EXERCISE 1. MICROORGANISMS IN THE ENVIRONMENT

Period 3

Procedure

1. Observe the plates prepared last period. Note and count the surface and sub-surface colonies. When counting the colonies, it is handy to draw a few lines with the wax pencil on the back of the plate to mark off a grid. The colonies can then be counted easily as you scan the sectors.
   
   
2. For each of the samples, you can determine the density of colony-forming units (CFUs) that were in the original (undiluted) sample if you know three things: the dilution of the sample, the amount inoculated into the plate (1 ml), and the colony count.
   
   

   For example, if you count 40 colonies on a plate which had been inoculated with one ml of diluted lake water, it follows that 40 CFUs had been in the 1 ml inoculum. As the inoculum was a 1/100 dilution of the lake water (i.e., "diluted 100 times"), there would have been 40 X 100 (i.e., 4000) CFUs per ml of the undiluted lake water. This result is expressed best in scientific notation: 4.0 X 103 CFUs/ml. The same method applies to the soil sample, but the answer is expressed as CFUs per gram of the soil. As the instructor will explain, we treat milliliters and grams as equivalents, for convenience. Bacterial quantitation will be dealt with more fully in Experiment 4 (with Appendix C), and you will note that we are always interested in the concentration of CFUs (the number in one gram or ml) rather than the total number in the entire sample.

   
   
3. Time permitting (i.e., with everything else having been done in Exps. 1 and 2 for today), macro- and microscopic observation of colonies can be made as in the previous period.
   

EXERCISE 2. MICROSCOPIC OBSERVATIONS OF BACTERIA

Period 3

Materials

• Staphylococcus epidermidis
  
• Pseudomonas fluorescens
  
• An UNKNOWN (Be sure to record the number!)

Procedure

1. On a clean glass slide, prepare heat-fixed smears of the three cultures, noting that these cultures are growing on a solid medium. Therefore the cells must be dispersed in a drop of water when preparing the smears, as a smear is always a dried suspension of cells. Note instructor's demonstration Take care not to make the smears too thick! S. epidermidis and P. fluorescens are your positive and negative control cultures (respectively) for your unknown.
   
   
2. Perform the gram stain procedure and note the gram reaction and cellular shape as in the previous period. Turn in your results for grading. Save your slide until your graded unknown is returned. (Remember to discard everything else as per the general clean-up directions.)