3rd EXAMPLE: A MORE COMPLEX CULTIVATION EXPERIMENT. (again, hypothetical)
Ok, let’s say your experiment above supported the fact that DE holds more water than verm. Now you want to take it to the actual cultivation step, so you need a new experiment with a new hypothesis.
#1. Establish your objective. What is it that you’re trying to establish? The objective is to determine whether DE is a suitable substrate additive in place of verm. Because we know it will hold more water than verm, we suspect that it might therefore retain more moisture in a final sterilized, colonized mushroom substrate than verm, which would of course provide the moisture needed to produce lots of fruiting bodies. So, we’d like to test whether DE can be used instead of verm in PF cakes.
#2a. Form a testable (measurable) hypothesis. Ok, so if it’s a “suitable” alternative, it has to be better for some reason – ease of use, cost or availability of materials, better mushroom production, etc. Let’s choose “better production” as our basis for investigating DE as an alternative to verm.
Remember that a hypothesis must predict something that can be measured objectively. A hypothesis that predicts “better mushroom production” is NOT testable - “production” has no established measurement system, can mean different things to different people, and the degree of “productivity” can often be a subjective decision. So, refine your hypothesis to evaluate something measurable that relates to production: how about, average total dry weight yield per substrate weight?
Now you can state your hypothesis: Because of the higher observed water-retaining properties of DE, a PF cake substrate made with DE in place of verm will produce, on average, a higher dry weight of harvested mushrooms per substrate weight, when compared to the typical PF cake containing verm.
#2b. Explain what type of controls you will use. Remember we need our baseline to determine whether this new, untested treatment really makes a difference. We need to compare it to the “normal” method when tested under identical conditions.
- So let’s establish the control (baseline/normal) condition: X grams of BRF + Y grams of verm @ field capacity.
- And then, the experimental (new/untested) condition: X grams of BRF + Y grams of of DE @ field capacity.
- You could also add in a negative control condition: X grams of BRF @ field capacity, no DE or verm added. (this allows you to see how BRF performs without any additives)
(Note that X and Y are variables; you can choose whatever values you want for X and Y, as long as they are constant across all conditions.)
Now, how many jars of each should you do??? Just 1 of each is surely not enough to remove chance from the equation. Let’s go with 5 jars of each condition (experimental, control, and negative control). And then, let’s ask that this entire experiment be repeated at least twice.
#3. Explain your methodology. Establish a strictly controlled method that will allow you and others to repeat, tweak, and perfect the various aspects of the experiment. Again, it doesn’t matter what method you use for your own personal experiment, as long as you MEASURE and RECORD everything and keep all parameters consistent for all of your samples! You should refer to a tek to get the details for your chosen method, and then tweak it to address your hypothesis. I won’t provide all the details of any TEK here since that’s not the point of this thread
Let’s propose the following hypothetical method:
a). Prepare basic BRF. Make sure that the texture/moisture/additive content of the final substrate is consistent for ALL conditions. Once each substrate type is mixed, place a weighed amount of completed substrate into each jar. Record the following:
- WEIGH the amount of BRF used to prepare the mix.
- WEIGH the amount of additive (DE or verm) + water for each condition.
- WEIGH the amount of completed substrate placed into each jar.
- NUMBER AND LABEL your jars so you know which is which !!!
Notice that everything is WEIGHED (not measured by volume) for consistency!
b). Sterilize and cool all prepped jars in an identical manner, in the same PC run if possible.
- RECORD the PSI on your PC, and TIME how long you maintained that pressure to sterilize.
- TIME how long the jars sat between completion of the PC run and when you actually inoculated them.
c). Inoculate the jars. Let’s say we’re using an LC prepared with tissue from an isolate. But you can use any isolate inoculum you want (slurry, etc.). *** NOTE: DO NOT USE MULTISPORE INOCULUM FOR A CONTROLLED EXPERIMENT, UNLESS YOU SPECIFICALLY WANT TO TEST A HYPOTHESIS RELATED TO GENETIC DIVERSITY. Using multispore introduces way too many uncontrolled variables and prevents you from gleaning anything meaningful from your results. ***
- HOMOGENIZE the inoculum first.
- MEASURE how much goes into each jar (be consistent).
d). Incubate the jars. The conditions (temperature, humidity, lighting, airflow, etc.) must be kept nearly identical for ALL of your jars. If any of these conditions fluctuate between the experimentals and controls, you’re introducing a whole new set of variables and, just like using multispore, it prevents you from gleaning anything meaningful from your results. However you decide to incubate, just be consistent!
- RECORD the temperature and humidity of the incubator/chamber and any other important things such as day/night fluctuations in temp, lighting, airflow, etc.
- TIME how long it takes the jars to fully colonize once introduced to the incubator.
e). Fruit the jars. Again, keep your conditions the same for all the jars!! Record your parameters … etc.
#4. Collect and analyze your data. THE FUN PART – RESULTS!!! Since you’ll recall we are measuring dry weight of harvested mushrooms per substrate weight, we must harvest fruits, dry them to standard “cracker dry” state, and weigh them - from EACH jar. You have 5 jars from each condition, so average the dry weight from those 5 jars to find the average dry harvest weight for the condition.
You might have noticed that “harvest per substrate” is a ratio, so you need to compare the average weight of the HARVEST to the average weight of the SUBSTRATE. Why did we set up this way? Because a substrate that is larger will naturally produce greater harvest, so we need to eliminate this bias from our results. Aren’t you glad you weighed the amount of substrate going into each of those jars in Step 3A????
(Note: This ratio is needed only because our hypothesis suggests a ratio. Just make sure the format of your results matches whatever parameter you included in your hypothesis!)
So, divide the average weight of the dry harvest for that condition by the average weight of the substrate in those jars.
- Experimental Cakes = 12g dry (avg) harvested from 150g (avg) cakes = [8g] / [150g] = 0.080
- Control Cakes = 8g dry (avg) harvested from 145g (avg) cakes = [8g] / [145g] = 0.055
- Negative Control Cakes = 4g dry (avg) harvested from 140g (avg) cakes = [4g] / [140g] = 0.029
Notice our results have no units (they cancel out). This allow us to compare the harvest per substrate simply by looking at the final ratio value we calculate.
#5. Determine whether the hypothesis is supported by your data. Recall our hypothesis: Because of the higher observed water-retaining properties of DE, a PF cake substrate made with DE in place of verm will produce, on average, a higher dry weight of harvested mushrooms per substrate weight, as compared to the typical PF cake containing verm.
So, did it actually do that???
We can see that the experimental cakes have a higher ratio of harvest to substrate weight (0.080) than the control cakes (0.055). In other words, the experimental condition w/ DE produces MORE harvest than the control w/ verm, given the same weight of substrate. Our data also shows that any additive (0.080 or 0.055) is better than NO additive (0.029), which is a nice supporting piece of information.
So, the conclusion: YES, our data supports the hypothesis, and YES, DE produces a higher harvest yield than the conventional verm in PF cakes, under the specific conditions tested.
HOORAY! Now it’s time to repeat the experiment and publish this as an accepted new TEK !!!!!
If you’ve managed to get this far, hopefully you can appreciate the value of this method in facilitating transparency and repetition. EVEN FAILED EXPERIMENTS ARE WORTH DOCUMENTING!!!! For instance, if the results in the last experiment had NOT supported the hypothesis, other experimenters could then tweak/refine/improve aspects of your method. Once it has been hashed out, the final methodology can be published as a “TEK,” and then other people can simply follow the TEK to get great results – thanks to all your hard work!!!!
Edited by Bombadil, 18 December 2014 - 11:13 PM.