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Invisiblemaynardjameskeenan
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Re: piximetre manual in english? [Re: Alan Rockefeller]
    #19464245 - 01/23/14 07:15 PM (10 years, 6 days ago)

Quote:

Alan Rockefeller said:
The numbers look good!

Regarding the photo, it's a bit grainy.  If the table is still you can turn down the ISO and get better quality images.



I'm using the eyepiece camera that Joust kindly sent me, I don't know if I change the ISO or not.
I can adjust the amount of light using the thingy that's between the light-source and where the slides are held, I don't know the proper wording for it.


--------------------
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May you be peaceful and at ease.
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OfflineAlan RockefellerM
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Re: piximetre manual in english? [Re: maynardjameskeenan]
    #19464284 - 01/23/14 07:22 PM (10 years, 6 days ago)

Quote:

maynardjameskeenan said:
I'm using the eyepiece camera that Joust kindly sent me, I don't know if I change the ISO or not.





I don't know either.


Quote:

I can adjust the amount of light using the thingy that's between the light-source and where the slides are held, I don't know the proper wording for it.





It is possible that if you give it more light, the computer will automatically lower the ISO.  Giving it more light will also reduce diffraction interference and depth of field, but if you give it too much light, you'll get a decrease in contrast.





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Invisiblemaynardjameskeenan
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Re: piximetre manual in english? [Re: Alan Rockefeller]
    #19465023 - 01/23/14 09:46 PM (10 years, 6 days ago)



8.16 4.78
8.12 4.80
7.92 4.98
7.52 5.12
7.82 5.08
8.16 5.44
8.05 4.78
7.97 4.82
8.25 5.03
7.83 4.53
8.34 5.00
8.71 5.19
8.40 4.86
7.50 4.37
8.12 5.31
8.08 4.82

7.5 [7.9 ; 8.2] 8.7 x 4.4 [4.8 ; 5.1] 5.5 µm
Q = 1.5 [1.6 ; 1.7] 1.8 ; N = 16 ; C = 95%
Me = 8.1 x 4.9 µm ; Qe = 1.6



--------------------
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Edited by maynardjameskeenan (01/23/14 09:50 PM)


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InvisibleByrain

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Re: piximetre manual in english? [Re: maynardjameskeenan]
    #19465049 - 01/23/14 09:50 PM (10 years, 6 days ago)

How are you making your sections?  Kind of looks like too much material, you would probably get better visibility if you used a smaller/thinner section.  If you want to look at cheilocystidia, just a little bit of the gill edge for example.


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Re: piximetre manual in english? [Re: Byrain]
    #19465138 - 01/23/14 10:05 PM (10 years, 6 days ago)

This shit is real!

I wish I had looked through a proper microscope, ever.


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Invisiblemaynardjameskeenan
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Re: piximetre manual in english? [Re: domesticgnome]
    #19465254 - 01/23/14 10:32 PM (10 years, 6 days ago)

Quote:

Byrain said:
How are you making your sections?  Kind of looks like too much material, you would probably get better visibility if you used a smaller/thinner section.  If you want to look at cheilocystidia, just a little bit of the gill edge for example.



I tried to take a small section of the true edge so I could look at the cheilocystidia but I don't think I did it right.
The gill slice was only about the size of a sliver, I don't think I crushed it with anything which would probably help.
I'm just rehydrating them with a tiny drop of water if that matters.
I'm going to try and see if Nomadbrad has more specimens of them because I sent out and cut up the only ones I had.
Quote:

domesticgnome said:
This shit is real!

I wish I had looked through a proper microscope, ever.



You should get one, they are great :smile:


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OfflineAlan RockefellerM
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Re: piximetre manual in english? [Re: maynardjameskeenan]
    #19465400 - 01/23/14 11:13 PM (10 years, 6 days ago)

Quote:

maynardjameskeenan said:
I tried to take a small section of the true edge so I could look at the cheilocystidia but I don't think I did it right.
The gill slice was only about the size of a sliver, I don't think I crushed it with anything which would probably help.





For cheilocystidia you want to use a very thin slice (approx 1 / 10th of a mm) that is as long as possible and as near the stem as possible, and ideally from one of the shorter gills.  A dissecting scope can help a lot when trying to make cuts like this.


You can stain it, but stain is not required.  It should be crushed, but not so much that the spores or cystidia break.

Rehydration of dried material can be done in one of 2 ways:

1) 70% isopropanol, then water
2) 5 - 10% KOH solution


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InvisibleJoust
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Re: piximetre manual in english? [Re: Alan Rockefeller]
    #19465648 - 01/24/14 12:28 AM (10 years, 6 days ago)

Quote:

Alan Rockefeller said:
Quote:

maynardjameskeenan said:
I tried to take a small section of the true edge so I could look at the cheilocystidia but I don't think I did it right.
The gill slice was only about the size of a sliver, I don't think I crushed it with anything which would probably help.





For cheilocystidia you want to use a very thin slice (approx 1 / 10th of a mm) that is as long as possible and as near the stem as possible, and ideally from one of the shorter gills.  A dissecting scope can help a lot when trying to make cuts like this.


You can stain it, but stain is not required.  It should be crushed, but not so much that the spores or cystidia break.

Rehydration of dried material can be done in one of 2 ways:

1) 70% isopropanol, then water
2) 5 - 10% KOH solution



3) use whatever the hell GSM is...


--------------------
~~~~~~***Psilocybin Mushrooms***~~~~~~
_________A Practical Guide To Psilocybin Mushrooms_________

:sporedrop:                      "Think about the species, not your scale". -NeoSporen                      :sporedrop:

"Mr. Joust, I see you don't actually partake in the psilocin, but it looks like it may partake in you!" -Gojira


       


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Invisiblemaynardjameskeenan
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Re: piximetre manual in english? [Re: Joust]
    #19478580 - 01/26/14 07:40 PM (10 years, 3 days ago)

Is this a Cheilocystidia?


I think these might be Basidium, I'm not sure though. It all looks like translucent blobs to me :shrug:


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May you be filled with loving kindness.
May you be well.
May you be peaceful and at ease.
May you be happy.



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Offlinesuchen
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Re: piximetre manual in english? [Re: maynardjameskeenan]
    #19478740 - 01/26/14 08:19 PM (10 years, 3 days ago)

Bottom left in the 2nd photo is a basidium. Not sure about the first photo.


--------------------
Rod Tulloss said:

The bulb is the bulb.

The volva is the volva.

They have a very long term realtionship, but they’re “just friends.”


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Re: piximetre manual in english? [Re: suchen]
    #19478851 - 01/26/14 08:48 PM (10 years, 3 days ago)

1 seems a bit large to be extended hyphae, my guess would be a cell that is unhappy, like Cheilocystidia that had been jacked up.


--------------------
~~~~~~***Psilocybin Mushrooms***~~~~~~
_________A Practical Guide To Psilocybin Mushrooms_________

:sporedrop:                      "Think about the species, not your scale". -NeoSporen                      :sporedrop:

"Mr. Joust, I see you don't actually partake in the psilocin, but it looks like it may partake in you!" -Gojira


       


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OfflineAlan RockefellerM
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Re: piximetre manual in english? [Re: Joust]
    #19479248 - 01/26/14 10:22 PM (10 years, 3 days ago)

You should do a crush mount when looking at cystidia.


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Invisiblemaynardjameskeenan
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Re: piximetre manual in english? [Re: Alan Rockefeller]
    #19479448 - 01/26/14 11:06 PM (10 years, 3 days ago)

Thanks for your help guys. :rockon:


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May you be peaceful and at ease.
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InvisibleByrain

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Re: piximetre manual in english? [Re: maynardjameskeenan]
    #19479495 - 01/26/14 11:18 PM (10 years, 3 days ago)

Don't worry about crushing it too much, its good experience to see broken spores, cystidia & cover slips at least once.  I like using a dissecting prod with a bent end for crushing, sometimes its good to crush while using the 4x & 10x objectives so you can see what you are crushing.  Also, use a headlamp or some equivalent light source while making sections, it really helps!


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OfflineAlan RockefellerM
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Re: piximetre manual in english? [Re: Byrain]
    #19479684 - 01/27/14 12:09 AM (10 years, 3 days ago)

Quote:

Byrain said:
I like using a dissecting prod with a bent end for crushing,




Oh that's what that's called.  That is what they use in the Guzman lab.

Quote:

sometimes its good to crush while using the 4x & 10x objectives so you can see what you are crushing.





I always do that,  It really helps when you can watch the cells get crushed.


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Invisiblemaynardjameskeenan
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Re: piximetre manual in english? [Re: Alan Rockefeller]
    #19487506 - 01/28/14 05:59 PM (10 years, 1 day ago)

What is the best thing(surface) to cut mushrooms on to make section for microscopy?
I've been using a plastic cutting board but it's really bumpy and kinda sucks.
I'm also having trouble understanding how I'm supposed to get a section 1/10 of a mm thin.
How do you hold it still enough to make such precise cuts?
I think I understand what I supposed to be looking for, it's just getting there I'm having trouble with.
Do any of you know of any good YouTube videos of someone preparing slides?
I'd like to see someone make some slide and not just read about, I think I probably learn better that way- visually.


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May you be filled with loving kindness.
May you be well.
May you be peaceful and at ease.
May you be happy.



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OfflineAlan RockefellerM
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Re: piximetre manual in english? [Re: maynardjameskeenan]
    #19488590 - 01/28/14 08:53 PM (10 years, 1 day ago)

I hold stuff in my hand when making cuts.  To get things super thin you use a dissecting scope and just chop against the ground.

But I usually cut by hand on a very slight angle, so one side is way too thick and the other side is impossibly thin.  Then cut off the thick side.

There is a nice video on youtube on making pleurocystidia mounts, but I didn't see it just now when I looked.


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Re: piximetre manual in english? [Re: Alan Rockefeller]
    #19489070 - 01/28/14 10:39 PM (10 years, 1 day ago)

Someone should make a tutorial video for fungi microscopy if there isn't already one. I know I'll have a billion questions when I get one. I remember a lot of the basics from school and when I had a little one as a child but that's it.


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Re: piximetre manual in english? [Re: nomadbrad]
    #19489298 - 01/28/14 11:30 PM (10 years, 1 day ago)

I hope this works and helps us all. I copy pasted the page The calibration (Changer d'étalon) through a translator and tried to link all the original pics on the page.

The calibration Piximètre

Convert pixels to unit length

The calculation of the length of an axis on an image drawn on the screen is performed by first pixels. Both ends of this axis are determined by their coordinates (x , y) relative to the screen (actually relative to the window that supports the image, but it is only a question of origin for the unit remains the same: the pixel). The axis is the hypotenuse of a right triangle . Its length is calculated by a square root ( Pythagorean theorem ) .

The purpose of calibration is to establish a relationship between pixels and selected unit of length (e.g., micron ) . For this it suffices to measure in pixels an object that perfectly knows the length in this unit !

The introduction of a magnifying glass that can expand or contract the scale corresponds to the change in this relationship by applying the multiplier of the microscope.

Very precise measurements of length , usually less than 2 % error (often with only 0.1 to 0.2 %) can be performed with Piximètre subject to having conducted a precise prior calibration.


Figure 1: Measuring a marker of known length .

Given that the images are mainly obtained using cameras Varifocal (zoom) , two separate operations are required to perform this calibration entirety:

initialization for each automatic autofocus camera used . Allow the calculation of image magnification for each zoom position used to shooting this operation aims ,
calibration of length itself, which aims to establish the relationship pixel - unit length.
These two operations of different kinds, are independent and can be performed in any order . You will perform the first operation once for each camera. The second can be repeated to set multiple standards ( see below) .

When images are obtained from cameras with fixed focal length (eg some cameras) or when they do not contain the necessary identification conditions shooting ( metadata) , only the length calibration is possible and necessary.

The Settings tab Piximètre calculator allows you to specify the nature of the images used , Normal (with metadata) compressed or otherwise .

It is strongly recommended that you read this note on the optical and measurement errors.



Initialization of automatic autofocus

Routine measurements and calibration of length, performed on pictures taken with one (or more ) digital camera. The intrinsic image magnification is a function of the position of zoom used to shooting , all things being equal as to the conditions thereof .

The initialization of the automatic autofocus Piximètre aims to establish the precise mathematical function that allows calculation of the magnification (see this operation here).

Piximètre uses the metadata contained in the photos ( but invisible to ) for the focal length used in the shooting. We will never heed to compress images which are intended . Indeed this can remove metadata they contain if the compression rate chosen is too high. Piximètre also easily handles images very large (the only limitation is the memory capacity of the computer used).

This initialization autofocus operation is performed once and for all, for each camera used , the pictures will now be correctly interpreted regardless of the focal length used for the shooting .

The autofocus Piximètre manages any number of devices different picture . Also, to work with images from several of them , it will be necessary to advance this initialization for each of them.

Piximètre also offers the possibility to directly import math functions in question, calculated and exported elsewhere ( see here). This is useful when a user wants to work with images that have been taken by others, with a camera that he does not have.

Also recall that Piximètre visually indicates the operation of autofocus images it processes (see here) .

Length calibration

What reference object and choose how to calibrate measuring Piximètre ? The answer to the first part of the question is simple: any visible object through the optical system used (eg microscope ) and are fully aware of the actual length ! This object reference is chosen so that its length is related to the scale subsequent to measure objects . For example, if you want to measure fungal spores whose magnitude is ten microns , you choose as a reference standard rule or an object of this magnitude rather than a double decimeter .

As to the second part of the question , just shoot this object to a defined state of the optical system . The calibration is then carried Piximètre :

by drawing an axis on the image of the object of known length ( Figure 1 , above) , and
indicating the exact length that represents the axis.
This operation is performed using the calibration Piximètre assistant . Note that if you previously initialized autofocus for the camera in question, here you can use any zoom for shooting. The correction will be made automatically. You can also use the magnifying glass and the lateral displacement of the image, essentially to magnify and position the image on the screen and increase the pointing accuracy .

Consider for example the case of a microscope :

Shooting through a calibrated micrometer eyepiece , ie you know the exact value of each division of his rule for the microscope objective used . The image of this rule appears in the photo and calibration is done by plotting Piximètre axis over all or a portion of the rule ( Figure 1 , above) and by indicating that it represents the exact length .

Shooting without a microscope eyepiece or through a non- ocular micrometer , ie you do not have the scale marked on the image. Piximètre must be calibrated independently of the images , using a scale of reference directly under the microscope objective . This is a different rule of the previous so-called object micrometer . It generally comprises a series of spaced apart divisions of 10 microns and is typically used to calibrate the optical microscopes . Here also the interval between tick marks is precise and well known . It therefore takes a photo of this rule and we proceed as in the case above.
It is important to note that in either case , you are shooting through an eyepiece micrometer or not, or directly in its place , behind the lens , and all things being equal, Photo value of a division of the scale reference depends on the lens used on the microscope, where the concept of state of the optical system introduced earlier. The calibration is valid only for a specific purpose.

Piximètre provides the ability to define and use different standards corresponding to different states of the optical system , such as the use of different objectives on a microscope.

The Calibration Wizard


     Figure 2: First calibration step .

The Calibration Wizard is activated by the " Calibrate " button in the measurement window on images or the "+" button that appears to the right. The "+" button triggers the first step . The " Calibrate " button is a shortcut that leads directly to the second stage ( Figure 3).

The first step (Figure 2 , below cons ) is activated by the button "+" specifies the operations to be performed :

Create a new standard or modifying an existing standard. You will see later that there are two types of standards and it is possible to name some of them to facilitate their use and be able , if necessary , change them.

Delete an existing standard.
The active icon tutorial on calibration.

The second step is based on the choices that are made . If the creation or modification is selected, the wizard helps you define a standard ( see Figure 3 below) . If removing one is selected it takes you directly to the operation. If both options are selected , the wizard helps you to first create ( or modify ) a standard and then the removal of another.

Here are below the calibration step , in three steps .


Figure 3: The calibration process at step # 3 .

The calibration process comprises three successive steps as shown in Figure 3 . You can open the reference image before or after the activation wizard , or even change during the process and possibly take it to step 1 (button " Resume" ) .

In step # 1 you choose the scope of the calibration. You may decide that the standard will be specific to this image, ie applied alone or generic contrary, ie applied by default to all images , open or future , who do not have already a specific standard ( see below ) . It appears here two standard types : specific or generic .

In step number 2 you draw a line on the ruler or object of known length, from the image . Note the length of the display of the axis in pixels ( 257.52 in FIG 3 ) . Also note that greater accuracy will be obtained by choosing a very long axis (decreased relative error inherent in the measurement) , subject, however, perfect linearity of the image. See here about it. You can also use the magnifying glass that improves the pointing accuracy and the lateral displacement of the image on the screen between each of the two points.

In step # 3 Finally, you specify the actual length of this axis in the appropriate unit (40 microns in this example where the object micrometer is 10 microns per division).

The Calibration Wizard , by default, has different units of length , but here you have the possibility to define new ones if those offered are not suitable. The choice " - Edit - " available in the dropdown list gives access to this feature.
The validation process ends when it comes to a specific calibration , this single image. The new standard is applied to the image and the axis is cleared thereon .

In the case of a generic calibration , apply by default on all images , the "OK" button presents a new step in the wizard asking to name the standard ( Figure 4).



Management requires more generic standards , in fact, their naming, which ensures their identification.

You can , for example , set a different standard for each microscope objective . Here, the new standard is named " X100 -995 " because the picture was taken under a microscope objective X100 with a Nikon 995 camera.

A list shows the generic standards already defined. One of them is permanent " Unity ( UN) ," the others were defined by the user. Selecting an existing standard allows its redefinition .

The "Finish" button validates the standard and ends the process . The new generic standard is assigned to all the images involved and , of course, the Formulator and comparison are updated .

If the standard name already exists , the wizard may, under the control of the user, replace its value with the new .

The "Delete " button to delete a selected list standard.

Choose a generic standard

Calibration standards are created as described above.

This is the measurement window Image (Figure 5 , below) that the user chooses the generic standard that will be applied to all currently open or future images and do not already referring to a specific standard. Remember that this standard is to choose according to the state of the optical system used in the shooting , in other words, in the case of a microscope, the lens used (one for x40 and one for x100 , not to mention the eye or the camera used).



yyyy Figure 5: Click on "generic Stallion " on the left shows the list on the right .
Figure 4 : Management of stallions names.
As shown in Figure 5, a mouse click on the link " Etalon generic " (or " Etalon Specific " ) shows the list of available standards and allows to choose one ( to the right).

It will be applied to all open images or future using a generic standard , ie which does not refer to a specific standard.

The generic standard selected remains valid as long as it has not been changed by the user.

Generic standard , specific standard

When you first open an image, Piximètre has no information on the optical system that is its origin. By default , therefore , the image is assigned the generic standard that is currently selected . This corresponds to the general case where most of the pictures are from the same optical system (eg they were all photographed with the same microscope objective ) .

The user must change standard or practice a specific calibration if this image is not the case .

The active memory Piximètre , when pressed, allows you to store all the parameters of an image at the time of its closure : its position on the screen, use the magnifying glass , all plots axes , etc. . and , in particular , the name and the value of the standard applied thereto and which allows the accurate calculation of the length of the axes .

At its subsequent reopening , if the working memory is always engaged, the image will return all settings and displayed in the exact condition it was in at the time of its closure. The standard length which allowed the calculation of the axes is compulsorily returned , regardless of the standards in the system , so defined . It becomes SPECIFIC to the image.

Thus, at its first introduction in Piximètre , an image is assigned by default to a generic standard , the user can maintain or, if necessary , change . But during its subsequent reopening this standard , whatever it was , is specific to the image and can not be affected by any modification of the generic standards , and his name is preserved. This principle ensures the conservation measures to images at their subsequent reopening .

In case the user deletes or redefined a generic standard that has previously created and used , all images of the active memory that reference it are transformed : the value of the standard course but kept his name amended by adding the "ex" prefix , since it no longer exists in the state. At its reopening with active memory triggered image finds the value of its initial standard but the name of it is then prefixed by "ex".


Edited by dodeski (01/28/14 11:39 PM)


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Invisiblemaynardjameskeenan
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Re: piximetre manual in english? [Re: dodeski]
    #19489348 - 01/28/14 11:40 PM (10 years, 1 day ago)

Holy shit, thank you! :rockon:
That's excellent man, very helpful.
:raisemyglass:


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May you be filled with loving kindness.
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May you be peaceful and at ease.
May you be happy.



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