EXERCISE 1. MICROORGANISMS IN THE ENVIRONMENT

Period 2

Materials

• Demonstrations of colonies of various species of bacteria and molds
  
• Tube containing 1 ml of a soil suspension (a 1/10,000 dilution)
  
• Tube containing 1 ml of lake water (a 1/100 dilution)
  
• 2 tubes of melted Plate Count Agar (PCA; 15-20 ml/tube) – in 50°C water bath
  
• 2 empty, sterile petri plates

Procedure

1. Before observation of the plates prepared last period, another plating method will be performed:
   
   
   1. For each of the two samples, dump the entire tube contents into an empty, sterile petri dish.
      
      
   2. Obtain two tubes of melted PCA from the water bath. Wipe off the excess water with a paper towel, and pour the contents of each tube into a petri dish sitting upright on the table, opening the lid just enough to pour out the tube. Mix the sample and medium in each dish with a gentle, swirling motion and let the medium solidify.
      
      
   3. Incubate the plates inverted at 30°C for 2-5 days.
      
      
2. Note the demonstration of colonies of various species of bacteria and molds. Keep the lids on the plates and observe the colonies through the top lid.
   
   
3. As time permits (i.e., with everything else having been done in Exps. 1 and 2 for today), the following can be done.
   
   
   1. Observe the plates from Period 1, noting the various bacterial and mold colonies. At this point, do not open the plates, especially if molds are present. (Any fuzzy or hairy colonies are probably mold colonies. Their spores are very easily dispersed into the air, causing subsequent contamination problems and perhaps allergic reactions as well!) Note: As a rule, we always observe colonies for their characteristics through the top lids of the plates. Very little information about colony characteristics and differences can be obtained by looking through the medium. Note the various shapes, sizes and colors of the colonies.
      
      
   2. From one or more of your plates which do not contain mold colonies, choose two or more different colonies and record their visible characteristics.
      
      
   3. OPTIONAL: For each of your chosen colonies, prepare a smear (with a drop of water) and stain by the gram-stain procedure (below). What is the gram reaction and morphology of the cells?

EXERCISE 2. MICROSCOPIC OBSERVATIONS OF BACTERIA

Period 2

Materials

• Bacterial cultures growing either in a liquid medium (Heart Infusion Broth) or on a slant of an all-purpose medium followed by suspension in saline:
  
  • Escherichia coli – "young" culture, incubated 12-15 hours
    
  • Bacillus cereus – "young" culture, incubated 12-15 hours
    
  • Bacillus cereus – "old" culture, incubated 2-3 days

Procedure

1. On one clean glass slide, prepare smears of the three cultures: Note the instructor's demonstration. Only when the smears have dried completely should the slide be heat-fixed.
   
   
2. Perform the gram stain procedure (reproduced below).
   

   As with any stained smear, definitive observations are made with the 100X, oil-immersion objective. Refer to the microscope directions, remembering to focus the slide initially with the 10X objective, moving then to the oil immersion objective. Keep in mind that the young cultures of B. cereus and E. coli are your positive and negative control cultures, respectively, for the observation of probable gram-variability of the older B. cereus culture.

3. Record your observations, noting the gram reaction (positive if purple, negative if red) and the cellular shape ("rod" or "coccus"). Is there any difference seen between the two cultures of Bacillus cereus? Is gram-variability evident for the "older" culture? Recall from the introduction to Experiment 1 that we can refer to old and young cultures but should not do so for individual cells.

APPENDIX G.2 – THE GRAM STAIN PROCEDURE

The gram stain is one of the most useful differential stains in bacteriology, including diagnostic medical bacteriology. The differential staining effect correlates with certain fundamental differences in the structure of bacterial cell walls as the instructor will explain.

For the most reliable results when performing the gram stain, certain precautions should be taken:

• The cultures to be stained should be "young" – incubated in broth or on a solid medium until growth is just visible (no more than 12 to 15 hours old if at all possible). With our lab periods being two or more days apart during the regular school year, we realize that short incubation times are not always possible. However, most of our organisms will still give reliable results for their gram reactions. One notable exception concerns organisms of the genus Bacillus: After about a day of incubation of the culture, the normally gram-positive (purple) reaction tends to give way to a gram-negative (pink) reaction as cell walls weaken and increasing numbers of cells become more easily decolorized. This "gram-variable" appearance should not occur if the effort is made to stain the culture as early in its lifetime as possible.
  
  
• The cultures to be stained should be grown on a sugar-free medium, if at all possible. Many organisms produce substantial amounts of capsular or slime material in the presence of certain carbohydrates. Such a condition often interferes with decolorization, and certain gram-negative organisms such as Klebsiella may give a "gram-variable" appearance.
  
  
• Smears must be thin, just barely visible (when dry) to the naked eye. Thick smears are not decolorized efficiently by the alcohol-acetone, and gram-negative cells can stain gram-positive.
  

Following is the traditional gram-stain procedure which we find reliable, as long as the precautions listed above are heeded.

1. Prepare and heat-fix a smear of the organism to be studied. Cover the slide with crystal violet. Allow one minute for this primary stain and then wash off (gently!) with a minimum amount of tap water, as an excess application of water tends to decolorize. Drain off most of the water onto a paper towel.
   
   
2. Cover the slide with iodine solution for one minute. The iodine acts as a mordant (fixer) and will form a complex with the crystal violet, fixing it into the cell. Rinse briefly with tap water, and then drain off most of the water.
   
   
3. Tilt the slide lengthwise over the sink and apply the alcohol-acetone solution dropwise – such that the solution washes evenly over the entire slide from one end to the other. Continue in this manner for about 15-20 seconds and then rinse immediately with tap water. If applied properly, the alcohol-acetone should decolorize cells with a gram-negative type of cell wall but not those with a gram-positive type of cell wall. Drain off most of the water.
   
   
4. Any decolorized, gram-negative cells need to be stained in order to be visible and differentiated from gram-positive cells. Cover the slide with the safranin counterstain for one minute. Rinse briefly. Gently (without rubbing) blot the slide dry.
   
   
5. For each smear, focus with the 10X objective, and then switch immediately to the 100X (oil-immersion) objective for the "official" observations. This is the standard operating procedure. Observe the cells for morphology (rod or coccus) and gram reaction. Regarding the latter, record each culture as "gram-positive" (purple cells) or "gram-negative" (pink cells).