Basic techniques of Microbiology

Introduction

Our first understanding of the genetics and biochemistry of living organisms was that of microorganisms such as yeast, mold, and bacteria. It was from the accumulated knowledge of the biochemical genetics of such organisms that molecular genetics arose. The general techniques for counting, transferring, and analyzing bacteria are essential to many different areas of biological research. While the equipment is simple, the techniques are not trivial. In the next two weeks, you will have to be able to carry out repetitions of these operations, smoothly, rapidly, and accurately.

Responsible handling of E. coli

The bacterial host used in most molecular genetics laboratories is Escherichia coli (E. coli). Since E. coli is a commensal organism of Homo sapiens, and a normal part of the bacterial flora of the human gut. It is not considered pathogenic and is rarely associated with illness in healthy individuals. Furhtermore, K-12-derived E. coli strains are ineffective in colonizing the human gut and lack the cell-surface "O antigen" respnsible for virulence. Adherence to simple guidelines for handling makes working with E. coli a nonthreatening experience.

  1. Always reflame the incoculating loop or cell spreader one final time before setting it down on the lab bench.
  2. Keep nose and mouth away from the tip of the pipet when pippetting suspension cultures to avoid any aerosol that might be created.
  3. Dispose all contaminated materials under the direction or your instructor.
  4. No smoking, eating, or drinking (or any evidence thereof) in the laboratory!
  5. Wash your hands before leaving the laboratory.

Many microbes are present on your workbench, hands, and transfer instruments. How is it possible to avoid contamination, the introduction of unwanted organisms into your pure culture?

Pure culture: contains only one kind of bacteria; it consists of the descendents from one bacterial cell (clonal population).

Pure cultures are essential if a biologist is to (1) identify bacteria with biochemical and genetic tests; (2) perform antibiotic sensitivity testing; (3) maintain stock cultures; or (4) use microorganisms in recombinant DNA technology.

Today, you will practice "sterile" or "aseptic" techniques - procedures that:

  1. protect the culture
  2. protect you and the environment.

How to streak starter plates

The goal in streaking starter plates is to obtain single, isolated colonies to be used in subsequent molecular genetics experiments. This is important to the success of these experiments.

  1. Label the starter plate E. coli and date it.
  2. Hold the wire-inoculating loop like a pencil and sterilize the circle at the end in the Bunsen flame until it glows red-hot. Do the same with the lower third of the wire next to the loop. Do not set the loop down.
  3. With your other hand, grasp the liquid culture of E. coli between your thumb and two fingers. Remove the vial cap using the little finger of the hand holding the inoculating loop. Avoid touching the rim of the cap. Quickly pass the mouth of the liquid culture through the Bunsen flame.
  4. Place the inoculating loop into the liquid culture. Remove the loop, flame the vial mouth, replace the cap, and set the liquid culture aside.
  5. Lift the top of the starter plate to perform the streaking.
  6. Glide the tip of the inoculating loop back and forth across the agar surface to make a streak across the top of the plate. Avoid gouging the agar. Replace the lid between streaks.
  7. Rotate the plate 90 degrees.
  8. Re-sterilize the inoculating loop.
  9. Stab the inoculating loop into the starter plate (away from the first streak) to cool it.
  10. Draw the loop tip once through the first streak, and without lifting the loop, make a tight zigzag across one-fourth of the agar surface 90 degrees relative to the first streak. Replace the lid.
  11. Rotate the plate 90 degrees.
  12. Reflame the inoculating loop, and cool it in the agar, as before. Draw the loop tip once through the last lines of the second streak, and make another zigzag streak in an adjacent quarter of the plate 90 degrees relative to the second streak. Replace the lid.
  13. Rotate the plate 90 degrees.
  14. Reflame the inoculating loop, and cool it in the agar, as before. Draw the loop tip once through the last lines of the third streak, and make another zigzag streak in an adjacent quarter of the plate 90 degrees relative to the third streak. Replace the lid.
  15. Incubate the plates upside-down overnight at 370C. Your instructor will put these plates in the refrigerator until the next laboratory period.

Bacterial growth

E. coli has simple nutritional requirements and grows slowly on a minimal medium containing (1) an energy source such as glucose, (2) salts such as NaCl and MgCl2, (3) the vitamin biotin and (4) thymidine. E. coli synthesizes all necessary vitamins and amino acids from these precursors. It grows rapidly in a complete medium, such as LB, in which yeast extract and hydrolyzed milk protein (casein) provide a ready supply of vitamins and amino acids.

A liquid bacterial culture goes through a series of growth phases. For approximately 30 minutes following inoculation, there is a lag phase during which there is no cell growth. The bacteria begin dividing rapidly during log phase, when the number of cells doubles every 20-25 minutes. As nutrients in the media are depleted, cells stop dividing and enter the stationary phase, with a concentration of approximately 109 cells/ml. During the death phase, waste products accumulate and the cells begin to die.

Optimum growth in liquid culture is achieved with continuous agitation, which aerates the cells, facilitates the exchange of nutrients, and flushes away waste products of metabolism. It can safely be assumed that a culture in complete medium (e.g. LB medium) has reached stationary phase, following overnight incubation with continuous shaking. A culture in the stationary phase will look very cloudy and turbid.

Serial dilutions

Serial dilutions are the easiest way to reduce the concentration of a bacterial culture. Before setting up the dilution, you must consult a protocol or the literature to decide on the dilutions you will need. 1:10 or 1:100 dilutions are the most commonly used in molecular genetics. Do your dilutions in the medium that your bacterial culture was grown in. Set the tubes up, and add the diluting medium (e.g. LB medium). Write the dilutions for each tube on a separate piece of tape and attach the label to the appropriate tube. Orient the test tube rack in the same way, every time, so you know which way you are working. Always pipet from left to right, to achieve decreasing concentrations. Set the tubes up to facilitate this. Discard pipet tips after you pipet into each tube!

In today's exercise, you will serially dilute your overnight bacterial culture in order to determine the total number of viable cells. Make sure you label all test tubes and plates. Incubate all plates overnight at 370C. Your instructor will place your incubated plates in the refrigerator until the next laboratory period!

EXAMPLE OF A SERIAL DILUTION