Growing Bacteria in a Lab: Experiments & Conditions

Despite the fact that bacteria are growing all around you, they can be surprisingly di cult to grow in a lab. This
lesson will explore some of the basics involved in culturing bacteria for experimentation.

Bacterial Coworkers
Mathematicians have to work with numbers. Marine biologists all get wet. When you decide to
pursue a career as a microbiologist, eventually you have to work with microorganisms. But,
performing experiments on living things far too small to see can be di cult. It is not as easy as
snorkeling to observe reef sh or putting white mice in cages. You have to plan and control every
aspect of your new pet bacteria's lives. At the same time, you have to diligently protect your
specimens from being contaminated by all the rogue bacteria that are all around you, covering
every surface, and colonizing your own body. In this lesson, we will hit on some of the basic aspects
of growing bacteria in a lab.

Culturing Vocabulary
One skill that every microbiologist has to master is how to properly grow bacteria without letting all
those environmental bacteria contaminate your work. But, let's get a few vocab words out of the
way rst. Bacterial culturing is a process of growing bacteria. Oftentimes, if you are trying to
identify a microbe causing a disease or investigating the populations in an environmental sample,
you begin with a mixed culture. A mixed culture is a bacterial culture made up of more than one
species of bacteria. You could have as few as two or as many as hundreds of species. It is your job
to take this mixed culture and isolate the one species of interest.

Mixed culture of bacteria

Notice above how many of the colonies look di erent? These are all di erent species. What you
want is to make that mixed culture into a pure culture. A pure culture is a bacterial culture made
up of only one species. Notice below that all of the growth looks the same.

Pure culture of bacteria

You want to have a pure culture so you know with certainty that any observations you make are a
result of that one species of interest and not some other unknown contaminant species. There are
many di erent techniques you can use to isolate a species, but most involve growing the bacteria
on or in media. So, what is media?

Growth Media
A medium (plural media) is a liquid or gel designed to support the growth of a bacterial culture.
The liquid or gel has to contain everything that the bacteria will need for growth and cell division.
Leaving out even one, seemingly insigni cant, ingredient from your medium can prevent all
growth. This can be a very frustrating problem, especially if you spent hours painstakingly setting
up your culture, only to see absolutely nothing growing. Today, most labs buy commercially
prepared dry media that contains all needed nutrients. All you have to do is add sterile water!

In addition to the nutrients and growth factors needed for life, there are many additional
compounds you can add to your media. For more information, see the lesson titled 'Di erential
and Selective Media in Microbiology.'

So, you want to make some media? First, you need to pour your dry media into a ask and add
sterile water. For some experiments, you can grow your cultures in this ask of liquid or use it to ll
several smaller culture tubes. If your experiment requires solid media, all you have to do is add
agar to the ask. Agar is a carbohydrate derived from marine algae that turns the liquid media into
a gel. As long as the agar/media mixture is hot, it remains a liquid. Once it cools, it hardens into a
suitable growth surface for bacteria. The hot agar can be poured into plates or into culture tubes.
Plates are one of the most common methods for growing bacteria. Solid culture tubes are
sometimes used for stab or slant cultures. Bacteria can be spread on the surface of the slant or
stabbed down into the gel, depending on what you need your culture to accomplish.
Growth Conditions
So, you have your bacteria and you have put it on your medium of choice, but that is not all you
need to know. Like you, bacteria require very speci c environmental conditions to survive. You
wouldn't survive very long in the freezer, sitting in stomach acid, or with no oxygen or food, and
neither will your bacteria. There are several key environmental parameters you need to set in order
to get the best growth rate for your bacteria.

Most bacteria require very moderate conditions. Anything too far on one extreme or the other will
be lethal. Some of the more common conditions you need to set include temperature, salt
concentration, pH, nutrients, and oxygen concentration. E. coli, one of the most commonly cultured
bacteria, is a normal inhabitant of your gastrointestinal tract. E. coli has evolved for thousands of
years to grow best inside your body, so it makes sense that you want to try and replicate these
conditions so your E. coli will grow at peak speed and e ciency.

Parameters like pH, salt concentration, and available nutrients are best set by adjusting the media.
You need to include the appropriate nutrients, add only the appropriate amount of salt, and use
bu ers to maintain the appropriate pH for your bacteria. Temperature and oxygen must be
controlled from the outside. Many bacterial cultures are placed in incubators or warm water baths
that ensure a constant temperature. Some bacteria like cool temperatures, while others like
warmer temperatures.

Oxygen concentration is often a bit trickier to control. Solid media in plates or slants will be able to
get enough oxygen from the air to support bacterial growth. If you're growing bacteria in a liquid,
the bacteria will quickly use up all the oxygen and die. Liquid tubes need to be shaken during
growth to allow oxygen to reach the entire volume, from top to bottom. But, not all bacteria can
survive in an oxygen-rich environment. These bacteria need to be sealed in containers that have
had the oxygen removed.

Keep in mind that it is very di cult to cookie-cutter bacterial growth conditions. There are so many
di erent bacterial species, media, and experimental goals that you really need to do your research
and customize your growth conditions for your needs. And, don't get frustrated if you fail. Despite
being everywhere and exploiting a huge range of conditions, when you actually want to grow
bacteria, it can be surprisingly di cult. And, of course, contamination is everywhere. Remember to
always be precise, take your time, and good things will grow eventually.

Lesson Summary
Every microbiologist eventually has to grow bacteria in the lab. Bacterial culturing is the process
of growing bacteria. Cultures can be mixed, containing multiple species, or pure, made up of only
one species.
Bacteria must be grown in a medium, which is a liquid or gel designed to support the growth of a
bacterial culture. The media must contain everything the bacteria need to survive and can be liquid
or solid. Agar is added to hot liquid media to make a gel used for culturing in plates, tubes, slants,
and stabs.

Bacterial growth also requires tightly controlled environmental conditions. Parameters such as pH,
temperature, oxygen concentration, salt concentration, and available nutrients must be speci cally
tailored to the bacterial species you are growing.

Remember, it is up to you to determine exactly what type of culture and the ideal environmental
conditions you will need. Every species of bacteria is unique, and what works well for one could be
lethal to many others.

Learning Outcomes
After you've completed this lesson, you might have the ability to:

Comprehend the process of bacterial culturing

Point to the di erences between pure and mixed cultures

Remark on the role of agar and media in bacterial culturing

Specify the environmental conditions that must be controlled when growing bacteria