This document outlines the requirements for growing bacteria, including moisture, space, a warm temperature, and an energy source or nutrients. It describes using an incubator set to 32°C and nutrient agar or broth to provide these conditions. Environmental samples or mother cultures can be used to introduce bacteria. Samples are diluted to avoid overgrowth and allow colonies to be counted. Sterile technique is important to avoid contamination. Bacteria are grown using streaking or spreading samples on agar plates, which are incubated for 24-48 hours to allow colonies to form.
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. 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. 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. 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.
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. 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. 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. 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. 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.
Editor's Notes
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.
