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Investigating the Uses of Backyard Bacteria

By Elisa Brako



Modified by Elisa Brako from "Selecting Soil Organisms" by Leslie Brinson in A Sourcebook of Biotechnology Activities , NABT, c1990, codirectors Alison Rasmussen and Robert Matheson III

Type of Entry:

  • class activity

Type of Activity:

  • hands-on activity
  • inquiry lab

Target Audience:

  • Biology
  • Advanced/AP Biology
  • Genetics
  • Ecology
  • Microbiology

Notes to Teacher:

This activity as detailed in the procedure would take a total of two days, but it does offer unique opportunities for inquiry based learning that could extend the experimental time (See "Extension" section). Note: Used petri dishes can be disposed of by autoclaving, or by other approved methods.

Preparation time needed: several hours

Class Time needed:

One class period to inoculate Petri dishes, follow up would require half a period to observe and count colonies


Abstract

The purpose of this lab is to recognize that the answers to some of society's industrial challenges may lie right in our own backyards. Diverse species of bacterial decomposers exist in nature that, through evolution, have developed numerous enzymatic systems allowing them to digest various organic molecules present in the soil. Some bacteria have the ability to digest proteins. Proteolytic enzymes are used by humans in areas as diverse as DNA analysis in forensics to more pragmatic concerns such as cleaning contact lenses and getting rid of stains on clothing. In this lab, students learn how to select for protein-digesting bacteria from various soil samples. After isolating the colonies, students are then encouraged to investigate ways to test the effectiveness of their specimens in breaking down protein stains on clothing.


Background

In this activity, students discover that the answers to some of society's waste and clean-up problems may be no further than the soil beneath their feet. Humans currently employ bacteria in numerous capacities from sewage treatment to creating foods such as cheese and yogurt to making chemicals such as acetone. Although biotechnology has allowed for the formation of important "designer bacteria", particularly in the fields of medicine and agriculture, students learn that nature has supplied us with a myriad of decomposers that can provide us with the biochemical breakdown of some materials without the expense and lengthy research and development associated with genetic engineering. This activity demonstrates how bacteria with specific enzymatic capabilities can be isolated with simple microbiology techniques. It will involve selecting for bacteria which contain proteolytic enzymes.


Project

Materials needed:

    for teacher preparation:

  • autoclave (* if not available, see alternative lab preparation below)
  • 30g nutrient agar
  • 1 L flask
  • 1L distilled water
  • 1 box "Parmalat" brand skim milk ("Parmalat" is basically sterile, can be found on grocery store shelves)
  • nonabsorbent cotton
  • magnetic stirrer
  • sterilized Petri dishes
  • stirring plate

    entire class:

  • balances
  • Bunsen burners

    for each lab group:

  • 95% ethanol
  • 3 sterilized Petri dishes prepared with skim milk agar
  • spreading rods
  • 3 sterile 1mL pipettes
  • 3 sterile test tubes with nonabsorbent cotton plugs
  • 3 sterile jars
  • sterile water
  • tape

Teacher Preparation

preparing the skim milk agar:

  1. Add 30 g nutrient agar to 1L of distilled water in a flask. Drop in a magnetic stirrer.

  2. Insert a nonabsorbent cotton plug and autoclave the mixture for 20 minutes.

  3. Remove from autoclave and allow flask to cool for 5 minutes

  4. Add one carton of "Parmalat" brand skim milk (approx. 1L) to the agar flask (not too soon or the milk will curdle)

  5. Stir on stirring plate for 1 to 2 minutes (too much stirring will cause bubbles)

  6. using aseptic technique, pour out the plates for all of the groups.

  7. Autoclave all glassware and enough sterilized water for all lab groups

*Alternative to preparing the agar*

Sterile agar can be purchased from supply companies, and the milk then added to the melted agar. Sterile skim milk agar can be purchased from Carolina Biological Supply Company's microbiology department It is sold in bottles so it would have to be melted down, then poured into plates.

Procedure

  1. Each group should start off with three samples of soil collected from different environments (a meadow, a wooded lot, etc). For the first sample, weigh out 1 gram of the soil and add it to 99 ml of sterile distilled water in a jar. Further dilute this mixture by removing 1 ml of the mixture and adding it to a test tube containing 9 ml of sterile distilled water. Mix thoroughly by swirling. Be sure to keep all tubes capped and employ sterile technique at all times.

  2. Use the pipette to remove 0.1 ml of this dilute soil mixture and put it on a Petri dish containing skim milk agar. Dip a spreading rod in the alcohol, hold it over a flame and cool it off by pressing it gently into the agar off to the side of the mixture. Use this to spread the diluted soil sample evenly onto the plate. Flame the rod when finished.

  3. Label this plate with the names of group members, date of inoculation, and soil source. Repeat procedure 1 and 2 with the two other soil samples. Stack the three dishes, tape them together, and store them upside down overnight at room temperature.

  4. The next day, check to see if there are any clear "halos" developing around the bacterial colonies. If so, record the number and position of such colonies in each dish ( sketches could accompany data chart)

  5. If no colonies are visible, record observations on the second day, etc.

  6. Your teacher will give you instructions for disposal after the activity is completed.


Method of Assessment/Evaluation

This lab could be organized into a formal lab report. Possible questions to be answered as part of the experimental analysis could include:

  • Was there varying success in isolating colonies from the different samples and if so what might account for the differences?

  • Why did it take some groups several days to develop protein

  • digesting colonies while others may have noticed them after 24 hours?

  • What did the clear "halo " indicate? Why didn't all bacteria have halos?

  • What do the bacteria contain that allow them to digest the proteins?

  • What are some of the protein sources that stain clothing and contact lenses? How can these bacteria be used in the cleaning industry?

  • What method could be used to isolate oil-degrading bacteria?


Extension/Reinforcement/Additional Ideas

Since the procedure employed in no way harms the bacteria, individual protein-digesting colonies could be re-cultured onto a new Petri dish and studied. Students could also perform a Gram stain on the colonies to determine some of their characteristics. They could test the efficiency of the bacteria in dissolving a protein stain on a cloth. (Albumin could be a possible protein source, and there are many indicator tests that could be used to test for the digestion of the protein). A comparison study could be done with some name brand laundry detergents. Motivated students could contact the manufactures of various laundry detergents and contact-lens cleaners to determine their enzyme source. In addition, student could devise various lab protocols for isolating colonies that can digest oil, starch, etc. and then test their effectiveness.


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