Exercise 14 - Monerans

Students study the diversity of the monerans and gain some experience with techniques in determinative microbiology. Products of fermentation are demonstrated.

EQUIPMENT

AMOUNT (Class of 24 with 8 groups)
Compound microscope	1/student
Oil immersion objective for demonstration	1/lab
Dissecting microscope	1/student
Incubator, 37 ° C	1/lab
Water baths, 37 ° C, 42 ° C, 45 ° C, and 80 ° C (can be improvised)	4/lab
pH meter	1/lab
Balance	1/lab
Hot plate	1/lab

MATERIALS

SOLUTIONS

Whole milk (grocery)
Nonfat dry milk (grocery)
Gram's stain kit (CBS#82-1050)*
Nutrient agar (CBS#78-5300)*
Thioglycollate medium (CBS#78-7800)*
Sterile 0.85% saline
Saline nutrient agar
Alcohol in beaker to wash slides
25% acetone in isopropyl alcohol (25 ml:75 ml)
India ink
Sterile water

PREPARATION

About Three Weeks before Lab

Order cultures, media, and stains as needed to arrive one week before lab.

Week before Lab

1. Saline preparation:

   0.85% saline

   8.5 g NaCl/liter water

   Package, 100 ml per bottle. Three bottles per student station are needed to perform serial dilutions. Autoclave.

2. Nutrient agar preparation:
   4.6 g nutrient agar/200 ml water

   Autoclave for 15 min at 15 psi. While warm (and liquid) pour into petri plates to depth of 5 mm.

   Saline nutrient agar preparation:

   12 g NaCl/200 ml water
   Add 4.6 g nutrient agar
   

   Autoclave and pour as in #2 above.

   Thioglycollate medium:

   Dispense sterile thioglycollate medium into sterile, capped culture tubes. Add enough medium to give a depth of 5 cm.

Take 1 gram soil and add to 100 ml 0.85% sterile saline. Gently agitate to suspend bacteria. Obtain two petri plates containing nutrient agar and two containing saline nutrient agar. Add 0.1 ml soil suspension to each plate and spread the bacteria with a hockey stick. Incubate one of each type of plate at room temperature and one of each type at 42 ° C. If you want to test the effects of drugs or pesticides on the growth of bacteria, use a cork borer to make disks 1 cm in diameter from sterile filter paper. Prepare appropriate dilutions of the test substances and dip the disks in the solution. Lay disks on the surfaces of the agar before incubating the petri dishes.

CLASSROOM SUGGESTIONS

This exercise is long and if time is limited you may want to do only selected activities.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. Points to consider:
   
   - pH-fruits are high in acid; meats and vegetables have a much lower pH.
   
   - Fruits are high in sugars (fructose); meats are high in proteins (the thioglycollate broth has a high concentration of peptone to assure good growth).
   
   - Heating to 100 ° C for twenty minutes should kill all bacteria and also destroy spores. Meats and vegetables are not usually heated for such a long period of time as they become unpalatable. They must be canned with a combination of heat and pressure (to drive the heat into the spores and destroy them). Inadequate canning procedures can result in the growth of Clostridium botulinum, a spore forming, anaerobic soil organism, which produces gas and neurotoxins.
   
   - Osmotic effects of high sugar concentration.
   
   - Anaerobic environment produced by sealing with paraffin or lids.
   Other methods of preserving foods?
   
   Pasteurization
   
   Salting
   
   Drying
   
   UV radiation - OK for surface sterilization but does not penetrate
   
   Gamma irradiation - uses Colbalt-60 as ionizing source; use on moist foods to produce peroxides in microbials cells
   
   Vacuum packaging
   
   UHT (ultra-high temperature) processing
   
   Pickling
   
   Spices often contain antimicrobials i.e., cloves, garlic, rosemary, sage
   
   Fermentation - dairy products
   
   Sodium nitrite - used to inhibit germination of Clostridium spores
   
   Ethylene oxide
   
   Freezing/freeze-drying
   
   etc.
   

   Leguminous plants such as peas, beans and peanuts form symbiotic relationships with Rhizobium sp. bacteria. The bacteria produce root nodules where atmospheric nitrogen is fixed into a form usable by the plant.

   Consider oxygen requirements- A skin abrasion would provide an aerobic environment - expect to see aerobic or facultatively anaerobic bacteria here. A deep puncture wound would provide an anaerobic environment (especially if it did not bleed significantly as blood would deliver oxygen to the tissues and also help flush the wound). Therefore, expect to see anaerobic or facultatively anaerobic bacteria here. Indeed, such a wound provides the perfect environment for Clostridium tetani, the causative agent of tetanus. The large intestine has the largest bacterial population of the body, consisting mostly of anaerobic species along with some facultatively anaerobic species. The possibility of a massive infection of the abdomen exists if the intestine is punctured, as happens with a ruptured appendix.

SUPPLEMENTAL MATERIALS

Bacteria, 19-minute film. Chicago, IL: Encyclopaedia Britannica Corp.
Bacteriological Techniques, 5-minute film. Boulder, CO: Thorne Film, Inc.
Bio Sci II, videodisc- contains representative photos of monerans. Dubuque, IA: Wm. C. Brown Publishers. See appendix.
Introduction to Bacteria, audio filmstrip. Burlington, NC: REX Educational Resources. #KF3012