PROCEDURES

PROCEDURES

Materials

Outside Materials Lab Materials

Garden shovel(s)                                                                                 10ml serological pipettes

Transect Sq. (0.5meters x 0.5meters)                                                         Bacteria Growth Plates

Centimeters/Inches Ruler                                                                 15ml Culture tubes with caps

Small Tub                                                                                              A 1- cc scoop

Soil Core Sampler(s)                                                                           Sterile Water

Plastic Sandwich Bags (3+)                                                                P200 micro-pipette with tips

Garden Gloves (optional {recommended}                                         Magnifying Glasses

Rubber Mallet(optional {recommended})

Lab Note book

Pencil/Pen

Bug Spray (optional {recommended})

Outsides Procedures

**Areas to search: Places with a large # in Mite Population**

1.      Collect all outside materials and place into the small tub.

2.      Choose 2 small areas, one near fresh water and another near dry soil.

3.      Place the transect square on the exact spot that you are investigating.

4.      With a soil core sampler, take 3 samples of soil that are 15cm deep by 2 cm in width in different sections of the transect sq.

5.      Place each sample into separate plastic bags that should be labeled A, B, and C and what site it came from. Place samples to the side. (These samples are going to be later tested.).

6.      After taking all three soil samples and placing them off to the side, take a shovel and began to excavate (dig) through the area. While excavating your area, search for mites.*

7.      Excavate 15 cm deep and .5 by .5 meters wide in your transect.

8.      Keep track of the # of mites that may be found in your lab note book.

   * Important Note: Wear gloves, there are other bugs than just mite

Lab Procedures

Serial Dilutions for Bacteria

1.      Use a clean new transfer pipette to add 10 ml to a 15 ml culture tube. Label the tube “10⁰“.

2.      Use the same pipette to add 9ml to a second 15ml culture tubes. Label the tube “10^-1 “.

3.      Repeat step 2 three more times to three additional 15ml culture tubes, only label them “10^-2”, “10^-3”, and “10^-4” respectively.

4.      Place 1 cc of your soil sample into the “10⁰“culture tube.

5.      Cap the tube and shake vigorously.

6.      Using a new clean pipette, remove 1 ml of the soil/ water mixture from the “10⁰” tube and place into the “10^-1” tube.

7.      Cap and shake vigorously.

8.      Using the same pipette in step 5, remove 1 ml of soil/ water mixture from the “10^-1” tube and place into “10^-2” tube.

9.      Cap and shake vigorously.

10. Using the same pipette in step 5, remove 1ml of the soil/ water mixture from the “10^-2” tube and place into the “10^-3” tube.

11. Cap and shake vigorously.

12. Using the same pipette in step 5, remove 1 ml of the soil/ water mixture from the “10^-3” tube and place into the “10^-4” tube.

13. You should now have a total of five cultural tubes.

14. Plate 100µl samples from all 5 tubes onto their own separately labeled Petri plates containing nutrient agar.

15. Allow to grow for 48 to 72 hours.

16. Examine each of the plates count the colonies starting with 10^-4, if less than 5 move on to 10^-3 and choose the plate with the lowest dilution that has at least 5 colonies to make your estimates of the number of bacteria in the original 1 cc soil sample using the following formula:

# Microbes in 1cc of soil = Colonies in sheet x 10^-2x10 ^{I
dilution # at which} ^{these colonies were found I}

^{Bacteria Plates}