Mark, is any of this selective media available in pre-poured plates? I’d love to be able to plate a mixed culture from a lambic and easily find the Brettanomyces colonies. Since I don’t have a pressure cooker, getting some pre-poured plates eliminates my primary barrier for entry into this part of the hobby. It’s been a while since I took Micro lab, but I’m pretty sure I can muddle through with plates and paperclips to start.
If you end up selling the tubes, I would be interested in participating.
I believe that White Labs sells pre-poured selective media plates, but I may wrong. As an RPh, preparing media from scratch, which involves starting with a base medium and adding reagents, should be a trivial process for you. I am not into wild yeast or beer spoilage bacteria other than eradicating these organisms from my brewery. Yeast, bacteria, and mold usually look very different on even a simple malt agar plate. However, selective media are much better at telling you exactly what is on the plate. For example, I used Lin’s Wild Yeast Medium to positively identify the wild yeast that was plaguing me when I was using Star San.
If you are interested in isolating Brettanomyces, here is a web page that focuses on Brettanomyces identification:
As an RPh, it will be much easier for you to obtain the reagents needed than an every day brewer. Many reagent suppliers will not ship to a home address. From our interactions, I am assuming that you work for an organization where the work involves more than pill counting; hence, you should be able to order reagents and have them shipped to your place of work as long as your employer does not mind.
Plates and slants are stored in a refrigerator at 4C/40F.
Correct
The process of inoculating a blank slant from a slant that contains a culture is known as “subculturing.” The amount of time that a slant will remain viable on a slant is strain dependent. Some strains will last two years without subculturing. Other strains, need to be subcultured every six months.
While many breweries use the master/working slant model. I do not brew enough to use this model, and any amateur brewer brewing with multiple strains will more than likely not use the master/working slant model. One the tenants in my brewery is that one should limit the amount of handling any yeast culture receives. A lot people who purchased yeast on slant form BrewTek did not subculture. They merely used the slants that they purchased for multiple inoculations. I do not recommend that model.
I posted this information earlier in the thread.
The use cycle is as follows
-
Pick the slant that has the most robust growth
-
Subculture two new slants from this slant (i.e., inoculate new blank slants with the loop using aseptic transfer technique)
-
Inoculate 20 to 40mls of autoclaved wort from the slant using aseptic transfer technique (I usually use more than one scrap)
-
Discard the slant that was subcultured and used to make the starter (i.e., the other old slant for this culture can be discarded at this point, or one can wait until the subcultured slants proof before discarding it)
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Incubate the slants (caps loose to allow for offgassing) and the 20 to 40ml starter
What will happen during incubation is that yeast will cover the surface of the agar solidified medium. I usually let the incubation period run until there is no change in the amount surface area covered by yeast. I then tighten the caps (they have to able to offgas while incubation), wrap a strip of Parafilm (a wax coated plastic wrap) around the cap and the tube, and write the culture number, date subcultured, and the generation from the original culture (the generation here is the slant generation, not the yeast cell generation because multiple generations of cells grow everything a culture is subcultured).
The 20 to 40 milliliters of sterile wort will usually need to be incubated for at least a day before being stepped up in volume. I usually let my first level culture grow for two days because I innoculate 40mls of autoclaved wort. If one is very clean (I mean anal retentive clean), 40mls can be stepped to 1L of boiled starter wort. That’s a 25:1 step ratio, so sanitation cannot be be lax (i.e., no “spray and go” Star San sanitation, in fact, no Star San period). I usually step 15:1, but I rarely grow a starter that is larger than 600ml due to my batch size being 3.5 gallons. A safer approach for someone just starting out is to use two steps. The first inoculation is 10mls of autoclaved wort. That culture is stepped to 100mls using autoclaved wort, and finally to 1L of boiled wort. That sequence got to be old fairly quickly, so I started bumping up the volume of the initial inoculation.
When growing a culture from such a small amount of yeast, we have to be mindful of what I am going to start to refer to as time-to-own, which is the time between inoculation and the culture owning the medium. The ratio between the medium volumes affects the time-to-own. The larger the step, the longer it takes the culture to own the wort. Extending time-to-own means that sterilization and sanitation become more critical.
For those who have not contacted me by PM, I need to know who is seriously interested in a 24-tube bundle. I have enough tubes for eight more people. I have had a request to assemble a starter kit. I willing to assemble a starter kit at cost, which includes the cost to ship the items to me. However, I do not want to be left holding the bag because I am not a business, nor do I want to be in the yeast culturing supplies business. This kit will be a shot deal because I am able to supply a limited number of new high-quality culture tubes from my personal collection of glassware. When they are gone, they are gone!
Here’s what I am proposing:
24 of the Kimble-Chase 45066A-13100 13x100mm culture tubes
A flint glass alcohol lamp
A basic nichrome loop (a basic nichrome loop will last a long time if not abused)
Agar powder
10 pre-sterilized plastic petri dishes (or pre-poured plastic plates)
You provide a pressure cooker capable of producing at least 13PSI above atmospheric pressure (15 minutes at 15PSI or 30 minutes at 13PSI).
The one thing that I am having difficulty acquiring without causing the price to rise too high are containers for first-level starters. I would love to include borosilicate glass media bottles, but the Corning 1395-100 media bottles that I use cost around $10.00 a piece in quantity 10, plus shipping. As I mentioned earlier, I used 4oz de-labeled baby food jars for a long time before before switching to media bottles. I would like to avoid including off-brand glassware. Corning and Kimble-Chase are the names to buy when purchasing laboratory glassware.
Anyone who is seriously interest in this project (because that is what it will turn out to be), please send a PM.
You’ve got a PM.
If they end up not being included in the kit and folks want to source media bottles, they’re avalable with prime shipping:
Single Corning 1395-100:
http://www.amazon.com/PYREX-100mL-Round-Storage-Bottles/dp/B004XR5T48/ref=sr_1_1?ie=UTF8&qid=1445254074&sr=8-1&keywords=Corning+1395-100
Pack of 10 Corning 1395-100:
http://www.amazon.com/dp/B004DGIGLW/ref=biss_dp_t_asn
I am on the fence with the kit idea. As I mentioned earlier, I am not a business, nor do I want to be in the yeast culturing supplies business. The only way that putting a kit together makes sense is if it can lower the overall cost, and the only way to do that is to shave shipping costs.
Here is a list of items and sources:
1.) 100 grams of microbiological grade powdered agar
I have had very good success with the stuff that Green Bio Research on eBay sells. A lot of the food grade stuff one that finds in health food stores and Chinese food stores is not as pure, so one end ups with non-agar particles in one’s media. Buying this quality of agar used to mean having to purchase 500 grams plus. It only takes 3.75 grams of agar to solidify 250ml of liquid media, so 100 grams will last a long time. I usually prepare 300ml of agar solidified media at time, but I am pouring plates and filling 20 larger tubes at the same time.
The rest of the items can be purchased from Carolina Biological Supply. This company deals primarily with professional educators and homeschoolers; therefore, their prices are very reasonable. They also sell in quantity one at a reasonable price.
2.) 5mm nichrome loop
I have been using aluminum handle, press fit loops for over twenty years. I like this design because there are no threads.
$2.95, purchase two because it is nice to have a backup
3.) Alcohol lamp
www.carolina.com/laboratory-burners/alcohol-lamp-with-metal-cap/706604.pr?prod_id=706604
$9.45
4a.) Pre-poured plates
Sabouraud agar is basically YPD without the yeast extract. It is a carbon (dextrose) and nitrogen (peptone)-based medium that is designed for growing fungi, and yeast are single-cell fungi.
Pack of 10 for $22.75
or
4b) Disposable Presterilized plastic petri dishes (you pour)
Pack of 20 for $5.95
or
4c.) Reusable borosilicate glass petri dishes (you sterilize and pour)
There are borosilicate and soda lime glass petri dishes. You want to purchase borosilcate glass. Pyrex is Corning’s trademarked name for borosilicate glass items. Kimax is Kimble-Chase’s trademark for borosilicate glass items. Glassware carrying either of these trademarks without the manufacturer name can be assumed to be manufactured by the trademark holder.
Corning 3160-100 100x10mm (my favorite size)
www.carolina.com/lab-dishes/pyrex-petri-culture-dish-100-x-10-mm/741156.pr?question=
$6.95 each
Corning 3160-101 100x15mm (a slightly deeper dish)
www.carolina.com/lab-dishes/pyrex-petri-culture-dish-100-x-15-mm/741158.pr?question=
$6.95 each
Carolina biological supply has a varied assortment of culture tubes, including Corning culture tubes. One word of advice is that one gets what one pays for when purchasing culture tubes. The first thing to go is the cap liner followed by the cap. The better quality Corning culture tubes sport a white marking spot. The Corning tubes with a blue marking spot are economy tubes. The Corning culture tubes with the white marking spot are from the Corning 9825 line of tubes. They are one step down in quality from the Kimble-Chase tubes shown above. They are fine tubes that will last a long time. The only difference is that they have plain rubber liners instead of PTFE-faced rubber lines. The Corning equivalent to the Kimble-Chase tubes shown in this listing is Corning 9826-13.
5.) Catheter Tip Irrigation Syringe (optional, but will make filling culture tubes easier).
$2.68
6.) Parafilm
Parafilm is used to seal culture tubes and plates. It is a stretchable plastic wrap that is coated with wax. I purchased a 2" x 250’ roll that will probably last my lifetime and that of my children. Carolina Biological supply sells Parafilm. You want the laboratory version of Parafilm, not the stuff that they sell for grafting. I cut off a 3/4" strip per culture tube, which means that it takes about fifteen inches to seal 20 cultures tubes. The tubes are sealed after they have been inoculated and the culture has been incubated long enough to cover the surface of slant (the cap must be loose, so that the culture can outgas during incubation).
This is cool. It seems like the only expensive item is the pressure cooker. What is the minimum size pressure cooker that will suffice?
Regarding the pre-poured plates, do they need to be used relatively quickly or will they last for a while if properly stored?
If you’re price shopping, check out White Labs lab supplies section on yeastman.com I can’t speak to the quality as of yet, but I just placed an order from them for some pre-poured plates to play around with plating and isolating colonies from mixed cultures. Prices were in the range of $15 for a 10-pack depending on the specific media you get.
While purchasing a la carte affords one the most flexibility, the downside is increased shipping costs. I suggested purchasing supplies from Carolina Biological supply because more than one items can be purchased from the site, resulting in reduced shipping costs (as well as taking me out of the loop).
While I would like to own a nicer and larger pressure cooker, I have been using the same T-Fal 6qt pressure cooker for twenty-three years.
You should be able to get around six months before the plates start to dry out if they are sealed with Parafilm and stored upside down.
Mark, my big question isn’t where to source things from, it’s what do I really need, and how many do I need? I kit isn’t necessary, and some guidelines for what is needed would be helpful.
It’s clear I was off base on the number of culture tubes I’d want. How many media bottles/baby food bottles yould you recommend? Reusable plates?
You need more culture tubes than anything else because they are the workhorses of an amateur brewery culture collection. Experience has taught me that the smallest number of culture tubes that a brewer should have is two dozen. A bank that contains more than a couple of cultures will be difficult to maintain if one is forced to prepare less than a dozen blank slants at a time, and one should keep two inoculated slants per culture.
To be completely honest with you, borosilicate glass Petri dishes are nice to have around for those times when you are out of disposable presterlized dishes, but I guarantee that you will eventually switch to using disposable presterilized dishes after the newness of the hobby wears off. At most, you will need half a dozen borosilicate glass Petri dishes.
If you are like me, you will probably pour half a dozen plates at a time, and end up throwing half away because you do not use them before they dry out. Your greatest plate usage will occur while establishing your bank because all of the cultures that are received in non-slant form will have to be plated for single colonies. I own twelve Corning 3160-100 Petri dishes (I purchased a pack new), and every poured plate that is currently in my blank media storage container was prepared using a disposable presterilized Petri dish. The difference in the amount of time that it takes to prepare plates in presterilized plastic dishes and glass dishes is like the difference between extract and all-grain time-wise.
You need at least one nichrome loop. They last a long time in an amateur brewery, but they are cheap enough that you can afford to own a backup.
You only need one alcohol lamp. You may or may not outgrow an alcohol lamp. While I own a couple of Bunsen burners, I still use a flint glass alcohol lamp most of the time due to its portability and much less intense flame. Some people are not patient enough to use an alcohol lamp. The loop has to be placed in the tip of the inner cone of the flame until it turns red hot. The flame on a Bunsen burner burns so much hotter that a loop turns red almost immediately after being placed in the inner cone, but it is also easier to burn oneself and burn one’s house down with a propane-fired Bunsen burner. I burn regular denatured alcohol in my lamp. Denatured alcohol is found in the paint section of most hardware stores, including Home Depot and Lowes. An alcohol lamp will also burn 190 proof Everclear with a nice blue flame, but 190 proof Everclear is illegal in Maryland.
I purchased a 10-pack of Corning 1395-100 100ml media bottles. I believe that the Kimble-Chase number for this size media bottle is KC14395100. I prepare 8 media bottles at time because that is the limit of my 6L pressure cooker. I will take me around six months to work my way through that many media bottles, but I am not going crazy with cultures.
Thanks for sharing this info with us Mark.
I think more importantly than size, I’ve heard you want to look for an American made model that will easily reach autoclave conditions (preferably 15 psi for at least 15 min). They are more expensive but worth the price because they are incredibly well made and you are assured to reach the proper temp/pressure conditions for sterilization. Chinese made pressure cookers will barely get you there.
Thought that you yeast guys might dig this:
Hope it posts correctly…
Cute
You are talking about the All American pressure canner/cookers. The only thing that keeps me from purchasing one of those pressure cookers is that they get scungy looking due to being made out of cast aluminum. It is merely an appearance thing, but I have a difficult time getting past it.
In practice, a 13PSI European standard pressure cooker will also work, but it extends the processing time to 25 minutes instead of 15 minutes. Thirteen PSI above normal atmospheric pressure delays boiling until water reaches 119C at sea level. At 1000 feet of altitude, the processing time at 13PSI extends to 30 minutes.
From: www.fao.org/docrep/010/ai407e/ai407e22.htm
The piece of autoclave tape shown below was inside of my 23-year-old 6L T-Fal stainless steel pressure cooker (the tape was wrapped around one of my 100ml media bottles). It is a European standard pressure cooker, which means that it only reaches 13PSI above atmospheric pressure. Autoclave tape looks like funny looking masking tape before it is subjected to autoclaving. The black lines form when the tape has been subjected to moist heat and pressure capable of sterilization.
The 15PSI for fifteen minutes rule only holds at elevations near sea level because autoclaving is altitude dependent. In order to determine the processing time for one’s altitude, one must first determine one’s air pressure using this page: Atmospheric Pressure vs. Elevation above Sea Level
The next step is to determine the temperature at which water boils given one’s air pressure and the pressure cooker PSI rating by adding the two values and looking up the temperature in Celsius on this page: Water - Boiling Points at Higher Pressures
The final step involves looking up the F-value in the F-value table associated with the temperature determined in the previous step, and then dividing 15 by that value. The result is the number of minutes of processing time that is equal to autoclaving at 15PSI at sea level.
For those who want to how the 15PSI at 15 minutes rule evolved, well, it is the amount of time at 15PSI that it takes to kill Bacillus stearothermophilus spores.
I don’t know if this is the one Mark has, but so far it’s the only pressure cooker I’ve seen that definitively states an ability to reach 15 PSI. Many of the other ones I’ve seen appear to reach 15 PSI, but don’t explicitly state that they do.
The model that I own was phased out of production long ago. The model that you have linked appears to have 10 and 15 PSI settings. My pressure cooker is actually a made in France T-fal-branded SA SEB 6L LNOX Type 3215 (T-fal model number 33215), which is why it is a 13PSI pressure cooker. It has a weight, but the weight spins instead of rocks. From what I can ascertain, the SEB manufactured pressure cookers were the original Sensor series. I purchased the model that I own because it was safer than the Presto pressure cookers that were sold during that point in time. I need to purchase a backup gasket before they are no longer available.