Feb 07, 2019
E. coli Plating Quantification
- 1University of Arizona
- 481b Laboratory
Protocol Citation: Kenneth Schackart, Kattika Kaarj 2019. E. coli Plating Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.xqhfmt6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 04, 2019
Last Modified: February 07, 2019
Protocol Integer ID: 19945
Abstract
This protocol describes how to serially dilute and plate cultured E. coli K-12 in suspension for quantification.
Guidelines
Labcoat and gloves must be worn at all times.
Materials
- Gloves
- Stock E. coli K-12 culture
- Pipette and tips
- 2 mL centrifuge tubes
- DI water
- Agar plate
- Inoculation loop
- Parafilm
Prepare Serial Dilutions
Prepare Serial Dilutions
Transfer 100 µL of stock solution to 2 mL centrifuge tube.
Add 900 µL DI water to the same centrifuge tube to make a dilution that is one-tenth the concentration of the stock solution.
Using 100 µL of the dilution you just made, make another dilution that is one-tenth the concentration of the second solution.
Repeat 6 times until you have a solution that is diluted by 106.
How to perform serial dilutions. X represents the number of 10-fold dilutions performed at each point.
Plate
Plate
Choose a dilution that you feel is appropriate and dispense 100 µL of that dilution on agar plate.
Evenly spread the cell suspension over the agarose gel with inoculation loop.
Note
This may be different from how you have streaked before. We are wanting the solution evenly spread so that we can count how many colonies are formed after incubation.
Wrap the agar plate's edge in parafilm.
Note
If you have not worked with Parafilm, ask a TA for help in wrapping your plate.
Wait 2 minutes, then flip the plate upside-down for incubation.
Label the plate with:
- Group number
- Date
- Dilution used (10^x)
Incubate
Incubate
Incubate at 37 °C over night.
Note
Performed by T.A.
Incubate
Incubate
Take image of incubated plate.
Note
Performed by T.A.
Include this image in the lab report results section.
Quantify Concentration
Quantify Concentration
Using the image of your plate, count the number of colonies formed. This can be done by hand or using an image processing software such as ImageJ.
Calculate the original E. Coli concentration using the following equation:
Include the result of this calculation in the lab report.