Laboratory Investigations in Microbiology

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Chapter 7: Culture techniques

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.

A colony of Pseudomonas with a spreading edgeBacterial 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 forA streak-plate of Serratia marcescens 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. 

Materials & Methods

A. Streak plate technique

Materials per person:
Procedure (This procedure is part of the Laboratory Skills Test)
Tuesday
  1. Observe quadrant streak plate demonstration
  2. Perform a quadrant streak using a "Mix" culture in TSB and a sterile TSA plate
    1. Mark your TSA plate on the bottom as seen in the diagram on the right:
    2. Aseptically pick up 1 loopful of bacterial broth from the mix (E. coli and S. aureus) 
    3. Lift the lid of the TSA plate with your non-loop hand and hold it at an angle just above the agar plate surface so it is between your face and the agar surface (acting as a sneeze-guard)
    4. Starting at the edge of the plate, streak your loop of broth all the way across quadrant #1 using a uniform zig-zag pattern. Do not double back. Try to cover as much area as possible. When you reach the center of the plate, remove your loop and close the lid.
    5. Flame-sterilize your loop and let it cool. With the sterile loop, start in the middle of quadrant #1. Drag the loop into quadrant #2, then do the zig-zag pattern inside quadrant #2 without crossing over into any of the other quadrants.
    6. Repeat step 5 for quadrants #3 and #4, always starting in the previous quadrant.
  3. Label your plate and return it for incubation
Thursday
  1. Examine your quadrant streak plate and record observations on data sheet

B. Pure culture technique (Thursday)

Materials 
Procedure (This procedure is part of the Laboratory Skills Test)
  1. Using your streak plate from the previous exercise, choose an isolated colony of each of your 2 bacteria from your mixture
  2. Using aseptic technique, transfer a portion of a colony to an agar slant. Hint: If your colony is large, take a portion from the edge of the colony. Inoculate the entire slant surface with a zig-zag motion from the bottom of the tube to the top.
  3. Repeat for your second colony from the MIX.
  4. Correctly label each slant and bring them up front to be incubated.

C. Broth culture

Materials
Procedure
Tuesday
  1. Label your broth: Lab section, your name, name of bacterium
  2. Aseptically transfer 1 loopful of bacteria from the slant to the broth
  3. Incubate until the next class period
Thursday
  1. Examine broth culture carefully (without shaking) for growth. Note where the growth occurs and describe. 
  2. To test your broth for purity, prepare a TSA streak plate from your broth

D. Spread plate (demonstration)

E. Pour plate (demonstration)

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.

Data Sheet & Review Questions (printable)

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