. 2. Rub it gently back and forth on the top 1/3 of the plate, not overlapping the streak. 3. Take a sterile loop and drag it from the place you rubbed into the adjacent 1/3 of the plate. 4. Re-sterilize the loop and go from the part of the plate you just rubbed across to the last clean spot.
    10. 10. Plating the bacteria Spreading a plate: 1. Add a small amount of liquid sample to your plate, usually 2 - 3 drops, or 100150 uL. 2. If using a glass spreader, make sure it is sterile, and cool. Place the spreader just in front of the area with the drops, and smear them in a circular path around the plate. 3. An alternate is to place sterile glass beads on the plate and roll them around until you feel like they have covered the area of the plate.
    11. 11. Incubating the sample: After the sample has been added to the plate, we say the plate has been inoculated. The plate should be labeled on the bottom with the date, initials of the person who plated the bacteria and sample name. The next step is to keep the plate in a dark, warm place for 24 to 48 hours. This step is called incubation. The next part of this lesson will discuss what we do with the bacteria once we have grown them.

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