in this video you will learn how to perform a new
Time-saving technique for serial dilutions and subsequent spot plating that involves the use of multi-channel pipettes
Here is the equipment you will need
Before you start make sure you calculate the dilutions yu'll be using so you know how much diluent and sample you need for each step
First you need to fill the wells in the mega block with your diluent poor enough diluent into a reservoir
set a multi-channel pipette capable of holding your desired diluent volume to the proper amount and
attach pipette tips to the pipette
In this example. We are using a diluent volume of 450 microliters
Insert the tips into the liquid in the reservoir and draw up
Ensure that all tips draw up the same amount
Then insert tips into the wells of the mega block where you want to transfer the liquid to and dispense
Repeat the step to fill all the wells you need for your dilution preparation
Next use a single-channel pipette to transfer the amount of each sample
needed to make the first dilution into corresponding wells in the first row
remember to change pipette tips between samples
You can label the rows with tape to help you remember where each sample should go
Before you make another dilution
take a 20 to 200 microlitre multi-channel pipette with tips attached
and set it to the right volume according to the dilution you want to make
Insert the tips of the pipette into the wells of the first dilution
and press and release the plunger button about 20 times to mix the contents of the well
then with empty tips draw up the amount of solution you need from the first dilution and
Transfer it into the second row of wells where you want to make the second dilution
Here the use of a multi-channel pipette and a mega block makes it possible to prepare serial dilutions
for several samples at the same time
Make sure you only attach the number of pipette tips to your pipette that matches the number of samples used each time
Change the pipette tips before you proceed to mixing the second dilution
Repeat the previous step several times to make as many delusions as you need
Always remember to change tips between dilutions to avoid carrying over any extra volume
Label an agar plate and draw a line down the middle of your plate at the bottom to divide the space in half
This allows you to play two replicates of the same sample on the same plate
Set the volume of a two to 20 micro liter multi-channel pipette to ten microliters
You can choose which dilutions you want to plate
here we made three dilutions for four samples, and we will plate the first and second dilutions of the first sample as an example
attach the number of tips on to your pipette that matches the number of dilutions you want to plate
and insert the tips into the wells that hold the dilutions needed
Draw up ten microliters of liquid and double check if the volume in each pipette tip is the same
With the tips touching the agar slightly press the plunger button lightly to release small droplets of sample onto one half of the agar plate
Continue dispensing in a straight line until the ten microliters of sample is all plated
Do not poke the agar with pipette tip and avoid dispensing large droplets of sample for easier colony counting
Repeat this step on the second half of the agar
After the plates are dry invert them and incubate as needed
Here is a sample plate after incubation
To begin colony counting start with the dilution roll that has the least amount of colonies
you need to count both halves of the plate separately
since the colonies might be small make sure you count all colonies in each droplet
if the count of the most diluted column is within the countable range
which is best between 10 and 80 colonies
record this value for further analysis
if it's out of the range move on to the next dilution column
and repeat the process until you obtain a plate count that's within the range
Thank you for watching