Laboratory Investigations in Microbiology |
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Bacterial growth on various culture media
Bacterial growth patterns depend on the type of medium they are in. Broth cultures go through the four stages of microbial growth sequentially: lag phase, exponential phase, stationary phase, and death phase. Because growth of bacteria in broths turns the broth cloudy, the extent of growth can be measured by spectrophotometry (Ch. 9). Observe some broth cultures, and you will also be able to discern a difference in the way various cultures grow in broths.
Bacterial growth on agar surfaces results in the formation of bacterial colonies. A bacterial colony is a visible accumulation of bacterial cells that arise from one single cell. Therefore, all cells in a pure colony are genetically identical (clones). Notice that bacterial colonies are not always uniform. The center of a colony often consists of cells in stationary or death phase, while the cells at the edges of a colony are actively dividing (exponential phase). This often causes the center of a colony to look different from its edges.
As we will soon see, microbial growth is affected by many factors, including temperature, pH, oxygen, and nutrient availability. When optimally adjusted, these growth conditions can support rapid bacterial growth. The following lab exercise will allow us to observe bacterial growth on various culture media and to practice the techniques for obtaining isolated colonies and preparing pure cultures.
Culture techniques
Streak-plate technique: The most frequently used method for obtaining colonies of bacteria on agar surfaces is the streak plate technique. The photo on the right shows a culture of Serratia marcescens spread across an agar plate using this technique. On the bottom left is the first streak, made with a loopful of broth culture. A second streak is made with a sterile loop that crosses into the first streak, spreading some of the bacteria out more (top left). A third (and sometimes fourth) streak spreads those bacteria even more, until in the end single bacterial cells are spread out. After incubation, the bacteria form colonies - visible piles of bacterial cells. Notice that the colonies that are most isolated are also the largest. Why do you think this is so? {Answer} You can also see that these bacterial colonies produce a red pigment. Colony characteristics such as color, shape, size, and margin can be useful in identifying a bacterial species.
Spread-plate technique: At times it is convenient to merely spread a sample of bacteria out on an agar plate using a sterile glass rod. This works well if the number of bacteria in a sample is relatively small - maybe 100 - 1000 cells. Spread plates distribute bacteria more or less equally across the agar surface, and more than 1000 colonies would make it difficult to isolate pure colonies.
Pour-plate technique: This technique is useful when bacterial numbers are to be determined. Instead of placing bacteria on top of the agar, they are mixed with the agar medium while it is still melted (~ 50C). Immediately thereafter, the mixture is poured into a sterile Petri plate where it hardens. Most of the bacteria will be embedded in the agar and will form smaller, denser colonies. This makes it possible to count several hundred colonies on one plate. However, it is more difficult to isolate pure strains and to observe the true colony characteristics. This technique will be demonstrated at a later time.
Preparation of pure cultures: Using a streak plate, spread plate, or contamination plate, pure cultures of each of the bacteria or fungi on the plate can be prepared if isolated colonies of the bacteria can be found. A isolated colony is one that is visibly uncontaminated and does not come in contact with any other colonies. In order to transfer bacteria from an isolated colony, aseptic technique is used. The tip of an inoculating loop is dipped into the colony (you don't need much of a colony - keep in mind that millions of cells can be clinging to a loop without you even seeing them), then placed onto the agar slant surface near the bottom of the test tube and gradually pulled out of the tube. By wiggling the loop back and forth as it is being withdrawn, a curvy line is formed on the agar surface. {I liken this to skiing down a ski slope - from the top of the slope (in the bottom of the tube) to the end (near the mouth of the test tube)}. After incubation, bacterial growth should cover the slant; although isolated colonies are rarely seen, the slant is easily inspected for contaminants that differ from the overall growth on the slant.
Answer to the question above: If you said that the isolated colonies have less competition for food, you are correct. Many colonies close together are much smaller since they must share the nutrients around them.
© 2003 - 2017 José de Ondarza, Ph.D.