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?)

Turn the plate another 90 degrees and sample some of the bacteria from the second streak and isolate as above. Turn the plate another 90 degrees and sample some of the bacteria from the third streak as above. Incubate the plates upside down (WHY?) for 24 hours at 30oC for 24 to 48 hours, remove and refrigerate plates. Observe the plates for individual colonies during the next lab section.

Figure 1. Inoculating and employing streak plate techniques to isolate individual bacterial colonies

Observation of individual colonies: In terms of colony characteristics, morphology, size, opacity and color completely describe the bacterial colonies that you have isolated. (See page 3.) Escherichia coli Bacillus subtilis

Staphylococcus aureus

Serratia marsescens

Second Lab PeriodAnalysis of bacterial colony characteristics: Bacteria have different colony morphologies and characteristics that are specific to the genus and species of the organism. Colonies can be classified by size, form, elevation, margins, color, texture, and opacity. With respect to opacity one can identify a given colony as opaque (cannot see through the colony), translucent (can see through the colony but cannot clearly see objects through that colony) or transparent (can see through the colony and can clearly see objects through that colony as if looking through glass). For example Bacillus subtilis produces colonies that are large with irregular borders, slightly raised with undulate margins, off white. They have a dull, rough and dry texture and are opaque. Figure 1 below shows some typical colony morphologies and characteristics.

Figure 2: Typical colony morphologies. A. form B. elevation C. margin

Atlas Microbiology text

One study hint for quiz preparation: There will be 2 practical components to next weeks quiz. You will be responsible for isolating 2 bacterial species from a mixed culture. Practice streaking for isolation on your own time. To this end, use a pen and a 3

sheet of paper containing a circle such that you can successfully repeat this exercise during the lab practical. Also, you will be responsible for completely describing a bacterial colony.