Starting a pure culture

Bacteria can, and often are, isolated from natural environments.   Since most bacteria reproduce asexually, a single cell will produce a clonal strain of E. coli. 

Different strains can have quite evolutionary histories, and so different behaviors. For example, strains that have been maintained in culture for many decades are generally quite different from strains recently isolated "from the wild", they have evolved and adapted to their laboratory environment.

We will use a well characterized strain of E. coli, K-12 MG1655.  Specific strains can be obtained from the American Type Culture Collection (ATCC) or the European Culture Collection Organization (ECCO) - both act as libraries for biological organisms.  

ATCC and ECCO supply microbial strains only to certified researchers, and it is illegal to redistribute them.

Even stocks from ATTC and ECCO can display heterogeneity.  To insure that we start our experiments with as homogeneous a population of E. coli as possible, we will isolate a single clone of cells.

Our clone will be a substrain of the original strain. It may well be somewhat different from substrains used in other labs.

To establish our substrain, we need to isolate individual bacterial cells. This is done on a physical surface, an agar-coated petri dish. Sterile petri dishes can be bought in a variety of sizes from scientific supply companies.

 
Making Agar Plates

Agar plates are made by adding agar-agar (a powder - 15 g/liter) to LB media. Agar is derived from seaweed and is a common ingredient in Chinese cooking.

Like gelatin (derived from animals), agar is used as a thickening agent.

Agar is composed of two polysaccharides: agarose and agaropectin. These molecules indigestible by most bacteria. The bacteria get their nutrients from the LB.


 

Autoclaving dissolves the agar and sterilizes the solution.

We now let the solution cool the solution to  approximately 60°C. LB-agar is liquid at this temperature and can be poured into sterile petri dishes.

As it cools, the agar sets to form a solid, transparent gel. Once set, it will not melt until heated above 65°C.
Send in the clones!

We have received lyophilized (freeze-dried) K-12 MG1655 E. coli cells in the mail from ATCC.

They came sealed in a glass vial.

Each vial contains ~109 bacteria, but we want only one!

How do we get it?

  
 
The technique is simple.   Where the colonies are well separated from one another it is likely, although not certain, that each is a clone, a population derived from a single "founder" cell.
 

 

Pick a well isolated colony and use it inoculate 5 mls of sterile LB in a sterile culture tube.

We will use this culture for our experiments measurements.

Watch the real thing

 
  • How do substrains appear? What causes them to be different from one another?
  • Often there are differences between the cells within a single clone - why?
  • How can you be sure that a colony was derived from a single cell?
Use Wikipedia | revised 02-Dec-2008