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STUDENT OBJECTIVE

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 Oil immersion objective for demonstration Dissecting microscope Incubator, 37oC Water baths, 37oC, 42oC, 45oC, and 80oC (can be improvised) pH meter Balance Hot plate	1/student 1/lab 1/student 1/lab 4/lab 1/lab 1/lab 1/lab
MATERIALS
Living cultures Anabaena sp. (CBS#15-1710)* Bacillus cereus (CBS#15-4872)* Bacillus megaterium (CBS#15-4900)* Clostridium sporogenes (CBS#15-4992)* Escherichia coli (CBS#15-5068)* Gloeocapsa sp. (CBS#15-1800)* Oscillatoria sp. (CBS#15-1865)* Pseudomonas fluorescens (CBS#15-5255)* Yogurt (grocery) Samples of soils from diverse habitats Prepared slides (sharing possible to reduce costs)	1/lab
Bacterial cell types (CBS#Ba029)* Merismopedia sp., whole mount (CBS#B6M)* Alcohol lamps Diamond pencils Bacteriological loops Microscope slides and coverslips Small paper cups Plastic wrap Autoclavable disposal bag (CBS#83-1630)* Beaker, 400 ml Beaker, 100 ml to hold slides Paper toweling Pipettes, 1 ml graduated in 0.1 ml Thermometers Petri plates Bacteriological culture tubes and caps	1/student 1/student 1/group 1/group 1/group 6/student 1/group 1 roll 1/lab 1/group 1/student 3/group 1/group 12/group 3/group

*Please refer to the Appendix for name and address of supplier.

SOLUTIONS

Whole milk (grocery)
Nonfat dry milk (grocery)
Gram’s stain kit (CBS#82-1050)*
Nutrient agar (CBS#78-5300)*
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.

3. Saline nutrient agar preparation:

Day before Lab

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^oC. 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

1. This exercise is long and if time is limited, you may want to do only selected activities.
2. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

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.

2. 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.

SUPPLEMENTAL MATERIALS

Identibacter Interactus CD-ROM by Alan Knopka. Dubuque, IA: WCB/McGraw-Hill.

A CD ROM tutorial entitled Viruses and Bacteria is available from CyberEd at http://www.cyber-ed.com/