isolation baterial

ISOLATION OF INDIVIDUAL BACTERIAL COLONIES ON SOLID MEDIA

Robert Koch developed a method for isolating pure cultures on solid media in 1883.

To this end he added agar (a solidifying agent) to liquid nutrient broth; the nutrient broth

supports the growth of a wide variety of microorganisms while the agar provides a solid

substrate on which bacteria can be mechanically diluted and therefore isolated as

independent colonies representing different bacterial species.

The isolation of independent bacterial species from various environmental sources

is important in all branches of microbiology since bacteria are ubiquitous and live in

microbial communities of mixed populations. Populations in microbial communities or

ecosystems may interact and cooperate in their efforts to obtain nutrients from the

environment with the waste products from one group of microorganisms serving as

nutrients for another. Alternatively, the metabolic wastes from fermentation processes of

some bacteria in a population (for example acid production) may provide a favorable

ecological niche for bacteria that prefer to grow at low pH.

This exercise is designed to teach you how to use solid media and streak plate

techniques to isolate pure bacterial colonies from a mixed population of bacteria. The

instructor will provide TSA plates (Tryptic Soy Broth mixed with 15% agar, heated to

sterilize media and melt agar then poured into a Petri dish) and a culture containing

Escherichia coli and/or Bacillus subtilis and/or Staphylococcus aureus and/or Serratia

marcescens.

Isolation of bacteria using the streak plate technique:

Label the bottom of a Petri dish (side containing the agar) (Why?) with your initials, date

and lab section. Your instructor will provide you with a test tube containing two or more

bacterial species. Mix the contents in the test tube by gently shaking the test tube. This

will ensure that all of the bacteria are in suspension. Aseptically remove some of the

culture from the test tube with a sterile cotton tipped applicator. Your instructor will

demonstrate this before you start your exercise.

Aseptically transfer the contents of the applicator to a small section of the plate (approx.

10% of total plate surface area) as shown by your instructor and in the diagram below.

(The cover of the Petri dish must never come in contact with the laboratory bench)

Flame your inoculating loop and allow the loop to cool in the air or by dipping the loop in a

sterile section of the media.

Turn the plate 90 degrees and sample some of the bacteria from the initial streak and drag

the loop across the surface of the bacteria in a zig-zag fashion. Make certain that you never

re-zig over a zag. Also make certain that you do not enter the previous sector more than

twice. (WHY?)