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Invisiblec10h12n2o
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c10's Agar Guide + Tips & Tricks (post #1000) * 43
    #24330957 - 05/18/17 09:00 PM (3 years, 4 months ago)

Disclaimer: i am a certifiable agar addict. lots of people ask me for the specifics of how i do agar, and rather than repeating myself every day, here is a convenient guide to the way i do agar, as well as some tips and tricks i have picked up. This is simply my post #1000 way of sharing what i have learned over 20k+ plates, and is not meant to be a comprehensive agar guide, bodhi's comprehensive agar tek does a fantastic job of that, and should be required reading for anyone starting agar. lots of what i do is less than ideal; i will try to point out where i am cutting corners, but i welcome any criticism, questions, additions, refutations, or discussion you have to add :smile:

c10's Agar Guide + Tips & Tricks

or M.A.G.A. : Make Agar Great Again!



couple weeks' supply of plates. make that mailman work for his money.


INTRO:
Agar is ubiquitous in professional mycology (or any form of microbiology for that matter). In the old days, when people were just figuring out the bulk teks, pros had been using agar for decades. For this reason, many people have been intimidated by agar and its prominent use on the cutting edge of mycology, thinking it was a tool reserved for people with proper labs and degrees, far beyond the scope of the average hobbyist. this was very misguided, because in reality making agar is no more difficult than making jello shots, and there are lots of agar techniques that will benefit even the newest of novices. agar removes all of the guess-work and virtually guarantees success. for this reason, these days we like to advise people to START with agar as soon as they enter the hobby

Quote:

Niccolo Machiavelli said:
The wise man does at once what the fool does finally.




do not be afraid of failing. agar gives us the chance to do culture work in very small spaces and run experiments with lots of data points. plates are CHEAP, so dont get attached to them. dont get discouraged by contamination. in fact, you can learn FAR more from failure than you can from success. if you think critically, you can learn a LOT from a contaminated plate: what techniques DONT work, what you are doing wrong, how contams behave on agar, how myc reacts to these stimuli, how extreme your clean technique needs to be, etc.. by comparison, ESPECIALLY with a species as hardy as cubensis, success can teach us relatively little. so learn to see your failures as opportunities to learn: make the most out of them. stay with it and you will be consistently successful, just be patient and persistent while keeping an open-minded 

Regarding SABs and FHs:


To do agar work, you are going to need to be somewhat familiar with clean technique, etc., and will need either a SAB (still air box) or a FH (laminar flow hood/cabinet). whichever route you go, its important to understand the basic principles of how the device works.

SABs:
For a SAB, the principle at work is STILL AIR, meaning you can NEVER get the SAB clean enough to do sterile work (it could be quite dirty and function just fine). STILL AIR (as opposed to STERILE BOX) is why a SAB works. in a SAB we are counting on gravity to pull particles/contams out of the air, and the SAB blocks interference/turbulence from outside.

For these reasons, it is crucial that you understand that contams are concentrated on the bottom of a SAB, and for this reason i STRONGLY recommend placing a damp towel in the bottom of the SAB and working on a cookie rack 2-3" above it. bodhi accomplishes the same thing by using some petris as spacers to raise his work surface off the horizontal floor surface. the idea is put some space between the entry to your clean culture dish (mouth of your petri) and the horizontal surface full of contams, and the towel helps trap contams and keep them from being stirred up by the movements of your hands.

this is also one of the reasons some people have better luck with no-pour/pastyplates than standard petris, since pp5 containers ALWAYS have a MUCH higher rim than petris (usually a matter of multiple inches) and thus have a wider space between the opening to the agar dish and the dirty floor. pouring petris in a SAB with no space between the dish you are workin in and the floor can easily go badly. that space can make a HUGE difference. over the course of about 5k plates without a towel+rack and another 15k with them, the single change of adding the towel+rack brought my success rate from around 94% to 99% clean (from 1/20 to 1/100)

if you need instructions for making a SAB,
check out bodhi's simple AF SAB tek

these are two SABs i built before i constructed my FH, a double hatch one (missing the towel, i hadnt learned that yet and was using contact mats to avoid slippage), and a totally custom one i built in this thread out of plexiglass i found on the side of the road :lol:

2 types of custom SABs i built


that custom one actually turned out really great, i LOVED that thing, it was built into a closet to leverage the walls for extra blocking of air currents and to be easily concealable, and it was plenty tall for bags :smile: . but looking back, i would advise going the simple AF route, then later building a custom SAB or FH to your needs if you still want one (basic SAB is all you need)

Flow Hoods/Cabinets:
in a laminar flow hood the air is passed through a HEPA (High Efficiency Particulates Air) filter which removes all airborne contamination to maintain sterile conditions. A laminar flow hood consists of a prefilter, a blower, and a HEPA filter.

so this is TOTALLY different from the SAB in terms of the principles being leveraged to allow for sterile work. in a SAB we rely on STILL AIR, but in a FH (flow hood/cabinet) we rely on a laminar column of sterile air. basically, you dont want to let anything potentially dirty get in between the sterile airflow and your clean work (like a petri dish)

my beloved flow hood


here are a few FH links for anyone wanting to build one:
c10s flow hood build
stonesun's FH tek
mushpunx FH build

i have REALLY been enjoying mine, it has been a joy to work with. i have literally only seen 1 plate contam since i built it (out of over a thousand) and it was one i dropped on the floor (outside the hood):lol:

Agar Prep:


here is how i prep agar, a liter at a time. i do a few things that arent exactly kosher, and will point those out.

getting materials together


like everything else we do in this hobby, it is CRUCIAL that we understand the point of what we are doing. when working with agar, we want a slightly nutritious medium which supports controlled growth in 2 dimensions. for this reason, nearly any kind of media the myc can digest can work fine (historically people have used cardboard and all kinds of stuff). These days, most people prefer either MEA (the standard in pro mycology), PDA (potato dextrose agar), or GSA (grain soak agar), but since we are using them to accomplish the same thing it doesnt really matter which recipe you go with as long as you have the right balance of agar-agar and sugar.

many of us like to use "half strength" agar with half the normal sugar content, to encourage myc to spread out and look for food rather than grubbing out and staying put, and make it easier for us to spot sectoring and other differences in mycelium characteristics. my preferred recipe is a "half strength" recipe of MEA (20g agar-agar, 15g light malt extract, 1L h2o), which i sometimes alternate with PDA or GSA.

i have experimented a LOT with different agar recipes and preparations, and found the simplest recipes to be the best for uniform results. peptone and yeast would often make the myc act weird, and eventually i quit supplementing with things like that because i realized that focusing on the nutrition of the agar (beyond what is required for healthy, consistent growth) kinda misses the point.

if you plan to be doing lots of agar work, and want to be able to draw meaningful conclusions about how one culture looks/acts vs another one, it is important that you standardize your agar recipe and prep (find something you like and stick to it), otherwise you wont be able to know whether you are observing a characteristic of the culture or a difference due to a different agar recipe

antibiotics are totally not necessary in this hobby, since you can clean up pretty much anything with well-timed transfers. i do keep gentamicin and chloramphenicol on hand, and occasionally use it, but it should absolutely not be a matter of course. the only thing i use it for is the final dish before making long-term master slants, to knock out any hidden bacteria before we put it into storage (just in case). i do not use antibiotics in the master slants, just the final plate before it, and usually dont use them even in these cases (they arent necessary). i have also used it before doing a2g (in the rare occasions when im trying to troubleshoot suspected hidden bacteria), or when a print is so incredibly dirty that the bacteria suppresses the myc from growing at all on standard agar (ive only seen this twice, and i think someone wiped their ass with the print).

i have ordered agar-agar from lots of suppliers, and dont see much difference in the bacteriological (EXPENSIVE) kind, the health food store kilo kind, and the telephone brand kind. as long as it is pure agar-agar, white/offwhite powder, it should be fine. i prefer to buy in bulk, but most often have ended up using telephone brand from the asian stores

old faithful


To make my standard MEA, i like to use a 2L beaker to mix everything in. First, i tare (zero) a food scale with the beaker on it, then weigh out 20g (ish, a little more/less wont hurt) of agar-agar like this:

weighing agar inside mixing beaker


next, i tare the scale again and weigh out 15g (ish) of light malt extract and mix the 2 dry powders together:

weighing malt extract inside mixing beaker


then add a small amount of cold water (tap is fine), just enough to mix the powders into a thick liquid. i like to use a glass stir rod with a rubber policeman on the end (from a lab supply store) to mix this suspension as evenly as possible:

concentrated agar suspension


and then add cold water until the total volume is 1 liter and stir well:

1L of agar suspension


now i add food coloring, which is usefult for 2 reasons: #1 it helps sort cultures visually for color coding, and #2 the coloration makes it much easier to spot healthy white mycelium and distinguish contaminants:

add food coloring (optional)


next i use a microwave to carefully boil the agar suspension and dissolve the mixture into a homogeneous solution. agar is VERY strange when it boils, it can easily start bubbling and boil over into a huge mess. for this reason, you need to keep a close eye on it and stir regularly. i like to get it to start boiling at full power, then reduce the microwave power down to 50% to maintain a controlled boil so it doesnt boil over. even at reduced power, i have to pop it open every few moments. i like to let it boil until i am sure everything has dissolved, then let it go a bit longer just to be sure

keep a close eye on this or you will have a huge mess


next, i take a towel and wrap it around the beaker so that i can handle it, and pour the contents through a funnel lined with cheesecloth, filling 2 1L media bottles halfway each (so they dont boil over). the purpose of the cheesecloth filter is to remove any clumps or foreign particles that might still be in your agar, to make sure everything is of an even consistency. this results in crystal clear agar that looks like glass, and makes pouring predictable. after filling, tighten the lids, then just barely crack them open so that steam can escape during the sterilization cycle

pouring agar through cheesecloth into media bottles, ready to sterilize


i like to sterilize my scalpel handles at the same time i do agar, usualy 8 or so at a time so that i dont have to do it often. i tear off a length of aluminum foil and fold it around a scalpel handle and fold it up into an envelope like shown. i then put a jar filled with these individually wrapped handles in the pressure cooker, with foil over the top so it doesnt fill up with water

individually wrapped scalpel handles for sterilization


these liter media bottles wont fit in my PC if i stand them straight up, so i have to get crafty to hold 2 of them. to accomplish this i stack 4 small jars against one edge and 2 jar rings along the other. i place the top side of the bottles onto the side elevated by the jars, and tilt the bottom edge against the rings, so that the media bottles do not come in contact with the edges of the pressure cooker

media bottles carefully loaded into pressure cooker


i then put hot water into the pressure cooker until it is close to level with the agar in the bottles, then close the lid and fasten it down. now we vent the PC by keeping the pressure valve open and turning the heat on until there is a constant plume of steam and reduce heat to medium-low to maintain this plume of steam. start a timer for 10 minutes and let it vent until the time is up


vent the PC for 10 minutes


when the 10 minutes is up, flip your valve (or apply your weight) and allow pressure to start building up. when it gets above 15 psi, start a timer for 20 minutes and maintain 15-18 psi for the duration of this time

PC for 20 minutes at 15-18 psi


WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC


now wait until it cools down to about 60 degrees Celsius. typically, i try to pour it before it starts gelling around 50 degrees. I like to use an infrared thermometer to check this, its really handy, but this is also the maximum where you can hold the bottle with your bare hands comfortably. so as if you dont have an IR thermometer, just wait until it has cooled down enough to pour comfortably

sterilized agar ready to pour


Pouring Plates:


now we move the agar to the our clean work area. im gonna be demonstrating with a FH, but same principles apply. you will notice, lots of people are successful even though they dont wear gloves, use a towel in their SAB, bathe (like ever lol), use iso, or whatever. this is because everyone's situation is different, and will have different requirements in terms of how extreme the sterile technique will need to be in a given environment. people dont necessarily have to do things perfectly to achieve success, and as you go you will learn which corners you can cut vs what is required in your environment. that said, when you are first working towards clean cultures/spawn, it is a good idea to be as clean as possible until you can reliably achieve clean spawn/cultures. so basically, when you see someone say they dont wear gloves, or dont use a towel, it doesnt mean you shouldnt (at least until you can establish that it doesnt matter in your case). its important to understand how these dynamics of sterile technique play out, and learn what works for you in your environment
 
work area for pouring plates


for a long time i would wear face masks, but my face always got sweaty/hot and uncomfortable after a while. eventually i started using a face shield, and i LOVE it. this thing is great for blocking your exhales (you can cough straight at the agar without worry) and also prevents hairs and skin cells from falling off your face. Another thing i like using is the black heavy duty gloves. these hold up a lot better than the standard gloves, especially over multiple uses. i reuse the same pair of gloves over and over, and wipe them down with alcohol before each session. i like to keep a rag soaked in 70% alcohol (this is dangerous so be careful) in a plastic bag in a container, so that the alcohol doesnt evaporate while im not using it. i pull this rag out and wipe things down with it regularly while working in the FH

face shield and alcohol rag wipe-down


now i pull out 60-80 plates worth of sleeves, and turn them upside down. using a retractable scalpel, i cut the end off each sleeve, then turn them right side up and pull the plastic sleeves off like this:

removing plates from sleeves


next i use the alcohol rag to wipe down my forearms and gloves, then carefully and thoroughly wipe the first media bottle. usually the lid is on very tight, so i use a strap wrench to loosen it, then i remove it by hand

strap wrench really helps break them open


then i center a stack of 20 plates and use the flat side of the bottle to line them up like so:

using the media bottle edge to line up the stack


next, i pick up the top 19 plates and the lid of the bottom one in my left hand, and pour agar into the bottom dish. if the plate needs to last more than long enough to transfer, i pour a layer that covers the whole floor of the dish, then replace the lid and move to the next plate (which is the kosher way to do it). usually, i try to maximize the number of plates i get per liter of agar, even at the expense of the agar possibly drying out in a few weeks (long after it has served its purpose). i do this by pouring about 1/3 to 1/2 the amount of agar required to cover the bottom of the dish, then i tilt the dish back and forth to make the agar cover the entire plate surface. this results in very thin agar medium which dries out easily, but also has some advantages (like not clogging up an LI needle with agar, since anything you inoculate with agar this thin will be almost pure myc, very little agar). picking up the plates is of course a possible contam vector, so i wouldnt recommend doing it this way until you can reliably achieve clean cultures (so that you will be able to tell if it causes problems). this is less risky in a SAB than a FH, since with a FH extra care needs to be taken to ensure that your hands dont get between the flow and your medium. another benefit of super thin plates is that transfers jump off onto receiving dishes faster, since there is less edge for them to colonize before it reaches the receiving dish. for this reason, even when using a standard thickness plate, i like to trim my transfers so that they are as thin as possible (cutting off the uncolonized agar underneath the myc) before transferring)

pouring plates super thin by using a tilting technique to spread the media. this is easiest when the media is relatively hot, because as it starts to gel and clumps form the surface will deform and wont be slick.


when i finish a stack, i grab it like this and move it to the side, then line up a new stack of 20 plates:

moving a stack of finished plates out of the way


about 30-50 plates in (depending on how thin i poured it) my first bottle of 500ml runs empty, so i repeat the alcohol wipe down and use the strap wrench to break it open before removing the lid gently by hand:

wiping down the second bottle


then i straighten up the stack again and resume the pattern: lift lid + stack, pour 1/3-1/2 plate, replace plate, lift again with the lid in place and tilt back and forth until there  are no more bare spots. i do this until the agar starts thickening up and i can see disturbances in the surface of the agar (where clumps distort what we want to be a level 2d field of media). at that point i revert back to the kosher method of pouring the entire plate with no lifting/tilting involved

pouring the rest of the plates


now i let these sit until they have set (normally the first ones are done setting and ready to be used by the time i finish the last ones), and use immediately OR bag up for future use. since ziploc bags are sterile out of the box, i like to use ziploc freezer bags to store ready-to-go plates until they are being used. this isnt normally more than a few hours to a day in my case though. these really thin plates are not suitable for storing for any long period of time and should be used asap (they will dry out)

Parafilm Preparation:


i absolutely LOVE parafilm. when i first got some in a yeast culture kit, i had no idea how it was supposed to be used, so i stretched out the 2 foot segment that came with my kit and promptly realized i had wasted it lol.... only a few years later did i realize the way parafilm is supposed to be used, and that everything i had initially had in mind was a HUGE waste of parafilm. a little bit goes a LONG way. alternatively, you can use saran wrap (bodhi prefers it), but i love the convenience of parafilm.

i like to keep a stack of prepared parafilm wraps ready to go, beside my scalpel, as this greatly speeds up my agar workflow. to set this up, i pull a few feet length of parafilm from the dispenser (the 4" wide kind), and fold it in half to determine the middle, and cut it in the middle (leaving half attached to the roll). next, use paper clips or clamps to fasten the piece you cut on top of the other, so that it is 2 layers deep and perfectly lined up. after that, i use a retractable scalpel to cut off 1/2" to 3/4" horizontal lengths, 2 at a time, and form a stack with the easy-peel ends all facing the same way. as i move up the length, i readjust the clips as needed

#1 cut a length in half, #2 clamp it 2 layers thick, #3 cut off sections 2 at a time


Working With Plates:



this is how i like my workspace to be set up, with scalpel tray, alc burner and butane bunsen,
stack of parafilm strips, 2 different recipes of plates, and a box of ziplocs. ready to work.


Preparing the Scalpel:
when i start working with the plates, i like to begin each session by opening a fresh scalpel handle from one of those foil envelopes (this is optional, flaming works fine too) and take out a fresh individually wrapped #11 scalpel blade. i like to make a small stand out of the aluminum foil from the envelope, where one end is rolled up to hold the blade end at an elevation that it is not touching anything. i put this improvised scalpel stand on the far right of my workspace

getting a scalpel handle and #11 blade ready, and making an improvised scalpel holder out of the foil envelope


EDIT: I have since upgradedto avoid open flames around so much alcohol vapor. I got a Bacticinerator and I couldnt be happier with it. Gets scalpels red hot in about 5 seconds and frees up both hands for labeling


this brings me to my flame setup. i absolutely love this Blazer brand butane bunsen burner. this japanese brand rocks in general (their torches, not their fuel), and i have for a long time been a fan of their Big Shot GT8000 for concentrates (it gets a ti nail red hot in less than 10 seconds, everyone who sees it goes out and buys one, its like a fucking lightsaber of flame haha). the butane bunsen burner is AWESOME for our purposes because it gets the scalpel red hot in about 2 seconds (it is also PERFECT for a hands-free quartz nail torch, so you can prep your dab while it is heating!!! truly awesome :rockon: ) . the only real downside is the piezo ignition, which (like all of these type igniters) will eventually wear out with too much use. i have been through 3 GT8000s and still own 2 (1 stolen, 2 ignition failed, 2 still work), and i have worn the ignition out on my first bunsen burner. for this reason, i like to use a combination approach, where i use an alcohol lamp on very low just to ignite the butane as needed. if i was only doing a few sleeves of plates at a time, i would probably just leave the flame on the whole time, but it would heat up after too long so i play it safe and just run it long enough to flame the scalpel as needed

using a butane bunsen burner and an alcohol lamp to flame a scalpel blade


next i take out a plate from the container/pile/stack to be transferred, and remove it from its individual plastic bag (more on this later), remove its parafilm wrapper, and visually inspect the culture (sometimes aided by a jewlers loupe or magnifying glass). to get a good look, i like to keep a light under my workspace rack which i turn on for backlight as needed, and i also hold the culture up to the light to get a good look

inspecting a culture in various lighting


ok so the kosher way of doing this next part (taking the sample to transfer) is to leave the plate on the worksurface, lift the lid slightly, and quickly slice out a sample 1-4mm square from the leading edge of the myc you want to transfer. i like to do something a little dicey, especially if i have cause to be concerned. i like to remove the lid, then pick up the dish and tilt it so that light reflects off of the surface of the agar. this allows us to see patterns, distortions, and blemishes which are otherwise invisible to us, and can help you spot a colony of bacteria that you might have otherwise unknowingly transferred with your myc. if you are going to do this, its important that you have slick, even, smooth agar to begin with, otherwise you cant really draw any conclusions from distortions in the reflective surface. unfortunately i couldnt get a good shot of what this technique of spotting hidden contams looks like, but hopefully yall get the picture

using reflecting light to identify hidden contams and irregularities in the agar surface while taking a sample


after that, we make the transfer to a fresh plate and label it with a sharpie (black for MS, red or blue for clones or isolates). i usually work with stacks of 8 plates at a time and start with the top and work down, opposite of pouring.  next we lift the lid slightly and then place the tissue sample onto the center. in the past, i used to stab the agar right through the center of my transfer tissue, as if to "nail it in place", but quit doing this for several reasons. mainly, it causes the growth pattern to be elongated around the blade trench (rather than uniformly circular), and also it doesnt do a good job of holding the sample in place (often makes it worse). it might seem a little counter-intuitive, but all you need to do is lay the piece of tissue down on the receiving dish, whether it is right side up, upside down, on its side, or whatever, does not matter. the surface tension between the two agar surfaces (or the myc and the agar if its upside down) will be more than sufficient to hold it in place, and the growth will be regular. if you try to readjust it a bunch of times or stab it or something, it can slide or fly all over the place, or even split into pieces, and make it hard to draw any conclusions from the shape of the resulting colony. Just dont drop your plates!

transferring a sample to the receiving dish


now i wrap the receiving dish in parafilm. first, i grab a piece from the pile and peel off the paper backing. next i pinch one end between my index finger and the dish, and rotate the dish thereby stretching the parafilm around the outside edges of the dish.

sealing a dish with parafilm


i like to put each plate into an individual ziploc sandwich bag, since they come sterile and it makes them more durable and easier to handle and inspect outside a sterile environment

individual ziplocs for each plate


Working with Spores:

since we fruit in open air, we can assume that spores are dirty. if someone sends you a spore print, you can assume it is going to have contams on it in addition to the spores you want to cultivate. this does not mean you got a bad print, it means you got a NORMAL print. the same thing applies to syringes. for this reason, when inoculating something with spores, it is a good idea to use as little as possible, since we only need a few spores and the more solution/spores we use, the more contams will be present as well. i recommend people never putting spores directly to grain, since it is so easy to culture the contams

a sterile jar of grain is basically the microbiological jackpot for anything that can digest it (fungi, bacteria, etc), so we need to take care to reduce the chances of culturing the wrong microorganisms. basically, ALWAYS germinate your spores first on agar before applying them to grain/lc/etc.

by using streaking techniques, you can easily clean up even the dirtiest of prints or spore syringes. you basically are using a loop to spread spores VERY thin across the surface of agar, creating various zones with different concentrations of spores. this is extremely useful for cleaning up a print, since you spread the contams thin while you spread the spores. this is also a great way to obtain isolates about 100 transfers sooner than you would get from a blob of spores, since the later zones have very few strains present in each colony. by doing one of these serial dilution style streaks, you end up with hundreds of colonies to choose from, of various genetic diversity, which gives you lots of chances to select a vigorous, organized culture

Quote:

In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria(fungi in our case). Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.

Streaking is rapid and ideally a simple process of isolation dilution. The technique is done by diluting a comparatively large concentration of bacteria to a smaller concentration. The decrease of bacteria should show that colonies are sufficiently spread apart to effect the separation of the different types of microbes. Streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. Aseptic techniques are used to maintain microbiological cultures and to prevent contamination of the growth medium.There are many different types of methods used to streak a plate. Picking a technique is a matter of individual preference and can also depend on how large the number of microbes the sample contains.

The three-phase streaking pattern, known as the T-Streak, is recommended for beginners.The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. The inoculation loop is first sterilized by passing it through a flame. When the loop is cool, it is dipped into an inoculum such as a broth or patient specimen containing many species of bacteria. The inoculation loop is then dragged across the surface of the agar back and forth in a zigzag motion until approximately 30% of the plate has been covered. The loop then is re-sterilized and the plate is turned 90 degrees. Starting in the previously streaked section, the loop is dragged through it two to three times continuing the zigzag pattern. The procedure is then repeated once more being cautious to not touch the previously streaked sectors. Each time the loop gathers fewer and fewer bacteria until it gathers just single bacterial cells that can grow into a colony.The plate should show the heaviest growth in the first section. The second section will have less growth and a few isolated colonies, while the final section will have the least amount of growth and many isolated colonies.




examples of streaked plates. different labs use different standards for streaking. colonies thin as the dish rotates counter clockwise


the way i like to do it is very similar to what is described above. basically, take your loop (bod's DIY loop tek) and scrape off a small amount of spores from a print (or drip a drop from a syringe directly onto the loop) and streak a side of the plate like below. then flame the loop again, DONT SCRAPE MORE SPORES, rotate the dish, and drag the loop from the area you streaked previously to streak zone 2, then flame and repeat

this is the streaking technique i like to use. it is tremendously handy for cleaning up spore prints, isolation projects, and even cleaning up old LCs


if you are using a spore syringe as your starting point (or a LC you are trying to refresh), you want to be super careful not to add any additional liquid to the plate, otherwise it will roll around and distort the streaking pattern. so make sure you dont drip over the plate, you shouldnt be putting any more liquid into the plate than what sticks to the loop

Conclusion:


so that is pretty much it!! a bit lengthy, but i think i covered most of what i wanted to cover for my post #1000. please let me know if yall have any questions or need clarification about anything :smile:

i also welcome any additional tips & tricks, feedback, criticism, discussion, debate, haters (:kiss:), or critical thinking :wink:

thanks everyone for everything yall have done to make this place such an incredible resource. im so grateful to live in an age where there is an online community to collaborate with on our hobbies :smile:

warm regards,
c10


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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Edited by c10h12n2o (02/14/20 05:27 PM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o] * 3
    #24330963 - 05/18/17 09:01 PM (3 years, 4 months ago)

:tldr:

but i will. this is awesome. happy 1000


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mushboy] * 1
    #24330989 - 05/18/17 09:08 PM (3 years, 4 months ago)

:superscience:      :hi:      :takingnotes:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mushboy] * 2
    #24330997 - 05/18/17 09:09 PM (3 years, 4 months ago)

Congratulations on post 1000! Great write up and  well documented with pics.  That's a +5  :mushroom: for ya! Keep the good stuff coming.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Just_A_Noob] * 1
    #24331001 - 05/18/17 09:11 PM (3 years, 4 months ago)

So this is why you've been so quiet lately!
What a post dawg!

Very good sir.
I concur.
Grate  write up, lots of info in once place

(keywords:agar, agar magic, amazing agar, easy agar, flow hoods, flowhoods, ahgar, Petri, plate, amazeballs, parafilm, recipe, suspension, incredible)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mynakedrat] * 1
    #24331014 - 05/18/17 09:18 PM (3 years, 4 months ago)

lol we should put you on SEO duty MNR :lol:

thanks guys :smile:

yeah this is why ive been so quiet. i got to post 99X and didnt have the guide ready yet so i was like damn... cant start any shit until i get this posted
:cryaboutit:

also my camera messed up right before i made this, so i had to use my backup camera instead of the one i bought for photographing this kind of stuff :/ its being repaired right now and should be back before too long


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C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


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"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o] * 1
    #24331233 - 05/18/17 10:52 PM (3 years, 4 months ago)

As usual, great info my friend. Well done


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Pipefitter537] * 1
    #24331269 - 05/18/17 11:07 PM (3 years, 4 months ago)

Wow! What a comprehensive and useful tek! I can't read it all tonight but be assured I will read it all. 

You are obviously very skilled.

Happy 1000th!!!  :birthday:  :birthday:  :birthday:  :birthday:  :birthday:  :birthday:  :birthday:  :birthday:  :birthday:  :birthday:  x  100


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Kenetic] * 1
    #24331380 - 05/18/17 11:50 PM (3 years, 4 months ago)

Great stuff sir!  Nicely illustrated too.

A big take-away for me is to buy a strap wrench, what a clever invention... no more battles with stubborn mason jar lids!

I will buy one tomorrow.  YASSSSSSS :thumbup: :smile:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mushboy] * 1
    #24331393 - 05/18/17 11:56 PM (3 years, 4 months ago)

Quote:

mushboy said:
:tldr:

but i will. this is awesome. happy 1000




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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Lobi] * 1
    #24333334 - 05/19/17 05:30 PM (3 years, 4 months ago)

much obliged my friends

:fuckyeahdance:

ive been planning on doing an agar tips thread for a long time, but figured since i do so many plates it might be a good idea to spell out my whole process and show what i have learned as far as what works and saves time on that scale (did 100-200 plates a day for several months lol). plus it gives people the chance to correct me on any dumb shit i do/think/believe :lol:

there are a few things i will add better pics of when i get my camera back from the shop (used my phone for all these :/ ), specifically the technique using light reflections to spot hidden contams on agar

i will try to update this to make it reflective of what i am currently doing as time goes on and i revise my technique :smile:


--------------------

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"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o] * 1
    #24334504 - 05/20/17 01:29 AM (3 years, 4 months ago)

I love your crystal-clear agar plates! :congrats:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o] * 3
    #24334565 - 05/20/17 02:02 AM (3 years, 4 months ago)

Just reading this made me feel smarter.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: WeavieWonder] * 1
    #24334572 - 05/20/17 02:05 AM (3 years, 4 months ago)

Great stuff. I has questions.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Kingdeviluke] * 1
    #24334657 - 05/20/17 02:57 AM (3 years, 4 months ago)

Tonight was the first time i used a print ive only used syringes... say the prints just show bacteria how would you go getting clean growth ?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Boogieman47] * 2
    #24334758 - 05/20/17 04:25 AM (3 years, 4 months ago)

fire away, all questions/comments welcome :smile: I appreciate yells feedback!

Quote:

Boogieman47 said:
Tonight was the first time i used a print ive only used syringes... say the prints just show bacteria how would you go getting clean growth ?




nice! I bet you'll get hooked and end up with a box or photo album full of prints (a lifetime supply of genetics to work with, in as many flavors as you like) to pull from as needed


I'm actually glad you asked that. I'm in the shower right now but I will update the guide to include my streaking techniques

but to answer your question: I would do a serial dilution streak, and watch it closely. in 99/100 cases, that will be more than sufficient to separate from contaminants, just by streaking and making well-timed transfers. alternatively you could just do a single dilution and streak, by scraping a tiny bit of spores onto a loop then dilute them into a tiny bit of sterile water, then just do a single streak pattern. either works great, but I prefer the method without water.


if that didn't work (it almost always does) I'd streak antibiotic agar


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o] * 1
    #24334775 - 05/20/17 04:42 AM (3 years, 4 months ago)

I just got some wire made a small loop and flamed it cooled in the agar and streaked the whole in a zigzag somewhat like that gif....

I have about 10 prints pans and cubes i have made tons of prints to send out but figured in my arrogant mind i wouldnt need to work with them lmao i ordered 14 syringes over a year ago 6 unopened and the rest are pretty full maybe 2 cc's if that ...


On each plate i dropped 5 drops in a 5 point star 2 plates per syringe 4 kinds and made 3 plates of prints with 3 varieties ...


I guess i was thinking a print will just be a booger of bacteria like the syringes sometimes do haha ..


Have you worked with black tea agar or charcoal?? Also my buddy has a silver water machine and said its antibacterial i was wondering if i was to make spore solution with it if it would help with cleaner syringes


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Boogieman47] * 1
    #24334839 - 05/20/17 05:48 AM (3 years, 4 months ago)

When tranfering how do you not make the agar stick to the hot knife if you don't stab the plate


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Kingdeviluke] * 1
    #24336286 - 05/20/17 07:43 PM (3 years, 4 months ago)

Quote:

Boogieman47 said:
I just got some wire made a small loop and flamed it cooled in the agar and streaked the whole in a zigzag somewhat like that gif....



loops are awesome, totally indispensable. for a long time i used to streak agar, but didnt really understand what i was doing. i would just do a basic streak pattern. at some point i made the connection that streaking is actually smearing something really thin, and then learned how to do the serial dilution streak like the gif. the biggest difference is the serial dilution streak involves scraping spores 1 time, streaking a side, then flaming, then smearing from the area you streaked before into a new area, then flaming again and repeating. the key is that you only scrape spores for zone 1, and each progressive zone has less and less spores per area. this is a really easy way to get isolates and highly organized cultures much faster (since anything from zone 4-5 is gonna have way less genetic diversity). you end up with something like this (sorry i dont have a more clear pic handy)


Quote:

Boogieman47 said:
I have about 10 prints pans and cubes i have made tons of prints to send out but figured in my arrogant mind i wouldnt need to work with them lmao i ordered 14 syringes over a year ago 6 unopened and the rest are pretty full maybe 2 cc's if that ...



i know the feeling man. thats why i made the album of my prints, so that i keep at least 1 of each and have the origin documented, etc. i keep extras in a box, in ziplocs, and send those out for trades. greatly reduces the risk of accidentally sending out my last one

when you streak like this, it really doesnt take hardly ANY spores... when i do a trade im happy if someone just gives me a swab of part of a print lol... its so easy to turn a few spores into as many prints as you want :smile: prints seem like the best long term way to store things. i dont trust syringes at all, even though ive had them last many years, there are just too many things that can go wrong, where with a print you can do whatever you want with it. really, a print is a lifetime supply for me. they are microscopic, we waste so many haha...

i would try to grow out all your syringe varieties (streak to agar > a2g or LI > grainspawn > mono) and make prints for long term storage. while you are at it, make slants out of any clones you particularly like :smile:

Quote:

Boogieman47 said:
On each plate i dropped 5 drops in a 5 point star 2 plates per syringe 4 kinds and made 3 plates of prints with 3 varieties ...



that sounds risky! when i am working with a syringe, i like to drip the solution directly onto my loop, then streak with that in the gif pattern (flaming between each step). you could also drip some into a sterile shot glass or something and run it through that. the point is that you only need a microscopic amount of spores, and the less the better.

since we can basically assume spores are dirty (whether print or syringe), the less we use the better. and if we can smear that tiny amount across the agar to thin it out even more, even better. also, we dont want to add ANY liquid to the plate if we can help it, because this will roll around and inoculate random spots with spores and contams, making it so that we cant infer anything from the shape. if we just use as little as possible, then it makes it easier to separate the good colonies from the bad since they remain distinct. any liquid on the agar surface is going to cause problems, so keep it to a minimum and spread out the contents

Quote:

Boogieman47 said:
I guess i was thinking a print will just be a booger of bacteria like the syringes sometimes do haha ..



thats a pretty good assumption to be honest :lol: they damn sure can. i assume spores=dirty. some prints are insanely dirty, just like some syringes (which are made from prints). id prefer a dirty print over a dirty syringe any day though, because in a syringe the liquid spreads the contams everywhere and gives them the h2o they need to reproduce. its much easier to work with a given amount of spores from a print, wheras i have no idea how many they put in a syringe. same process applies to cleaning up either one though: streaking and well-timed transfers

Quote:

Boogieman47 said:
Have you worked with black tea agar or charcoal?? Also my buddy has a silver water machine and said its antibacterial i was wondering if i was to make spore solution with it if it would help with cleaner syringes



i havent. i did experiment with my agar recipe a lot, but i eventually realized that i was asking the wrong questions (thinking about antibiotics, nutrition, etc). with agar all you need is a slightly nutritious medium that allows for growth in 2 dimensions at a controlled rate. trying to "optimize" the nutrition completely misses that point.

i do use antibiotics occasionally, but very very rarely. 99/100 times, well timed transfers and basic MEA or PDA is all you need to clean up a print, and if you use a streaking technique it can be done in 1 transfer from pretty much anything, no matter how dirty. the only time i use antibiotics is occasionally on the final plate before making long-term master slants (the final plate, not the slant itself, for that i use standard full strength MEA), and if a print is so filthy that the bacteria is keeping the spores from germinating

id say the better question to ask would be why you want to be working with syringes at all? all i use them for is sucking up LI. i doubt i will ever touch a spore syringe again, besides what i get in trades, etc.. what exactly do you have in mind for syringes that couldnt be more effectively done with agar and g2g? i could very well be missing something :lol:

Quote:

Kingdeviluke said:
When transferring how do you not make the agar stick to the hot knife if you don't stab the plate




good question, i need to make that more clear in the guide. we are talking about 2 different things: in the guide i was referring to after i take the transfer and set it into the receiving dish. i used to (and sometimes still do from habit) place the transfer on the receiving dish and then stab the transferred tissue, piercing it and the agar in the receiving dish, as if to nail it in place. this often caused the transferred tissue to split, and distorted the colony growth pattern because of myc cells on the blade, which often cause the colony to grow out of the gash made by the blade. these days, instead of that, i try to just sit it down and move on, since the surface tension holds it just fine and the stabbing doesnt really help

what you are asking about is something different. i heat the blade red hot in the butane bunsen burner and then set it down onto that little aluminum foil tray i make each session, which holds it at an angle so that the blade doesnt contact anything. i time things so that as soon as i make a transfer the next thing i do is flame the scalpel, then put it down on that tray, then i label, parafilm, and bag the donor and recipient plates, and get out the next donor plate and unwrap it. by the time i am done with all that, the blade is always cool enough to do the next transfer without problems.

the exception to this is when i have a plate that i am taking multiple transfers from, since that doesnt give the blade long enough to cool down between transfers. in that case, i do drag the hot blade through the receiving dish to cool it down. this is also a handy way for me to id cultures, since i know that any plates that have that scar from cooling the scalpel were done in a series from the same donor

does that make sense guys? let me know if yall have any questions about anything :smile:


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C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o] * 1
    #24336637 - 05/20/17 10:20 PM (3 years, 4 months ago)

I like the distinction you make into the differences of using a flowhood in comparison, and contrast to a SAB.
The different color markers representing the different types of culture is cool, too.
You're a hoss for picking up all 19 plates at a time to pour.
I need to get me a nice swivel chair, and some lighting like your's for my DIY lab/clean room, and spawning area.

Excellent write up!  :super:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: hamloaf]
    #24336706 - 05/20/17 10:57 PM (3 years, 4 months ago)

Nice 1000th post. One day I will have cool toys n stuff. Need to finish fixing my house and then sell it first. Getting close.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Pastywhyte]
    #24336897 - 05/21/17 12:50 AM (3 years, 4 months ago)

thanks for the feedback guys :headbang3:

i am gonna go back shortly and add some more on streaking and some more distinctions between SAB and FH technique that i forgot to mention

what toys are on your wish list pasty?


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"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24336909 - 05/21/17 12:56 AM (3 years, 4 months ago)

psh your custom SAB is on my wishlist lol https://www.shroomery.org/forums/showflat.php/Number/23557396/fpart/1/vc/1
the plexi one that is.

I wouldn't do a hood until I could make a room with as close to seamless hygienic floor and walls as I could manage.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: bodhisatta]
    #24336938 - 05/21/17 01:13 AM (3 years, 4 months ago)

lol that thing was pretty handy,i didnt really take as many pics as i should have, it had a hinged lid with lights built in and shit.. it was surprisingly comfortable for doing bags, i could even do bag to bag in it

i actually found the big piece of plexiglass for that thing on the side of the road haha... my gf thought i was insane the way i pulled a U turn for "a piece of garbage" :lol:

i had around a 99% success rate with plates in that thing after i started using a towel + rack (over 15000 plates)... not so great with jars and bags though... probably sometimes because of how hard i had to struggle to unscrew lids before i got a strap wrench (that thing is clutch). more like 90%

that custom SAB was built into a closet, so that i could close it and it totally disappeared :lol: it was super covert

i still have to build the table for my FH, i plan to build it into a really big closet, with a custom table underneath. my coffee table has an e-nail built in, im gonna build one into this table as well, and lots of storage for tools and stuff :cool:

i expected similar success rates with my FH, but so far over a couple thousand plates i have only seen 1 with ANY contam (and i dropped it on the floor :lol:). ive only made 14 grain jars so far with it, and they all look great. been kinda amazed at how well this thing works, i expected to see at least 1 plate contam every few hundred, ive had to adjust my workflow and do less plates (since i dont have to plan on ANY contaminating)


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"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o] * 2
    #24336951 - 05/21/17 01:18 AM (3 years, 4 months ago)

what do you do with these hundreds of plates if you only did a few grain jars lol.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: bodhisatta]
    #24336967 - 05/21/17 01:29 AM (3 years, 4 months ago)

My wishlist is for a room without carpet and tile floor to do lab work. Have a flowhood and dissection scope. Erbach would be nice along with a 100 cc borosilicate spring loaded syringe with a 3 inch 14 ga stainless luer lock needle with cross cut tip. A walk in GH would be sweet. You know the little things.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Pastywhyte]
    #24337001 - 05/21/17 01:52 AM (3 years, 4 months ago)

Quote:

bodhisatta said:
what do you do with these hundreds of plates if you only did a few grain jars lol.




only a few grain jars since building my flow hood, ive only had it a few weeks... i moved recently, and unexpectedly, and instead of building a new SAB i built a FH.

i did hundreds in my SABs, and lots of bags. since getting that FH built ive refreshed the cultures i want to work with, started a bunch of new stuff, and continued a bunch of isolation projects (some isolates, some 1 to 80 transfers in)

Quote:

Pastywhyte said:
My wishlist is for a room without carpet and tile floor to do lab work. Have a flowhood and dissection scope. Erbach would be nice along with a 100 cc borosilicate spring loaded syringe with a 3 inch 14 ga stainless luer lock needle with cross cut tip. A walk in GH would be sweet. You know the little things.




a man of fine tastes :smile: glad i asked, never thought about one of those syringes.. ive been shopping for a dissection scope lately... i think it was mushpunx who said he got an erbach and lab blender for $70!!


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24337022 - 05/21/17 02:10 AM (3 years, 4 months ago)

Shit I have been looking at places that custom create syringes. I even priced out one that would be everything I wanted, was over 300 bucks. But the shit would last forever. Maybe when the tax return comes in.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Pastywhyte]
    #24337217 - 05/21/17 04:57 AM (3 years, 4 months ago)

You guys be joshin too much


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OfflineManifoldPrime
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Boogieman47] * 1
    #24337390 - 05/21/17 08:42 AM (3 years, 4 months ago)

Beautiful write up c10! :rockon:

I've only been doing the Pastywhyte Ezi LC jars for the past few months, I've all but forgotten how to do petris :hehehe: You've convinced me to pull the trigger on getting a 500pc order, I was wondering if it was worth it but clearly I need to step up my game.

I'll be following this tek to the T when I do my next batch of petris.

You seriously deserve TC status!

Make
Agar
Great
Again!


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Edited by ManifoldPrime (05/21/17 08:45 AM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: ManifoldPrime] * 1
    #24341493 - 05/22/17 10:03 PM (3 years, 4 months ago)

UPDATED.

haha thanks buddy, i appreciate it :smile:

:rofl: MAGA: make agar great again, i fucking love it!! thats going in the subtitle :smile:


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


Edited by c10h12n2o (05/22/17 10:04 PM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24341508 - 05/22/17 10:10 PM (3 years, 4 months ago)

:takingnotes: phones at 1% so I'll read later. nice new avatar C10 :lol:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: van hatton]
    #24341540 - 05/22/17 10:26 PM (3 years, 4 months ago)

ok i finally read your post.


:rockon:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mushboy]
    #24355310 - 05/28/17 12:52 AM (3 years, 3 months ago)

:thumbup: good info thanks :thumbup:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Sappbelly]
    #24374239 - 06/03/17 06:42 PM (3 years, 3 months ago)

UPDATED


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24374455 - 06/03/17 08:07 PM (3 years, 3 months ago)

Nice post man!


--------------------

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Edited by mushpunx (06/03/17 09:01 PM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mushpunx]
    #24375352 - 06/04/17 02:54 AM (3 years, 3 months ago)

thanks buddy :smile:

im gonna be updating this again soon with more pics and some additional tips, and possibly some videos :smile:


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24414276 - 06/18/17 09:05 AM (3 years, 3 months ago)

I just used that streaking method for the first time, and loosely followed this guide as a refresher to whip up a batch of B+ colonies.

Will post updates ITT.

Thanks C10!


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: ManifoldPrime]
    #24414558 - 06/18/17 11:11 AM (3 years, 3 months ago)

Nice write up man. I'm gonna try that streak pattern tonight I like it. Good info thanks.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Dabadoodledoo710]
    #24423337 - 06/21/17 11:50 AM (3 years, 3 months ago)

informative write-up, thanks for mentioning the part about straining the agar for clarity. i had heard of this being done but couldn't find any info on how.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Panarchist]
    #24423523 - 06/21/17 01:01 PM (3 years, 3 months ago)

:takingnotes:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: oontribe]
    #24424207 - 06/21/17 05:41 PM (3 years, 3 months ago)

thanks guys :smile:

im a bit behind on all my chores (working with waaaaay too many species , not to mention races and cultures lol), but i have a nice update for this coming as soon as i get the chance. gonna add lots more pics of examples, distinctions between SAB and FH techniques, cloning, maybe some video demonstrating how to execute particular steps, etc...

im spawning an assload of reishi, king oysters, and cord m, testing a bunch of different substrates and preps

let me know if yall have any questions about anything, want clarification on something, or want to request that i cover a specific topic in subsequent updates :wink:


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24425993 - 06/22/17 09:50 AM (3 years, 3 months ago)

You should do a journal / log of your grows, I really enjoy reading through grow logs to pull cool ideas and whatnot. And you have some interesting varieties going there (What is Cord M?)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: ManifoldPrime]
    #24426013 - 06/22/17 10:01 AM (3 years, 3 months ago)



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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: foragedfungus]
    #24426017 - 06/22/17 10:05 AM (3 years, 3 months ago)

Oh the zombie shroom, lmao I posted that in the 100 facts thread. I should memorise latin names better:facepalm:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: ManifoldPrime]
    #24440018 - 06/27/17 07:03 PM (3 years, 2 months ago)

Wow, this is incredibly helpful, thank you. I'm just dipping my toes into agar and will be referring back to your guide regularly!

:bow2:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: NattyDR]
    #24440107 - 06/27/17 07:32 PM (3 years, 2 months ago)

I reference this once a day at least lol


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: jkz]
    #24440193 - 06/27/17 08:00 PM (3 years, 2 months ago)

thanks guys :smile: im glad yall find it useful :smile:

there is a lot of stuff in there that i REALLY wish had been made clear to me when i first started. im a critical thinker, so i take everything apart and ask a million dumb questions in my head about everything, and that can be really overwhelming when you first start agar: there are just so many applications, so many factors involved. some of the things that took me a while to figure out could benefit a newbie from day 1

i have some planned updates i want to add, including some videos (as soon as my camera gets back from the shop), with lots of distinctions between SAB and FH technique, and more on streaking techniques, but ive been playing catchup with all my projects... recently started doing plant tissue culture and good god that shit is complicated... lots of overlap though, i really look forward to learning more about it and applying some of the techniques to our hobby

yall are always welcome to bump this thread with questions, requests, clarifications, racism, pornography, flames, idk :smile: (though mods might)

this is a work in progress. id like to keep it somewhat current with my techniques, because i am always trying and testing new things i read in lab manuals, textbooks, and papers, and will be happy to share anything that others might find useful. i love the thought of newbies getting to start with all the info i wish i had when i started :smile:


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24461479 - 07/06/17 11:18 AM (3 years, 2 months ago)

:takingnotes:cheers lad


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: bill bixby]
    #24499787 - 07/21/17 11:42 PM (3 years, 2 months ago)

This is awsome definitely moving on to agar next it's insane how these teks have helped me progress in my mycology thank you for your time sir..


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mushboy]
    #24533743 - 08/05/17 11:51 PM (3 years, 1 month ago)

Nice words and pictures in great order.:rockon:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: wtfcrazymofo]
    #24573576 - 08/23/17 07:07 AM (3 years, 1 month ago)

Clearly a lot of effort went into this, would be 5 shrooms rating if I hadn't already done so!


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: lovelaughlibs]
    #24573671 - 08/23/17 08:40 AM (3 years, 1 month ago)

what brand of face shield, and what brand on the reusable black gloves?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24581368 - 08/26/17 04:09 PM (3 years, 1 month ago)

I spent the time to read this, great post.
Can't believe the time that was spent just to put this together.
Great job C10, thanks for the help.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: optic1]
    #24581384 - 08/26/17 04:24 PM (3 years, 1 month ago)

Where the fuck u at c10?  Hope everything is ok.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Pipefitter537]
    #24586423 - 08/28/17 10:32 PM (3 years, 30 days ago)

Excellent write up, I'm just getting started and have been doing a ton of research. I really picked up a ton of information from this post.

I like the information about serial dilution, that brings a whole new dimension and logical thought process to something as simple as smearing spores on agar.

This is something I can read, and reread, and continue to pick up on little techniques. Very well written and logically thought out. Easy to follow. A lot of information in the comments as well.

When you say you use "full strength" agar in your long term slants, what exactly is your recipe? 20g agar, 30g light malt extract per 1000ml water? Also curious as to what size tubes you personally use.

I would love to read your thoughts on long term storage and how you make and store your slants and prints and keep them viable. The photo album is an excellent idea. I know there are plenty of other teks about it out there (franks). But I'd like to hear the processes and equipment you've settled with, and how you maintain your library. If there is anything you do different. Or any tips or tricks you have on the subject from your experience. (C10's storage guide?) Lol

Thanks again, keep up the exceptional work!


Edited by Sphink (08/28/17 11:00 PM)


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OfflineManifoldPrime
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Sphink]
    #24586945 - 08/29/17 03:36 AM (3 years, 30 days ago)

Quote:

20g agar, 30g light malt extract per 1000ml water




Thats a little strong, usually youd use 20g malt and around 2-4 g dextrose. It would work, but generally speaking you want your nutrient content to be on the low side, you want the myc to work for its dinner and spread out faster :biggrin:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: ManifoldPrime]
    #24587481 - 08/29/17 11:30 AM (3 years, 30 days ago)

Quote:

ManifoldPrime said:
Quote:

when you say you use "full strength" agar in your long term slants, what exactly is your recipe? 20g agar, 30g light malt extract per 1000ml water?




Thats a little strong, usually youd use 20g malt and around 2-4 g dextrose. It would work, but generally speaking you want your nutrient content to be on the low side, you want the myc to work for its dinner and spread out faster :biggrin:




Yes, for petri's you want nutrients on the low side to encourage rhizomorphic growth and sectoring. C10 did a good job explaining that in the original post and suggests "half strength" agar. But for master slants you would want the opposite, no?

wouldn't you want it to grub out and stay put to avoid unnecessary cell division? Thus the refrigeration ect...


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24636430 - 09/16/17 11:03 PM (3 years, 11 days ago)

hey U4EUh, nice writeup, hope all is well, nobody heard from you in a while

put together a couple things from what you condensed, thanks much

again, hope all is well


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mekannic]
    #24831774 - 12/07/17 01:28 PM (2 years, 9 months ago)

hey thanks, great post and nice written. i have a question, if i follow your recipe and also make dishes thin like you it will be possible to let them sit in the fridge for 1 month and later use them with success?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Psychedeliciouss]
    #24836907 - 12/10/17 01:53 AM (2 years, 9 months ago)

If you wrap them with parafilm and store them in something airtight, E.G a ziplock bag or a tupperware, should be fine. Would also show if your technique isnt good considering any plates that would contam due to tek would do so over a month.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: ManifoldPrime]
    #24836909 - 12/10/17 01:59 AM (2 years, 9 months ago)

Quote:

ManifoldPrime said:
Would also show if your technique isnt good considering any plates that would contam due to tek would do so over a month.




Hes talking about storing cultures in the fridge


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: ManifoldPrime]
    #24836986 - 12/10/17 04:24 AM (2 years, 9 months ago)

thank you :smile:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Psychedeliciouss]
    #24889755 - 01/04/18 03:00 PM (2 years, 8 months ago)

Thanks for this!


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Snickelfritz]
    #24895955 - 01/07/18 11:02 AM (2 years, 8 months ago)

Great read booked for later studies


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mushboy]
    #24913146 - 01/14/18 12:26 PM (2 years, 8 months ago)

This is awesome!  Thx for the details:thumbup:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Pablo1211]
    #24941070 - 01/25/18 03:08 PM (2 years, 7 months ago)

Gonna be getting into agar, and I especially liked the streaking technique, gonna use that, Thanks for the experienced info.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #24950258 - 01/29/18 12:48 AM (2 years, 7 months ago)

Thank you for this. First time grower here and decided to start with spores on agar. This has been my bible and hasn't failed me yet.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: w33dh4x]
    #25116373 - 04/05/18 06:57 PM (2 years, 5 months ago)

c10h12n2o, I think I just found my hero.
Started a couple grain bags and some BRF jars a week ago. To keep me occupied while they colonize, I decided to jump straight into agar for hands on experience and put some use to my left over syringe.
Learned a few things right off the bat; My 64q SAB is to short. My petri stand to keep space between the cloth on the bottom and my petri dishes is too high along with my 1000ml slimline media bottle.
Gonna have to make some adjustments, thinking a home made plexi glass one may be the way too go. Next step, knock one up and see how my other dishes grade my sterile technique. "Like a blind man in an orgy ya gotta feel things out", I guess.

Great write up, fuel to my newfound fire, thank you for sharing! :thumbup:


--------------------
-The heaviest thing one will ever carry is a thought-
-"Like a Blind man In an orgy you gotta feel things out.".-
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Edited by R.I.P.Zappa (04/05/18 06:59 PM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #25149190 - 04/18/18 09:37 PM (2 years, 5 months ago)

C10,
First of all let me praise you for the clear and concise TEK you’ve provided and let you know that I realize, how much time and effort you’ve put into doing it. Just amazing!

Now, first an excuse:  I’m (as of this coming June) 80 years old.  I’ve just started cultivating psilocybe cubensis in the last few months and I’m having a difficult time navigating the Shroomery board and absorbing all the information available.

I’ve spent the last several hours reading your TEK and a number of others. At this point I seem to be in overwhelm  I’ve finished PCing a few agar dishes using Gladware plastic mini-containers and I have a spore print ready to use.

Here’s my question: What’s the best method of getting the spores from the spore print onto the agar?  I don’t seem to find in your TEK how you’re transferring the spores onto the agar.  It’s probably just me.  I’ve started to slow down in my comprehension these last couple of years.  Are you just using an inoculating loop to scrape some spores from the print?

There’s no need to write something out and waste your time. If you could just point me to the right post.  I’ve tried searching and just haven’t been able to come up with a clear procedure.

I noticed that somewhere in your description you mention not to drop any liquid from a syringe onto the petri dish as that just leads to contamination and spreading the mycelium growth indiscriminately across the agar.

Any advice or suggestions you might provide would be greatly appreciated.

Thanks in advance,
JB


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: bison2] * 1
    #25149503 - 04/18/18 11:06 PM (2 years, 5 months ago)

These may help. Gotta do allot of digging to fill in the blanks. Sometimes ya find the answer to what you are looking for in a thread not even related to what you are looking for. Good thing is you never run out of stuff to read and often find somthing new.

Here is a good one for how to use the search engine better.
https://www.shroomery.org/forums/showflat.php/Number/24270830#24270830

How to use the sites features
https://www.shroomery.org/forums/showflat.php/Number/23745550

Heres is a bunch of compiled agar thread links.
https://www.shroomery.org/forums/showflat.php/Number/22721954/vc/1#22721954

Simple example:
I copy pasted your, "spore print onto the agar", from your sentence,
"Here’s my question: What’s the best method of getting the spores from the spore print onto the agar?", onto the search bar on the top and came up with this.
https://www.shroomery.org/forums/showflat.php/Number/20296483#20296483

Could also ask in these two threads as well.
https://www.shroomery.org/forums/showflat.php?Cat=&Board=83&Number=25145624&fpart=1&PHPSESSID=
https://www.shroomery.org/forums/showflat.php?Cat=&Board=2&Number=24679216&fpart=1&PHPSESSID=

There is more than one way to skin a cat and there is allot of info and helpful folks here.
Welcome and :goodluck:


--------------------
-The heaviest thing one will ever carry is a thought-
-"Like a Blind man In an orgy you gotta feel things out.".-
-When we agree about our hallucinations, we call it “reality".-
-If you defy authority because your told to, that's no better than blindly trusting authority.-

psychonautwiki.org

How it should & shouldn't look - NEW CULTIVATORS GUIDE
BOD's Easy AF OAT prep tek.
Principles of mushroom growing for beginners


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: R.I.P.Zappa]
    #25255177 - 06/08/18 12:49 AM (2 years, 3 months ago)

Nice! just chiming in to track this thread. :mushroom2:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: k5hd2y]
    #25255235 - 06/08/18 01:57 AM (2 years, 3 months ago)

this thread is dead for the most part. c10 hasn't been around for a while. it's a good right up though


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #25465191 - 09/16/18 07:29 PM (2 years, 11 days ago)

Very detailed,,thanks for taking the time to share it.
Happy I found this post, will bookmark it for sure.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Casi Loco]
    #25466229 - 09/17/18 04:26 AM (2 years, 11 days ago)

Super helpful write up c10h12n2o, great to see so many pix


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Scrubbs]
    #25534235 - 10/13/18 12:28 PM (1 year, 11 months ago)

thanks for sharing! Great wealth of information there :smile:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #25565207 - 10/25/18 03:37 AM (1 year, 10 months ago)

How can I utilize the agar dishes to inoculate PF cakes ?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Casi Loco]
    #25566225 - 10/25/18 01:27 PM (1 year, 10 months ago)

this thread is DEAD my friend. agar to brf cake is not a normal way of doing this but you could (plausbly) just put it on top with no verm layer. better yet would be using clean agar to make a clean lc and then noccing up the cake with the lc. or... make an LI out of the agar and use that. bottom line though is brf cakes are for beginners who are NOT using agar. there is nothing wrong with still using brf cakes but grain spawned to bulk will give you better yields. brf cakes are for people who are still just using spores. spore syringes aren't all to clean and brf cakes are really good at mitigation bacterial infection.

... get yourself some wild bird seed (or other grain) and noc that shit up proper. just my two cents.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Casi Loco]
    #25566682 - 10/25/18 04:39 PM (1 year, 10 months ago)

Quote:

Casi Loco said:
How can I utilize the agar dishes to inoculate PF cakes ?




Make a cake with a grain jar lid (has filtered gas exchange), skip the dry verm barrier since your lid is now the filter. Then inoculate it like a grain jar.

Now for me to sound mean but if you have to ask i would stick to the beginner methods, get feet wet. And when all these terms and techniques make sense and are not so overwhelming and confusing then its time to branch to "advanced" methods.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: bodhisatta]
    #25567006 - 10/25/18 07:08 PM (1 year, 10 months ago)

Or fuck it, do everything at once. It can work. :cool:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: stareatclouds]
    #25567243 - 10/25/18 08:24 PM (1 year, 10 months ago)

Good write up, thanks for that. Can anyone give me any more info of what kind of malt will work? Light malt, opposed to normal malt? I don't know much. Thanks.

Edit: https://www.woolworths.com.au/shop/productdetails/35176/saunders-malt-extract

This has some info in the pics about what's in it, would it be suitable?


Edited by Caster (10/25/18 08:41 PM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Caster]
    #25567356 - 10/25/18 09:11 PM (1 year, 10 months ago)

Both light and regular malt will work, but don't use dark. This is what I use.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: stareatclouds]
    #25567841 - 10/26/18 12:04 AM (1 year, 10 months ago)

Quote:

stareatclouds said:
Both light and regular malt will work, but don't use dark. This is what I use.




Thanks a heap! That's good to know.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #25593391 - 11/05/18 10:55 AM (1 year, 10 months ago)

Sorry if this is a stupid question, but i'm only on my third set of plates and I feel like my technique could definitely use some work, but i'm having a hard time finding the answers to my questions. Before you streak your plates, what's your method for removing the spores from the print? Do you add a drop or two of sterilized water to the print then swipe it? If so, do you stir them up in the water first before you swipe it? I put a little too much water on the print to loosen them from the foil on my last set, and it's now running on the surface of my Agar, so it's probably about to be contam city up in here. Also, the spores are in big clumps on my Agar instead of spread out (I used the zig zag method) Lessons learned I guess. That one was a PE6 print though, so I really hope I didn't trash those by accident :frown:


--------------------
Can someone point


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: dmar]
    #25593403 - 11/05/18 10:59 AM (1 year, 10 months ago)

Watch my video its in my links


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: dmar]
    #25593408 - 11/05/18 11:02 AM (1 year, 10 months ago)

c10 hasnt been around for awhile but heres what i do:

i always keep a few syringes of sterile water handy. making them is easy but with prints i take a sterile swab, wet the swab with the syringe of sterile water, then dab a tiny spot on the print to pick up spores. then swipe it to as many dishes as i need.

ive dropped whole chunks of spores onto agar. all blackish and gross looking but it will still germinate. souldnt ruin anything.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: mushboy]
    #25595008 - 11/06/18 12:33 AM (1 year, 10 months ago)

Quote:

bodhisatta said:
Watch my video its in my links



I'll go check that out, thanks Bod.

Quote:

mushboy said:
c10 hasnt been around for awhile but heres what i do:

i always keep a few syringes of sterile water handy. making them is easy but with prints i take a sterile swab, wet the swab with the syringe of sterile water, then dab a tiny spot on the print to pick up spores. then swipe it to as many dishes as i need.

ive dropped whole chunks of spores onto agar. all blackish and gross looking but it will still germinate. souldnt ruin anything.




So I PC a jar of water then take it into the SAB and open it up and fill the syringe, but I haven't tried a swab yet. When you say sterile swab, is that essentially just the Cotton tip applicators with the wooden tip that you can get at Walmart? Also, what's the advantage there when compared to using an inoculating loop?


--------------------
Can someone point


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: bodhisatta]
    #25595022 - 11/06/18 12:41 AM (1 year, 10 months ago)

Quote:

bodhisatta said:
Watch my video its in my links




I just watched the video. You don't use any water whatsoever, so when you cool the loop down in the dish, some Agar sticks to it, which helps pull up the spores when you scrape them? :ooo:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: dmar]
    #25596527 - 11/06/18 05:38 PM (1 year, 10 months ago)

Why can't you use dark malt?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #25756721 - 01/20/19 07:20 PM (1 year, 8 months ago)

Could I boil the agar in a pot instead of using the microwave? I’m assuming it’s fine just double checking


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Fireneeb]
    #25756738 - 01/20/19 07:25 PM (1 year, 8 months ago)

yes you can. this thread is dead as shiznit though. you'll probably get more help posting in an active agar thread.

watch it closely though. the shiz likes to boil over and make a huge mess


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: verum subsequentis]
    #26262423 - 10/19/19 07:55 AM (11 months, 3 days ago)

should inoculated agar be stored in darkness or on the shelf?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Samalander]
    #26262932 - 10/19/19 01:23 PM (11 months, 2 days ago)

Living things like light. Also, this thread is older than my great granny's panties. There are quite a few active agar threads going. Pop into one of those and you'll learn all kinds of cool shit.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: verum subsequentis]
    #26263384 - 10/19/19 04:04 PM (11 months, 2 days ago)

great! thank you:thumbup:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Samalander]
    #26332454 - 11/20/19 01:00 AM (10 months, 2 days ago)

tnx


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: verum subsequentis]
    #26463609 - 02/01/20 02:40 PM (7 months, 21 days ago)

Hello friends, sorry for missing some questions,  I got super busy and had to take a hiatus from the forums for a while

The good news is that I'm back :smile:

So feel free to post any questions or share any new tricks you've learned

Much love


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26486623 - 02/15/20 09:19 AM (7 months, 8 days ago)

Good to have you here again and thanks for taking the time to write this.
Wish I had read this sooner.
Two days ago I did my first plates and just swiped a Z with the inoculation loop. The thing is that I ran it through the agar not on top of it... but I think that will work out somehow.
Thanks again.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: nosf3r4tu]
    #26486876 - 02/15/20 12:56 PM (7 months, 7 days ago)

That'll happen sometimes. It's fine.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: nosf3r4tu]
    #26487112 - 02/15/20 03:43 PM (7 months, 7 days ago)

Happy to help :smile:

What was your agar recipe?

I really love the streaking technique posted here, REALLY speeds up the process of isolating cultures and cleaning contams. I've gotten an isolate in 3 transfers like this (!)


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26487293 - 02/15/20 06:08 PM (7 months, 7 days ago)

How do you know fur sure that it was an "isolate"


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: verum subsequentis]
    #26487696 - 02/16/20 12:17 AM (7 months, 7 days ago)

Well I guess technically I dont know for sure, havent had its dna analyzed or anything. but it certainly looks and performs like one, probably 10 transfers in, and back and forth from 3 slants, and through multiple grows

But I agree with your point, I've seen stuff randomly start sectoring on later plates after looking perfectly even for a few transfers

Though I've seen Much less of that from MS since I started using that streaking technique for spores, since its basically a serial dilution on the plate, and there are very few spores present in the final zones.

On the plate in question, I even used less spores than normal, and diluted them farther in water before streaking, so very little germinated since they were spread very thin


--------------------

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"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


Edited by c10h12n2o (02/16/20 12:26 AM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26487783 - 02/16/20 02:51 AM (7 months, 7 days ago)

Quote:

c10h12n2o said:
Happy to help :smile:

What was your agar recipe?

I really love the streaking technique posted here, REALLY speeds up the process of isolating cultures and cleaning contams. I've gotten an isolate in 3 transfers like this (!)





PDA, just potato flakes, honey and agar.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26488127 - 02/16/20 12:06 PM (7 months, 6 days ago)

Glad you're back too!

Quote:

c10h12n2o said:
Happy to help :smile:
...cleaning contams. I've gotten an isolate in 3 transfers like this (!)



Does this mean, T1 thru T3? And if so does that mean you are using the same streaking technique with myc?

And maybe I can sneak this question in, no one seems to want to touch it.
My GT isolates keep looking "just fuzzy", am I just isolating contam?
           


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Inthepit]
    #26488398 - 02/16/20 03:08 PM (7 months, 6 days ago)

Quote:

nosf3r4tu said:
Quote:

c10h12n2o said:
Happy to help :smile:

What was your agar recipe?

I really love the streaking technique posted here, REALLY speeds up the process of isolating cultures and cleaning contams. I've gotten an isolate in 3 transfers like this (!)





PDA, just potato flakes, honey and agar.




How much agar agar?

It's easy to accidentally stretch the surface, especially if you are using low amounts of agar


Quote:

Inthepit said:
Glad you're back too!

Quote:

c10h12n2o said:
Happy to help :smile:
...cleaning contams. I've gotten an isolate in 3 transfers like this (!)



Does this mean, T1 thru T3? And if so does that mean you are using the same streaking technique with myc?

And maybe I can sneak this question in, no one seems to want to touch it.
My GT isolates keep looking "just fuzzy", am I just isolating contam?
           



I'm pretty sure this was all documented in a thread somewhere... basically someone was saying it was impossible to get an isolate in less than 10 transfers, so I figured I'd see how fast i could do it, just for fun

I started by taking a few spores from a loop, mixing it in a small media bottle on a stir plate w magnetic stir bar, then used the solution to do serial dilution (took .1ml from the 50ml and used it to make another bottle of 50ml, repeated that 2 times I think). Then i used the most dilute one to streak 5 plates. Then I transferred all the colonies from later streaking zones, and kept transferring till i thought I had an isolate. Most took about 7 transfers but I got lucky on one

So yes, it was 3 transfers away from the streak plate. I did more just to verify

Not streaking with myc, taking tiny transfers the size of a big salt grain using a 15C scalpel blade

About your question:
Have you ever grown it out? 

They definitely look weird.. what kind of agar recipe are you using? Do all your plates look like these or just these cultures?

I cant tell from the pics, does it look powdery at all? The first and 2nd pics kinda do but last one not so much. I've had lots of pan cyan myc look like that 3rd pic

Some myc just refuses to go rhizo on agar, but I'd be surprised to see that across that many cultures . How many transfers in are you from the original print/clone?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26488440 - 02/16/20 03:40 PM (7 months, 6 days ago)

5 g potato flakes
10 ml honey
9g agar agar
450 ml water

I didn't scrach it I went all the way through it. To the bottom.
This is my first attempt at agar and I've learned my lesson:) probably still more to come. I think I have enough spores to play with till I get my first prints.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26488491 - 02/16/20 04:15 PM (7 months, 6 days ago)

Very helpful post indeed. I do have one question though. If performing the 3 streak inoculation with swabs instead of a loop, would you use a new swab for each new streak? Or could you possibly rotate the swab after each streak?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Zakkery]
    #26488556 - 02/16/20 05:00 PM (7 months, 6 days ago)

I dont like using swabs directly to agar, I know it works, I just cant consider them sterile. I use individually wrapped swabs from medical supply places so I guess they are sterile, I just love using a loop

When I'm using a swab I normally scratch it with my loop above zone 1, then streak that area to spread them out, then flame and streak the next zones (using bacticinerator or flame between zones)

That way I'm not introducing food for contams onto my swab, and less chance of introducing contams from my swab onto the agar

Also I usually do 4 or 5 zones for the streak. You could probably do the same with a new sterile swab, but unlike a loop, most spores would probably end up on the swab rather than the agar, so idk how well it would work. I'd just make a loop and either scrape it like I do or swab section 1 then use the loop to spread it out and create the other zones


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26488563 - 02/16/20 05:08 PM (7 months, 6 days ago)

Quote:

c10h12n2o said:
I dont like using swabs directly to agar, I know it works, I just cant consider them sterile. I use individually wrapped swabs from medical supply places so I guess they are sterile, I just love using a loop

When I'm using a swab I normally scratch it with my loop above zone 1, then streak that area to spread them out, then flame and streak the next zones (using bacticinerator or flame between zones)

That way I'm not introducing food for contams onto my swab, and less chance of introducing contams from my swab onto the agar

Also I usually do 4 or 5 zones for the streak. You could probably do the same with a new sterile swab, but unlike a loop, most spores would probably end up on the swab rather than the agar, so idk how well it would work. I'd just make a loop and either scrape it like I do or swab section 1 then use the loop to spread it out and create the other zones





:takingnotes:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26489413 - 02/17/20 06:32 AM (7 months, 6 days ago)

Quote:

c10h12n2o said:
I dont like using swabs directly to agar, I know it works, I just cant consider them sterile. I use individually wrapped swabs from medical supply places so I guess they are sterile, I just love using a loop

When I'm using a swab I normally scratch it with my loop above zone 1, then streak that area to spread them out, then flame and streak the next zones (using bacticinerator or flame between zones)

That way I'm not introducing food for contams onto my swab, and less chance of introducing contams from my swab onto the agar

Also I usually do 4 or 5 zones for the streak. You could probably do the same with a new sterile swab, but unlike a loop, most spores would probably end up on the swab rather than the agar, so idk how well it would work. I'd just make a loop and either scrape it like I do or swab section 1 then use the loop to spread it out and create the other zones




Great thanks for the info. Ive got a bunch of packaged sterile swabs too but I'll take your advice and add making a loop to my 'to-do list'.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26489467 - 02/17/20 07:27 AM (7 months, 6 days ago)

Thank you for such a detailed response!

using a 15C scalpel blade
Awesome scalpel, when I actually grow something, I'll be spending on equipment.

About your question:
Have you ever grown it out?

Sorry I don't know what that means. My guess, what I was thinking, is to just put some in a jar and let 'er rip! LOL Then if it goes 100% colonized, G2G it out.

They definitely look weird.. what kind of agar recipe are you using? Do all your plates look like these or just these cultures?
Well I started with the SAGARI philosophy, but it seems like the agar is really wet. 4g agar, 7g potato, 50% oat broth, for 500mL. Next batch mebbe 8g agar, 7g potato...Oh and I strain the potato bits out because they're distracting. Can hot agar go thru a coffee filter? Oh and some food color which the PESA loves to eat.

I cant tell from the pics, does it look powdery at all? The first and 2nd pics kinda do but last one not so much. I've had lots of pan cyan myc look like that 3rd pic
No not powder, more like cotton ball.
I know this GT refuses to go rhizo. The PESA and TOC with the same agar are fine.
            That T1 went to Oats ACKCHUALLY!   
PESA has gone to Oats and is about 80% col and lookin goooood!
TOC is slower and mebbe soon to Oats. Since Oats are so controversial here's what I'm using.

Some myc just refuses to go rhizo on agar, but I'd be surprised to see that across that many cultures . How many transfers in are you from the original print/clone?
It looks like I'm at T5 and needing to go T6. My labeling system matured as the plates evolved.
   
Funny thing, this pin is my first mushroom in 45 years! OMG!
   

So to anticipate your answers:
  1. Ya, jar that bitch!
  2. No, dummy, you can't get hot agar to go thru a coffee filter. Filter the crappy potato first.
  3. Ya SAGARI is for a certain way, just stick with 8 to 10g agar, and the broth is ok.


Edited by Inthepit (02/17/20 08:39 AM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Inthepit] * 1
    #26489531 - 02/17/20 08:44 AM (7 months, 6 days ago)

Quote:

Inthepit said:
Some myc just refuses to go rhizo on agar, but I'd be surprised to see that across that many cultures . How many transfers in are you from the original print/clone?
It looks like I'm at T5 and needing to go T6. My labeling system matured as the plates evolved.
   
Funny thing, this pin is my first mushroom in 45 years! OMG!
   





So not sure how you are labeling, but I don't see how you are on T5 at all unless there are a lot of plates missing from that pic.  If you have a germ plate, the first transfer off that is T1.  Then a transfer from that particular T1 plate to a new plate is T2 and so on. 


And hot agar will go through a coffee filter just fine.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: InfiniteDreams]
    #26489537 - 02/17/20 09:00 AM (7 months, 6 days ago)

Hey thanks for that, really appreciate all the help!

So not sure how you are labeling, but I don't see how you are on T5 at all unless there are a lot of plates missing from that pic.  If you have a germ plate, the first transfer off that is T1.  Then a transfer from that particular T1 plate to a new plate is T2 and so on.

Well ya, the photo was the one from Germ plates and it isn't up to date with later TX's.


And hot agar will go through a coffee filter just fine.

Oh cool, gotta try that!


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Inthepit] * 1
    #26489934 - 02/17/20 02:39 PM (7 months, 5 days ago)

I would use fine cheesecloth rather than a coffee filter. There are pics of the way I do it in the guide.  Never tried a coffee filter , cheesecloth does the job just fine


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26490078 - 02/17/20 03:59 PM (7 months, 5 days ago)

Quote:

c10h12n2o said:
I would use fine cheesecloth rather than a coffee filter. There are pics of the way I do it in the guide.  Never tried a coffee filter , cheesecloth does the job just fine




Thanks, but the wifie says she hasn't been able to find it here. Prolly try online.
Anyway I'm gonna take the GT to T6 and will see. Thanks again


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26490080 - 02/17/20 04:00 PM (7 months, 5 days ago)

Quote:

c10h12n2o said:
I would use fine cheesecloth rather than a coffee filter. There are pics of the way I do it in the guide.  Never tried a coffee filter , cheesecloth does the job just fine




I must have followed your guide.  Great success and would recommend!


--------------------
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: InfiniteDreams]
    #26490474 - 02/17/20 07:08 PM (7 months, 5 days ago)

I added my bacticinerator to the guide a few days ago. Cant stress how much I love that fuckin thing. Got it for less than 40$!!!

Yeah cheesecloth is at most grocery stores but you can get the extra fine stuff cheap on Amazon

Anytime, happy to help :smile:


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26492212 - 02/18/20 07:56 PM (7 months, 4 days ago)

Quote:

c10h12n2o said:

WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC


c10




How do we do this with a PC that does not have a pressure gauge?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: PNat]
    #26492585 - 02/19/20 12:06 AM (7 months, 4 days ago)

Good question... I'm not sure, I have never actually used one without a gauge

I cant think of a way to guarantee that there would be positive pressure without a gauge. Also you dont want too much pressure ofc. Sorry but I cant think of a good way to do that without a gauge


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26492647 - 02/19/20 01:15 AM (7 months, 4 days ago)

THANK YOU c10 for answering my question, I guess I will skip that part.  I also want to say that I really APPRECIATE you for this agar guide and the tips & tricks that you provided.  I just started, so there are so much to learn and you guys in this forum help me out a bunch.  Again THANKS for the guide.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: PNat]
    #26492756 - 02/19/20 03:54 AM (7 months, 4 days ago)

Happy to help, let me know if you have any more questions:)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26492809 - 02/19/20 06:16 AM (7 months, 4 days ago)

Any input on these PE plates?

Started from a swab.

Only 1 plate showed any growth so transferred to 3 plates then took 3 samples from the best two plates and below is the result.

Are these good for grain now or should I transfer some more?

Plate 1:


Plate 2:


Plate 3:


Thanks


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: PNat]
    #26492831 - 02/19/20 07:07 AM (7 months, 4 days ago)

Quote:

PNat said:
Quote:

c10h12n2o said:

WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC


c10




How do we do this with a PC that does not have a pressure gauge?





Just an idea..  You could time how long it takes for your pressure cooker to go from your usual sterilization pressure(15,16,17, etc) to 0 by starting your timer when you remove it from the heat and then stopping it when you hear the seal fall(the seal is officially called an Air Vent Cover Lock). 

Then in the future you can just walk into the kitchen and 2 minutes before it's due to fall and then remove the weight when it does.

Frankly though, simply listening for the seal to fall and immediately going to remove the weight is probably sufficient.


--------------------
Everything i say is completely hypothetical...



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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Hobbyist]
    #26493110 - 02/19/20 11:35 AM (7 months, 4 days ago)

Quote:

Hobbyist said:
Quote:

PNat said:
Quote:

c10h12n2o said:

WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC


c10




How do we do this with a PC that does not have a pressure gauge?





Just an idea..  You could time how long it takes for your pressure cooker to go from your usual sterilization pressure(15,16,17, etc) to 0 by starting your timer when you remove it from the heat and then stopping it when you hear the seal fall(the seal is officially called an Air Vent Cover Lock). 

Then in the future you can just walk into the kitchen and 2 minutes before it's due to fall and then remove the weight when it does.

Frankly though, simply listening for the seal to fall and immediately going to remove the weight is probably sufficient.




That's a great idea!!!  I will definitely try that method and I was thinking about this all night.  Would wrapping a foil on the media lid take care of the sucking pressure?


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Grumio]
    #26493324 - 02/19/20 02:08 PM (7 months, 3 days ago)

Quote:

Grumio said:
Any input on these PE plates?

Started from a swab.

Only 1 plate showed any growth so transferred to 3 plates then took 3 samples from the best two plates and below is the result.

Are these good for grain now or should I transfer some more?

Plate 1:


Plate 2:


Plate 3:


Thanks




They are probably fine . They dont look GREAT, but personally I've never gotten great looking pe myc on agar. I know other people do all the time, might just be the genetics I've worked with. That said, they've always fruited fine

But it really depends on your goal. If you are fine with MS, go for it

Quote:

Hobbyist said:
Quote:

PNat said:

How do we do this with a PC that does not have a pressure gauge?





Just an idea..  You could time how long it takes for your pressure cooker to go from your usual sterilization pressure(15,16,17, etc) to 0 by starting your timer when you remove it from the heat and then stopping it when you hear the seal fall(the seal is officially called an Air Vent Cover Lock). 

Then in the future you can just walk into the kitchen and 2 minutes before it's due to fall and then remove the weight when it does.

Frankly though, simply listening for the seal to fall and immediately going to remove the weight is probably sufficient.




That's a great idea, assuming the seal fails while it still has positive pressure. And if it doesnt, just subtract a minute or so from the wait time

Also you'll probably have to factor in the volume of water and maybe ambient temp

Quote:

PNat said:
Quote:

Hobbyist said:
Quote:

PNat said:
Quote:

c10h12n2o said:

WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC


c10




How do we do this with a PC that does not have a pressure gauge?





Just an idea..  You could time how long it takes for your pressure cooker to go from your usual sterilization pressure(15,16,17, etc) to 0 by starting your timer when you remove it from the heat and then stopping it when you hear the seal fall(the seal is officially called an Air Vent Cover Lock). 

Then in the future you can just walk into the kitchen and 2 minutes before it's due to fall and then remove the weight when it does.

Frankly though, simply listening for the seal to fall and immediately going to remove the weight is probably sufficient.




That's a great idea!!!  I will definitely try that method and I was thinking about this all night.  Would wrapping a foil on the media lid take care of the sucking pressure?




The whole point of that trick is to NOT have any negative pressure, you want to do it while it's got positive pressure so that you dont have to worry about anything getting sucked in. That said, I still use foil, but it's mostly to prevent stuff from getting too wet


--------------------

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"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


Edited by c10h12n2o (02/19/20 02:14 PM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26495598 - 02/20/20 06:40 PM (7 months, 2 days ago)

I also want to say thanks to you c10 for this guide. Getting back into the hobby and getting back into agar. Building a new still air box today as my former was just too small and cumbersome.
Just got 500 plates delivered and a media bottle.
I started some spores on no pour pastys a few weeks ago so i have some transfers to do, plus start some new plates with the streaking technique.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: JohnnieYen]
    #26495633 - 02/20/20 06:59 PM (7 months, 2 days ago)

Happy to I could be helpful! Let me know if you have any questions about anything


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


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"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26504912 - 02/26/20 03:51 PM (6 months, 27 days ago)

C10 this was a great write up. Also found some really useful information here. I love the idea of using colored plates based on culture varieties.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Sockadin]
    #26504919 - 02/26/20 03:53 PM (6 months, 27 days ago)

Thanks, I'm glad you found it helpful :smile:


--------------------

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"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26570393 - 04/01/20 06:53 AM (5 months, 24 days ago)

I could use some help to correct whatever I did to get this black mold.
My first guess is the from the water I was misting with.
Possibly I could avoid the black mold by misting with bleach?
:mushdance:   
A little history of the tubs; first they were too wet, haven't dialed in the proportions yet.
Then I took the lids and put them sideways, but I believe the tubs got too dry. Then I started misting heavily.
So basically over correction all around, noob style!

And I wonder how I got it, but there's lots of mold here in the tropics.
No one drinks the tap water here!


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Inthepit]
    #26571577 - 04/01/20 06:26 PM (5 months, 23 days ago)

Have those tubs flushed yet? Or is this before the first flush?

At what point did the mold show up? I'd definitely clean that asap before it starts spreading spores


--------------------

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"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26571607 - 04/01/20 06:48 PM (5 months, 23 days ago)

Yes, 2nd and maybe 3ird. They were rather random looking compared to the first flush.
     

And the shoeboxes have been turned into a garden patch
And washed with soap and bleach.
I noticed the mold just recently.

But, in the future would your bleach mist be beneficial?
Bleach mister TEK formula

Thanks for your time! :hatsoff:


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Edited by Inthepit (04/01/20 06:56 PM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Inthepit]
    #26571614 - 04/01/20 06:54 PM (5 months, 23 days ago)

No problem.  I think a lot of that is spores, but that one pic does look more like mold


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"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26652266 - 05/06/20 07:55 AM (4 months, 20 days ago)

When I put my agar mixture into the microwave to disolve it down, it just gels up the agar and everything else is liquidy. So i cant really filter it. Am I not boiling/microwaving it enough? I do around 10min all together.


--------------------
“Today a young man on acid realized that all matter is merely energy condensed to a slow vibration, that we are all one consciousness experiencing itself subjectively, there is no such thing as death, life is only a dream, and we are the imagination of ourselves. Heres Tom with the Weather.”
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: HoneyBadger9321]
    #26652281 - 05/06/20 08:10 AM (4 months, 20 days ago)

Quote:

HoneyBadger9321 said:
When I put my agar mixture into the microwave to disolve it down, it just gels up the agar and everything else is liquidy. So i cant really filter it. Am I not boiling/microwaving it enough? I do around 10min all together.




I'm not entirely sure I understood your question but basically when microwaving you need to almost bring it back to boil (with some mixing,shaking in the middle to ensure consistent heating).  It does not warm up evenly so you have to shake/mix it a few times (lid closed).

Agar has to reach >85C before solid chunks start to melt and due to how microwave oven works it never is 100% even. Then at around 32-38 it starts setting again so you have a window between 60C(too hot to handle for most people, also you get more condensation but other than visibility  that it is fine to have it) and 35C to pour it.

For my 1L media bottle (about 800ml of agar in there) I do about 6min x 600W, shake,  followed by 3 min at 600W and then I repeat it in 30s-60s chunks until all chunks have melted and the liquid is uniform. I find that using maximum power (900W in my case) produces too localised heat in places where waves end up concentrating more so it can almost burn by accident.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: MrBovineJoni]
    #26652284 - 05/06/20 08:12 AM (4 months, 20 days ago)

Quote:

MrBovineJoni said:
Quote:

HoneyBadger9321 said:
When I put my agar mixture into the microwave to disolve it down, it just gels up the agar and everything else is liquidy. So i cant really filter it. Am I not boiling/microwaving it enough? I do around 10min all together.




I'm not entirely sure I understood your question but basically when microwaving you need to almost bring it back to boil (with some mixing,shaking in the middle to ensure consistent heating).  It does not warm up evenly so you have to shake/mix it a few times (lid closed).

Agar has to reach >85C before solid chunks start to melt and due to how microwave oven works it never is 100% even. Then at around 32-38 it starts setting again so you have a window between 60C(too hot to handle for most people, also you get more condensation but other than visibility  that it is fine to have it) and 35C to pour it.

For my 1L media bottle (about 800ml of agar in there) I do about 6min x 600W, shake,  followed by 3 min at 600W and then I repeat it in 30s-60s chunks until all chunks have melted and the liquid is uniform. I find that using maximum power (900W in my case) produces too localised heat in places where waves end up concentrating more so it can almost burn by accident.




Wow, beautiful. Thank you it helps a lot.


--------------------
“Today a young man on acid realized that all matter is merely energy condensed to a slow vibration, that we are all one consciousness experiencing itself subjectively, there is no such thing as death, life is only a dream, and we are the imagination of ourselves. Heres Tom with the Weather.”
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: MrBovineJoni]
    #26652285 - 05/06/20 08:14 AM (4 months, 20 days ago)

The microwaving takes up a lot of the work time. Is it possible to just skip this step. The microwaving is to achive crystal clear agar, correct?


--------------------
“Today a young man on acid realized that all matter is merely energy condensed to a slow vibration, that we are all one consciousness experiencing itself subjectively, there is no such thing as death, life is only a dream, and we are the imagination of ourselves. Heres Tom with the Weather.”
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: HoneyBadger9321]
    #26652297 - 05/06/20 08:23 AM (4 months, 20 days ago)

Quote:

HoneyBadger9321 said:
The microwaving takes up a lot of the work time. Is it possible to just skip this step. The microwaving is to achive crystal clear agar, correct?



Sorry, I might have misunderstood your question.


So there are 2 steps where microwave is used in this TEK.

1) Initial preparation to cook agar in the bottle before pressure cooking. This ensures that the contents of bottle are uniform and agar is evenly mixed in water. This step is very important and not what I described in my previous post above. if you skip this step you might get permanently lumpy/chunky agar if some agar bits don't properly dissolve before PCing.

2) Reheating of already PCed and cooked agar bottle (stored in fridge) to use for pouring. This is what I described above.


Edited by MrBovineJoni (05/06/20 08:24 AM)


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: MrBovineJoni]
    #26652620 - 05/06/20 11:56 AM (4 months, 20 days ago)

Right yes! I was referring to the first (before PC-ing). I just cant seem to dissolve it completely and chunks still remain when i filter it. Maybe i am not doing it long enough. I had a look at RRs agar videos and there he does not cook it before PC-ing. Takes a lot of time for me and seems that the results are the same, just a little bit cloudy agar. I might be wrong tho?


--------------------
“Today a young man on acid realized that all matter is merely energy condensed to a slow vibration, that we are all one consciousness experiencing itself subjectively, there is no such thing as death, life is only a dream, and we are the imagination of ourselves. Heres Tom with the Weather.”
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: HoneyBadger9321]
    #26652673 - 05/06/20 12:29 PM (4 months, 20 days ago)

Quote:

HoneyBadger9321 said:
Right yes! I was referring to the first (before PC-ing). I just cant seem to dissolve it completely and chunks still remain when i filter it. Maybe i am not doing it long enough. I had a look at RRs agar videos and there he does not cook it before PC-ing. Takes a lot of time for me and seems that the results are the same, just a little bit cloudy agar. I might be wrong tho?



I see, that makes sense. To be honest I'm not sure about that chunks/sediment in the agar as I have never had it happen for me but I have seen plenty of people have it in their agar. Perhaps it is the matter of TEK or just the quality of ingredients.

What I usually do ( I started basing my initial agar work on C10s guide) for my media bottle (1L - safe to use for up to 850ml of mixture, otherwise it might boil over):
a) weight dry ingredients. In my case it is 12g of LME (light malt extract) & 16g of agar agar powder for ~800ml of water.  Mix them.
b) carefully transfer dry mixture to the media bottle and add about 100g of water, tightly close the bottle and shake the living shit out it .
c) Add the rest of the water (so it makes ~800ml or so) and shake once more. There are bound to be some chunks and flecks floating (for me they seem to be from the LME powder not the agar. LME absorbs water from the air like crazy so it does tend to clump up.
d) (optional) Add a drop of food colouring (I like green but it is up to personal preference).
5) Put the bottle cap on (it should not be tight so some of the steam can escape) and place in a microwave. Blast it on max for a while and keep shaking every few minutes (lid tightened for that!).
6) I do this until all of agar has dissolved (the temperature of the agar here is close to boiling point so wear gloves). However, be careful once the agar starts bubbling in places (and close to being fully dissolved) - as the cap is not super tight at this point. Agar boils over incredibly quickly so if you turn away for 20s you can end up cleaning your microwave. The result is crystal clear agar for me except for some bubbles on top from all the shaking (it will disappear after PCing).

7) Done - put the foil over the cap and place in a PC.

Steps 1-6 don't take longer than a total of 15minutes for me.


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: HoneyBadger9321]
    #26653033 - 05/06/20 04:09 PM (4 months, 19 days ago)

Quote:

HoneyBadger9321 said:
When I put my agar mixture into the microwave to disolve it down, it just gels up the agar and everything else is liquidy. So i cant really filter it. Am I not boiling/microwaving it enough? I do around 10min all together.




Before it boils i stir it with a stir rod, once or twice

Once it starts boiling i turn the power down to 50% , stir it, and try to maintain that boil for a minute or so. Super carefully,  because it boils over very easily

I wouldnt say these steps are mandatory, but it definitely seems to help get crystal clarity and perfect uniformity.

Sounds like you are just omittimg the intermittent stir. Also make sure you use cold or lukewarm water for the initial mix before boiling. If the water is too hot it will clump up

Whole process of measuring, mixing, boiling, straining, and foiling takes me about 10 minutes total for 1 liter of agar


--------------------

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26689881 - 05/23/20 09:38 AM (4 months, 3 days ago)

hello, just started using this site seriously, this is my first time trying agar out and honestly I'm really satisfied with how healthy the mycelium looks and how easy contaminates are to deal with!



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Life, is art, and art is life. 
Since art is subjective to the viewer, there is no such thing as good or bad art, seeing as those are concepts humanity invented to control the masses.
Therefore, I feel as though I am damn well entitled to my opinion and nobody could prove me wrong otherwise."
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: TigerToxins]
    #26692327 - 05/24/20 12:21 PM (4 months, 2 days ago)

Yep. Agar is the shiz


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: verum subsequentis]
    #26693217 - 05/24/20 07:33 PM (4 months, 1 day ago)

Nice!

It really helps a lot w success rates. It quite addictive, i think i need agar rehab haha


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C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


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"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26694564 - 05/25/20 11:40 AM (4 months, 1 day ago)

I'm perfectly satisfied with my agar habbit. No rehab for me


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: verum subsequentis]
    #26695320 - 05/25/20 06:43 PM (4 months, 17 hours ago)

Yeah lemme rephrase that:

My gf thinks i need agar rehab


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C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26919355 - 09/05/20 03:30 PM (21 days, 21 hours ago)

Thank you so much for posting this exhaustive exposé on agar technique.  Like many of the other TEKS contained in this forum, this provides a fast-track for a beginner like me to obtain successful results.  The work that it took to prepare the pictures, the write-up, and the experience required to make this happen are noted and appreciated.


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