Growing bacteria in vitro

Traditional methods for studying the growth and behavior of microbes rely on the ability to grow the organism as a monoculture; a monoculture contains one and only one type of organism. 

This is not always easy and in some cases, it may not even be possible.   Many organisms are very particular about their growth requirements and may be dependent upon products made by other organisms

It has been estimated that we know how to grow as monocultures only a small percentage of the prokaryotic species that exist in nature.

Methods have been developed to identify organisms within their normal habitat, without having to be able to grow them in the lab (or "in culture"). 

These techniques are useful in defining biological diversity and identifying previously unknown pathogens.

Here is an example of biofilm
click on it to learn more!

For E. coli, however, growth conditions are well known and very simple. 

E. coli is a chemoautotroph; it can synthesize all it needs to grow from water, simple inorganic chemicals and an energy source. It can extract the energy it needs through the oxidation of organic compounds.  

Organisms that capture the energy of light are known as photoauxotrophs, those that extract energy from inorganic compounds, known as chemolithoauxotrophs.  

Starting sterile: If you want to study the behavior of a single organism, you need to know that there are no other organisms in your system.  A sterile system contains no living organisms or viruses. 

To do that you must sterilize all of the instruments, containers and materials you plan to use.  

Laboratory containers used to be made of glass.  Glass containers can be sterilized by baking them at high temperatures;

In modern labs, plastic containers are more common.  Nevertheless, we still refer to organisms grown in the lab as grown in vitro, which means in glass.

Plastic containers are sterilized using chemicals or radiation.  Most "plastic ware" comes from the manufacturer sterile and is discarded after use

Growth media: Early on, scientists such as Redi, Spallanzani and Pasteur used simple solutions, such as beef broth or green infusions (tea), to grow microbes.  

They boiled their media (the plural of medium) to sterilize it.

Boiling does not kill all organisms, however – some thermophilic bacteria flourish at temperatures well above 100°C

Moreover, many organism generate spores, which can survive boiling, as well as drying, high levels of radiation, and other rather nasty conditions.  Spores are so hardy that it has been suggested that they can travel through interstellar space. 

A more effective method for sterilizing media is the use an autoclave, essentially a pressure cooker.

Increasing the pressure increases the temperatures that can be attained, without boiling.

In an autoclave, media is heated to 121°C at a pressure of 20 atmospheres.

Media that has been autoclaved will generally remain sterile as long as the container in which it is place is itself sterile and airtight. 

For simple media, autoclaving is the method of choice.   Some more complex nutrient broths contain molecules that are destroyed at the temperatures used in an autoclave.

These liquids can be sterilized by passing them through using special filters.  These filters have pore sizes so small (> 0.2 µm in diameter) that organisms and spores cannot pass through them.

These filters will not remove all bacterial viruses however, many of which are smaller than 0.2 µm. In fact viruses were first classified based on the fact that they can pass through such filters!

An E. coli can synthesize everything it needs from a single simple carbon and energy source and inorganic salts. This type of media is referred to as minimal media

For most routine studies, E. coli are grown in a rich broth, usually Luria-Bertani medium or LB for short.   In LB, it grows quite fast.

The growth of a bacterial culture is typically divided into three phases, known as lag, exponential and stationary phases.

During lag phase, the bacteria are adapting to their new environment.  In exponential phase, they grow as fast as possible.  As resources are exhausted and waste produces accumulate, growth slows and the culture enters stationary phase.

• If two organism depend upon one another to grow, how would you approach the problem of growing them as monocultures?
• Which molecules does E. coli not have to synthesize when grown in LB, compared to minimal media?
• Both types of media contain glucose, what is is used for?
• Why would the presence of other (unknown) organisms and viruses confuse studies on the growth a specific organism?
• Why did you let the autoclaved media cool before you add the organism you what to study to it?
• Some organisms, known as hyperthermophiles, grow at temperatures above 100ºC.  How is that possible?   What determines the upper temperature limit for survival?