BISC209/S12:Lab Basics

LAB BASICS: Serial Dilution Technique :

This technique is useful for obtaining countable numbers of bacterial colonies by diluting a highly concentrated culture. The protocol described here is for serial 1:10 dilutions in a final volume of 1ml and is easily adjusted for any volume or dilution series desired. Generally, selected dilutions are plated in duplicate or triplicate. This procedure is based on using a 1/100 stock (10^-2) of a Soil Extract (1 gram soil in 100 ml diluent)

1. Label tubes 10^-3, 10^-4 etc. through 10 ^-7 or carry out to whatever dilution is appropriate.

2. Label destination plates of appropriate solid medium with the dilution and identifying information. Because your goal is to obtain 30-300 well isolated colonies on a plate, generally only the 10^-5 through 10^-8 dilutions are plated; however, the choice of dilutions to plate will vary. Often dilutions are plated in duplicate or triplicate.

3. Slightly dehydrate the medium on each plate by cleaning the laminar flow hood, turning on the fan, placing the plates in the hood, and positioning the covers so they are ajar for 10 minutes or until the medium surface shows no visible moisture.

4. Pipet 0.9 ml of sterile water using the same sterile 1 ml pipet into the appropriate number of tubes labeled in step 1.

5. Using a P200 micropipet, transfer 100 μL of a 1:100 dilution to the 0.9 ml diluent in the tube labeled 10^-3, mix well by vortexing or shaking vigorously.

6. Using a new tip, transfer 100 μL ml of the 10^-3 dilution to the 0.9 ml diluent in the tube labeled 10^-4. Mix well. This makes a 10,^-4.

7. Continue sequentially in this manner until you have carried the dilution to an appropriate end, usually 10^-7 or 10^-8.

8. Use your P200 μL pipet and a new tip to transfer 100 μL of the most concentrated dilution that you want to plate (most often this is the 10^-4 dilution)and dispense it to the center of an appropriately labeled agar plate. Use any of the spreading techniques below.

SPREADING TECHNIQUES:
The easiest, and least environmentally friendly, way to spread your inoculum evenly around your plate is to use a sterile, plastic, disposable spreader. This is also the safest method since no open flame is used. Gently push the dispensed sample two or three times clockwise around the dish, and then several times counterclockwise. Don't press too hard as force will cause the microorganisms to collect at the edge of the spreader, resulting in uneven distribution.

Using a glass spreader:
You will need a reusable, glass spreader and a petri dish of 95% alcohol. The spreader is sterilized by briefly dipping it in the alcohol and passing it through the flame of your Bunsen burner. (The Bunsen burner is not used to heat sterilize the spreader but only for igniting the ethanol.) After the spreader has been flamed, it should be moved through the air as little as possible. In order to cool the spreader, the cover of the Petri dish is lifted just enough to get the spreader under it. Briefly touch the spreader to the agar avoiding the dispensed dilution (touching the hot spreader to the microbial solution may kill cells.) Use the sterilized spreader to gently push the dispensed sample two or three times clockwise around the dish, and then several times counterclockwise. Don't press too hard as force will cause the microorganisms to collect at the edge of the spreader, resulting in uneven distribution.<

Using sterile glass beads
Find a flask of presterilized glass beads. Remove the cap, pass the lip through the flame and shake 10-20 glass beads onto your inoculum. Place the cover on the agar plate and place your fingers lightly on the center of the plate cover. Using a circular motion, move the beads around the plate until the inoculum is evenly distributed all over the surface of the plate. Pour the now contaminated beads into a small beaker of disinfectant so they can be sterilized for reuse.

9. Repeat step 8 for 10^-5 through your last dilution.

10. Allow moisture to be absorbed into the agar before inverting and incubating at the appropriate temperature.

Basic Dilution Help

There is a helpful animation of making dilutions and solving dilution problems available at:
[| Link to Wellesley College Biological Concepts & Lab Dilution Tutorials at http://www.wellesley.edu/Biology/Concepts/Html/dilutions.html]