Gametophytes per mL - painting vs spray bottle method
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Hello, sorry if this is in one of the guides or has been answered already but I couldn't find it. All info I could find was for spores or by weight. I am blending the gametophytes and then counting them using a hemacytometer. How many gametophytes per mL should I be aiming for when painting onto a spool and how many when using a spray bottle? Thanks!
@levi_44 We portion gametophytes by weight (.1 gram/100ft of seedstring) which is a more efficient/consistent way of getting even biomass applied to the seedstring.
Our method is documented here and there is a protocol for weighing gametophyte biomass at the end of the less.
@toby_sheppardbloch I don't have the equipment for the weight method but thank you.
@toby_sheppardbloch I don't have the equipment for the weight method but thank you.
@levi_44 Hi there! The "industry standard" (AFAIK) is either weighing or measuring the density with a spectrophotometer (Optical Density).
Establishing a reliable seeding standard via the weighing method is very tedious, finicky and prone to contamination. That's why I'll always recommend the other method, measuring/counting particles.
You mentioned that you can count gametophytes with a hemacytometer, I recommend to make the method robust. Make a dilution gradient and measure each sample at least thrice to verify the accuracy of the method and the machine.
The gametophyte density side is very much based on probability if there is no established optimal seeding density. Some seed spores, others gametophytes or sporophytes. Each method requires in house experimentation and research.
First you'll have to establish the sporophyte(plantling) density that you would like to have per meter of line. Lets say it needs to be 6000 sporophytes per meter. Based on the basics of biology, you'll need at least a male and female gametophyte for 1 sporophyte. Then you'll need to account for fertility rates and death due to stress caused by handling/preparation.
This would result in a formula (very simplified) like this: (desired density * 2) * fertilization factor * death factor * line length
Desired density = Your desired sporophyte density per meter.
2 = 1 female and 1 male gametophyte
Fertilization factor = the rate of fertility. If 50% of the gametophytes fertilize you would need twice as many gametophytes to reach the desired amount of sporophytes. Based on the 50% scenario you would fill in a 2 in the formula.
Death factor = the rate of death. I usually account for 20% of gametophyte death after mechanical processing. Based on 20% death you would fill in 1.25 in the formula.
Line length = The amount of line on 1 spool. E.g. 1 spool is 100 meter of line, fill in 100 in the formula.
So, that would be around 30,000 gametophytes for 6000 sporophyes per meter. Approx. 3 million gametophytes for 1 spool.
The water volume depends on the fluid absorption of the line. E.g. if one spool with line absorbs 100 mL of liquid you would need to concentrate the 3 million gametophytes in 100 ml (30,000 sporophytes per milliliter).
Fluid absorption can be tested easily by applying measured amounts of water on the spool. When the spool starts to drip it should be fully saturated. Repeat this on at least 3 spools to get a reliable and workable volume.
From my own experience I recommend a density between 4000 to 16000 sporophytes per meter. But this is very much based on the desire to create a very densely grown spool, that is also going to weather on sea.
I hope this helps! If you have any more in depth questions you can always send me an email.
Cheers!