A Library of Bacteria

Creating a library plate is one of the first steps to finding antibiotic producing isolates by collecting, isolating, and culturing independent colonies to look at.

The setup:

Setting up a library plate is essential to documenting and processing bacterial isolates. It is a starting point for further testing.

1. Label a new LB agar plate with your initials, the date of culturing, and the soil sample ID

2. Trace a 4x4 grid using a grid template as guidelines for placing colonies

3. Label the axes of the grid. A-D, 1-4 to "name" the colonies in each square

   
   
   
   

In your lab notebook...

Proper lab notebook setup will help you compare the original colony morphology to the library plate inoculation, ensuring the intended colony is growing

1. Create a grid in your lab notebook that matches the grid of the LB agar plate

2. Record information on the morphology (see chart below) and the color of each colony transferred from the serial dilution plates onto the library plate

   1. Tip: Take any extra notes that might be necessary, including: which serial dilution each colony was taken from, possible inhibition, possible contamination from inoculation, etc.

      
      

Selecting colonies:

Pick colonies of different shapes, sizes, and textures. This library plate is a way to gather different bacteria in isolated colonies to further test for antibiotic production. Remember: don't pick a colony that appears to have spread over a large portion of a serial dilution plate!

1. Look for well-isolated colonies on your serial dilution plates

2. Things to look for: any that appear to be inhibiting the growth of other colonies, uniquely shaped/colored colonies, and any that are generally interesting.

   1. Tip: DON'T pick a filamentous colony that has spread and/or grown on top of other colonies!

   
   

   

   See how large and overgrown this is? Do not pick a colony of this morphology or size!

Transferring a colony onto the library plate:

Isolated colonies are essential to seeing how each individual colony interacts against ESKAPE safe relatives in further screening steps. Mixed colonies would lead to inaccurate results.

1. Flame an inoculation loop for 2-3 seconds, allowing it to cool for 15-20 seconds after

   1. Tip: To speed up the transfer process, consider using multiple loops to alternate heating/cooling times

2. Gently touch a colony with the loop, only transferring a barely-visible amount onto one square of the grid.

   1. Tip: Ensure to inoculate as close to the center of the square as possible, colonies spread when the grow!

3. Continue transferring individual colonies onto individual grid squares

   
   

Here's an example of a library plate I made with 16 colonies!

Incubate at 26ºC for 24-48 hours to allow for growth.

Assessing your library plate:

1. Make sure that no colonies are touching each other

   1. IF multiple colonies are touching one-another, a new library plate may be needed to salvage the colonies that were not overtaken by another

2. If some colonies did not grow, your inoculation loop may have been too hot while inoculating, or the colony may have needed more time incubating. Make note in your lab notebook of any colonies that did not grow on the library plate

3. Note any morphological differences in the original colony on the serial dilutions versus the growth on the library plate

Creating a library plate can be fun! YOU get to decide which colonies seem interesting and keep a catalogue of what is being worked on. They are a great visual tool to screen multiple diverse bacterial colonies, comparing them to one another directly. Using the tips and protocol, you are ready to make a successful library plate!