Serial Dilution

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Definition[edit | edit source]

A serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M ... Serial dilutions are used to accurately create highly diluted solutions as well as solutions for experiments resulting in concentration curves with a logarithmic scale. A tenfold dilution for each step is called a logarithmic dilution or log-dilution, a 3.16-fold (100.5-fold) dilution is called a half-logarithmic dilution or half-log dilution, and a 1.78-fold (100.25-fold) dilution is called a quarter-logarithmic dilution or quarter-log dilution. Serial dilutions are widely used in experimental sciences, including biochemistry, pharmacology, microbiology, and physics.

(from Wikipedia)

The serial dilution protocol which I will paste below is a step by step guide which I wrote of the technique for serially diluting spore solution to lessen the amount of spores present in the inoculant solution so as to increase the chance of isolating (transferring/fragmenting) monokaryotic mycelium from the germination plate.

Isolating a monokaryotic culture (monokaryon) will allow you to use it in any cross pairing you wish. Success of said pairings are soley dependant on mating type and pheromone compatibility.

Serial dillution protocol[edit | edit source]

Materials needed[edit | edit source]

• 100ul-1000ul Pipette

• Sterile pipette tips

• Sterile microcentrifuge tubes

• Sterile syringe and needle

• Autoclaved water

• Plate spreader(to spread spore solution)

• Spores and spore scraping tool

• Agar plates

Label your microcentrifuge tubes "1x" , "10x", "100x" & "1000x". I also label one "S" for spores and "W" for water. Scrape a small amount of spores into the "S" microcentrifuge tube and draw in some sterile water into your sterile syringe and fill the "W" microcentrifuge tube (500ul-1000ul) Set pipette to 100ul and put 100ul of water from the "W" tube into each tube (1x, 10x, 100x & 1000x). Now put 100ul from the "W" tube into the "S" tube, and shake it around a good bit. Now set your pipette to 10ul and take 10ul from the "S" tube (concentrated spore solution) into the 1x tube. Shake (or pipette up and down) and repeat. Take 10ul from the 1x tube to the 10x tube. Repeat this for the 100x and 1000x as well. I recommend changing your pipette tip at least once before filling the 100x or 1000x tube to lessen the amount of carryover spores. Now each spore solution within each tube (1x-1000x) is diluted to its name.

Now set your pipette to 50ul and suck up spore solution, spray the plate and use your plate spreader to spread to spore solution around as much as possible over the surface of the agar. Label your plates according to the corresponding tube. Using 50ul for plates allows you to inoculate 2 plates for each 100ul of diluted spore solution.

After germination begins you can check your plates under a microscope to look for clamp connections to confirm you have a monokaryon. Also best to look for other structures like conidia to identify whether or not what you're looking at is mold.