How to Achieve Isolated Colonies

Plating for isolated colonies or streak plating on agar media is a common practice in a microbiology lab. It is a basic skill that you will need to fine tune and find your own method that works for you.

To explain streaking, it is a method used to isolate a pure strain of a microorganism, typically bacteria, from a sample or experiment. It is done using a sterile plating tool such as a metal loop, plastic loop or a cotton swab on a sterile agar plate. The goal is to achieve isolated colonies in the 4th quadrant of your plate in a healthy quantity. This will give you many microorganisms to work with for future testing.

To achieve isolated colonies, you must first divide your agar plate into 4 quadrants.  This is how I like to divide up my plate and how I was taught throughout University and College. The 4th quadrant should be your largest as it is where you will achieve your isolated growth.

                                               
                                     My amazing Paint skills on display[/caption]

You will first need to sterilize your loop if it is metal. If using a pre-packed plastic swab or loop, you can start as soon as it is removed from the package but I will explain as if you were using a metal loop as it is the most common practice.

To sterilize your loop, place it in a Bacti-Cinerator or flame. This will insure there is no carry-over from past samples that might contaminate your isolation.
                                          
                                                            Method for Colony Isolation

Steps:

1. Flame the loop and wire and streak very tight lines in quadrant A (like plate 1 of diagram.


2. Reflame the loop and let cool.


3. Streak into quadrant B by passing loop through quadrant A twice before zigzagging in quadrant B (like plate 2).


4. Reflame the loop and let cool.


5. Streak into quadrant C by passing loop through quadrant B twice before zigzagging in quadrant C (like plate 3).


6. ** Here you can flame or not flame depending on your technique. I tend to have "light hands" so I do NOT flame as I would not achieve isolation otherwise. If you have "heavy hands" you might want to flame here.**


7. Streak into quadrant D covering as much area as possible to achieve isolation.


8. Label the plate and incubate it inverted.

Practice is the only way you can get better at streak plating and I recommend doing as many plates as possible so you can become great at isolation. You do not want the microorganism to start isolating in the 2nd or 3rd quadrants because it means you were too "light handed" or did not carry over enough of the colonies.

First quadrant is too large and is isolated too early!

Good luck fellow Microbiologists!

-K