4: Obtaining Pure Cultures from a Mixed Population

THE POUR PLATE AND SPIN PLATE METHODS OF ISOLATION

Another method of separating bacteria is the pour plate method. With the pour plate method, the bacteria are mixed with melted agar until evenly distributed and separated throughout the liquid. The melted agar is then poured into an empty plate and allowed to solidify. After incubation, discrete bacterial colonies can then be found growing both on the agar and in the agar.

The spin plate method involves diluting the bacterial sample in tubes of sterile water, saline, or broth. Small samples of the diluted bacteria are then pipetted onto the surface of agar plates. A sterile, bent-glass rod is then used to spread the bacteria evenly over the entire agar surface in order to see isolated colonies. We will use this technique as part of the plate count method of enumerating bacteria.

USE OF SPECIALIZED MEDIA

To supplement mechanical techniques of isolation such as the streak plate method, many special-purpose media are available to the microbiologist to aid in the isolation and identification of specific microorganisms. These special purpose media fall into four groups: selective media, differential media, enrichment media, and combination selective and differential media.

There are literally hundreds of special-purpose media available to the microbiologist. Today we will combine both a mechanical isolation technique (the streak plate) with selective and selective-differential media to obtain pure cultures from a mixture of bacteria. In future labs, you will work to isolate and identify pathogenic bacteria using many additional special-purpose media. 

To see examples of different types of growth and interpretation of results on various special media types, please see this excellent tool created by Cindy Arvidson.

MEDIA

One plate of each of the following media: Trypticase Soy agar, Columbia CNA agar, and MacConkey agar.

ORGANISMS

During this lab you will be given a broth culture containing a mixture of Gram-positive and Gram-negative bacteria. Over the next three labs you will attempt to obtain pure cultures of each organism in your mixture and determine which two bacteria you have. Today you will try to separate the bacteria in the mixture in order to obtain isolated colonies; next lab you will identify the three bacteria in your mixture and pick off single isolated colonies of each of the three bacteria in order to get a pure culture of each. The following lab you will prepare microscopy slides of each of the three pure cultures to determine if they are indeed pure.

Procedure:

1. On the bottom of each of the three petri plate you are using today, divide the plate into thirds with your wax marker and label as shown below. This will guide your streaking.
2. Although Trypticase Soy agar (TSA), which grows both Gram-positive and Gram-negative bacteria, is not normally used as an isolation medium, we will attempt to obtain isolated colonies of the two organisms in your mixture by using strictly mechanical methods. Often, however, one bacterium overgrows another in a mixture and by the time you spread out the more abundant organism enough to get isolated colonies, the one in smaller numbers is no longer on the loop so you may not see single colonies of each on the TSA next time.
3. Streak your mixture on a plate of Trypticase Soy agar.
4. Streak the same mixture for isolation on a plate of Columbia CNA agar (selective for Gram-positive bacteria). 
5. Streak the same mixture for isolation on a plate of MacConkey agar (selective for Gram-negative bacteria and differential for certain members of the bacterial family Enterobacteriaceae).
6. Incubate the three plates upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period. 

Bacterial Colony Morphology

Bacteria grow on solid media as colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.

In the identification of bacteria and fungi much weight is placed on how the organism grows in or on media. This exercise will help you identify the cultural characteristics of a bacterium on an agar plate - called colony morphology. Although one might not necessarily see the importance of colonial morphology at first, it really can be important when identifying the bacterium. Features of the colonies may help to pinpoint the identity of the bacterium. Different species of bacteria can produce very different colonies.

In the above picture of a mixed culture, an agar plate that has been exposed to the air and many different colony morphologies can be identified. Nine obviously different colonies are numbered: some colony types recur in various areas of the plate (note # 3 and # 4). Not only are pigment differences seen, but also size, edge, pattern, opacity, and shine. Two circles have been drawn around merging colonies, where the species of the 2 colonies are different. Trying to pick a bit of one of those adjacent colonies increases the chances of picking up another mixed culture, consisting of the 2 species that were merged together. ALWAYS pick a well-isolated colony when subculturing.