Building a home QC lab... questions

[passedpawn]

That all sounds correct to me. I don't know why the triple lines are there. I use them as landmarks / boundaries to know where the heck I am on the plate.

 

[ajdelange]

And that is exactly what they are there for.

 

[butterpants]

The manufacturer made (makes?) a kit for this microscope. Conceivably a third party could make one that is compatible but I am not aware of any. The substage condenser is removed and replaced with a wheel that contains aperture plates for each of several objective strengths and the rest of the kit is a set of phase objectives and a telescope (eyepiece) for aligning the zone plates.

The conversion kit is still made. Branded Cambridge Instruments which I believe is some hybrid of post B+L/Leica conglomeration. 875$ I was quoted from microscopecentral. Looked around for a used one but it seems once the scope is converted, people just leave it and sell the whole package.

It's in my future but kills my lab budget too much at the moment....

 

[butterpants]

First off guys, thanks for making this thread not only informative but a lively discussion as well. I was afraid initially there would be little interest. I'm glad that wasn't the case. I've found literally every post to be helpful, which is crazy because that never happens.

Ok so I'm actually buying stuff now! I've got a stupid long laundry list of items i'm thinking of. Hopefully you guys can help me narrow it down a bit....

The microscope was bought. Bausch&Lomb galen 3. Ebay 300$. I just couldn't pull the trigger on bargain optics even if the price point was appealing. I spent a lot of time behind a bolt gun and understand the value of glass. Doesn't have phase contrast (for now) which is a bummer but I can add that later.

Autoclave purchased as well.....American Sterlizer, again ebay 300$. 25quarts I believe and it's the model with its own heat source so you just plug it in. I can't wait to cram my Therminator in there.....soooo dirty

Hemacytometer and pipettes listed earlier from Cynmar. I already have an Acculab VIC123 so precision weighing is not a problem.

For the questions....

1) Are you guys using an incubator for plate growth? If not room temp is ok?

2) Thinking of getting a better pH meter while Cynmar is in my sights. The pen style I have from Hana keeps going tits up. Thoughts?

3) For staining/fixing are their certain slides that are preferred? What thickness of coverslips to get?

4) How are you cleaning your optics? Alcohol and lens paper?

5) Which housing tubes should I use for slants? How long as you guys getting away with freezing them at household fridge temps?

6) Glassware.....between borosilicate bottles for keeping sterile wort, beakers for mixing agar, flasks for propagation and graduated cyls what count and sizes are mandatory or need multiple of? Stoppers as well or only tinfoil? The biggest batches I ferment are 12 gallons.

7) Thinking of a stirplate with a heating element. Anyone doing this?

8) Do they make Bunsen burners that will run off compressed camp fuel bottles or even larger propane tanks? I really don't like alcohol lamps (if you've ever had a crazy ******* throw a Molotov cocktail at you, you'll know why).

9) Methylene Blue and gram stain kit....any other stains needed?

10) For the guys pouring their own plates.....into plastic or glass? I was initally thinking of just buying prepoured but the shelf life worries me. Think i'd rather have a bag of WLN/WLD and just be able to make them as I go since I have the autoclave. I'm assuming (hoping) the dry media has an extended shelf life.

That's it for now

 

[passedpawn]

9) Methylene Blue and gram stain kit....any other stains needed?

Alkaline Methylene Violet. I bought it from White Labs. I think it was a better viability indicator. I used it and the blue stain.

 

[butterpants]

ok great, thanks.

For White Labs ordering do I need to create an account at yeastman? Everytime I go to try and browse the products there it promps me to that page....

 

[ajdelange]

2) Thinking of getting a better pH meter while Cynmar is in my sights. The pen style I have from Hana keeps going tits up. Thoughts?

https://www.homebrewtalk.com/f128/ph-meter-calibration-302256/

Looks as if the Milwaukee MW102 and the Hach Pro+ are the contenders in the near $100 market at this point. Hach is still shipping some defective units but making good on them willingly. The Milwaukee has a separate probe if that is appealing.

 

[butterpants]

thanks aj. yes the seperate probe is nice

 

[nhamilto40]

For a propane lab burner search Cynmar for "burner lp". lp in this context means propane and/or butane at 11 inches water column. Cost is $10-60 depending on type/output.

You will need an appropriate regulator for you gas source. Disposable camping bottles or a grill tank will work but the small disposables may freeze when used with a Meker.

 

[passedpawn]

ok great, thanks.

For White Labs ordering do I need to create an account at yeastman? Everytime I go to try and browse the products there it promps me to that page....

Yes, that's what I did.

 

[butterpants]

Text books/Lab manual recommendation?

Brewing Microbiology by Fregus Priest was mentioned a few places I looked. Amazon has a 2013 softcover edition for 75$.

I'm the type of person who easily reads a text then can go put it into practice, so for me a lab manual is just in the cards...

 

[helibrewer]

Text books/Lab manual recommendation?

Brewing Microbiology by Fregus Priest was mentioned a few places I looked. Amazon has a 2013 softcover edition for 75$.

I'm the type of person who easily reads a text then can go put it into practice, so for me a lab manual is just in the cards...

Once you get yourself organized, write yourself some simple protocols to use everytime you do a particular task, including the aseptic techniques to use. For the hemacytometer make sure to note your counting techniques, i.e. which 2 sides of a square you count cells that are touching the line or not. We used the center section (25 small squares) and counted each corner square and the center square ignoring cells that touched the left and top lines, then averaged those 5 sections, which is why written protocols help even if it is just you doing the work.

 

[trentm]

First off guys, thanks for making this thread not only informative but a lively discussion as well. I was afraid initially there would be little interest. I'm glad that wasn't the case. I've found literally every post to be helpful, which is crazy because that never happens.

Ok so I'm actually buying stuff now! I've got a stupid long laundry list of items i'm thinking of. Hopefully you guys can help me narrow it down a bit....

The microscope was bought. Bausch&Lomb galen 3. Ebay 300$. I just couldn't pull the trigger on bargain optics even if the price point was appealing. I spent a lot of time behind a bolt gun and understand the value of glass. Doesn't have phase contrast (for now) which is a bummer but I can add that later.

Autoclave purchased as well.....American Sterlizer, again ebay 300$. 25quarts I believe and it's the model with its own heat source so you just plug it in. I can't wait to cram my Therminator in there.....soooo dirty

Hemacytometer and pipettes listed earlier from Cynmar. I already have an Acculab VIC123 so precision weighing is not a problem.

For the questions....

1) Are you guys using an incubator for plate growth? If not room temp is ok?

2) Thinking of getting a better pH meter while Cynmar is in my sights. The pen style I have from Hana keeps going tits up. Thoughts?

3) For staining/fixing are their certain slides that are preferred? What thickness of coverslips to get?

4) How are you cleaning your optics? Alcohol and lens paper?

5) Which housing tubes should I use for slants? How long as you guys getting away with freezing them at household fridge temps?

6) Glassware.....between borosilicate bottles for keeping sterile wort, beakers for mixing agar, flasks for propagation and graduated cyls what count and sizes are mandatory or need multiple of? Stoppers as well or only tinfoil? The biggest batches I ferment are 12 gallons.

7) Thinking of a stirplate with a heating element. Anyone doing this?

8) Do they make Bunsen burners that will run off compressed camp fuel bottles or even larger propane tanks? I really don't like alcohol lamps (if you've ever had a crazy ******* throw a Molotov cocktail at you, you'll know why).

9) Methylene Blue and gram stain kit....any other stains needed?

10) For the guys pouring their own plates.....into plastic or glass? I was initally thinking of just buying prepoured but the shelf life worries me. Think i'd rather have a bag of WLN/WLD and just be able to make them as I go since I have the autoclave. I'm assuming (hoping) the dry media has an extended shelf life.

That's it for now

Looks like you got a great deal on the microscope and sterilizer. I'll give my opinion on some of your questions:

1) For yeast culturing I use one of my temp controlled chest freezer's when possible. It just speeds things up a bit as ale yeast really like temps ~80F. However they will do fine at room temp; I try to find a place where the temp is consistent. It seems you are interested in using selective media to identify/isolate bacteria. If so, a controlled temp device becomes more important. It's fairly easy build your own "hot box", if you have a working environment that stays below your desired culturing temp as keeping the temp up is all you have to deal with.

2) A. J. response +1

3) For viability staining it's common to do this with your hemocytometer as you count you can tally the live and dead cells a derive a percentage. Fixing? I don't do this at home as often the chemicals required are very dangerous to work with and I haven't had a purpose, yet. That said, for observing fixed specimens, I think your standard slide and cover slip are adequate. I am curious about what specimens and why you are interested in fixing.

4) Could be a mistake, but I just wipe frequently with a soft cloth like one that comes with a new pair of eyeglasses, I think it's microfiber. Kimwipes with alcohol if necessary. Lens paper is probably better. And I keep the cover on my scope at all times it is not in use.

5) I use 15ml disposable centrifuge tubes (aka Falcon Tubes). This may not be the best choice for longevity of your culture because I suspect the the plastic may allow some air to enter the tube and air is one of my biggest concerns. I reslant every 6 months and my cultures have always been viable at that time (have gone as long a 9 months). And I have maintained the my first isolate in good condition since 2008 (although I have lost a couple along the way). The reasons I choose Falcon tubes is for their versatility, low cost, and ease of use. I also use them for; the first step of a yeast culture, isolating yeast from beer or wild yeast from fruit. Many are sold as sterile and some can be autoclaved. I have not tried freezing in a household freezer, although many have reported good results.

6) I like to use home canning jars for keeping sterile wort. they are relatively inexpensive, come in a variety of sizes, available at most grocery stores, and work great. For media, I use 500 ml "media bottles" available from Cynmar. While most instructions for preparing agar include boiling before autoclaving, the numerous labs I have been associated with skip this step and mix up the ingredients in a media bottle and autoclave. Certainly for wort agar and YPDA this process works fine, however, there may be some types of media that would benefit from pre boiling. If you choose media bottles, be sure to get the one with a pouring ring, the ring makes pouring much easier with less mess. BTW, 200ml of media will pour ~10, 100mm x 15mm plates. For a graduated cylinder, I have a 100ml and wish for a 500ml. I think tinfoil is the best for a flask closure for propagation. Your flask size will depend on your yeast yield and of course you batch size and gravity. Yeast strain may have an effect on yield. For my Belgian strains, in a well controlled environment (air and temp), I can produce ~300 million cells per ml. Keep in mind that lab produced yeast can be pitched at around half the recommended rate compared to re-pitched yeast. I use 400,000 to 750,000 cells/ml/degree plato for a final cell density as a rule of thumb and for my Belgian Strong Pales (~1.07 OG), I go with 600,000. So for my 10.5 gallon batches, I use ~1400ml culture. I believe I get better aeration of the culture by using 2, 2000ml flasks containing ~700ml each on a orbital shaker. I also believe this would be optimal on a stir plate as well. Aeration, in my opinion, is the key to healthy yeast and high yields.

7) I have often thought a heated stir plate would work well but not sure about temp control.

8) I get by with a wickless alcohol burner. Bunsen burners have definite advantages.

9) I only have experience with methylene blue. Works fine, but with lab produced yeast, it just reassures that the culture is >99% viable. If you are re-pitching, some type of viability stain would be valuable.

10) I have no experience with selective media. For wort agar and YPDA, I mix and pour my own using disposable petri dishes. These ingredients have a long shelf life and at 50 cents for a disposable dish and 14 cents per plate for agar, this is affordable. On the other hand, glass plates would allow you set the plates in you sterilizer and relieve the worry of contamination. Warthaug (aka Bryan) has a protocol for this.

 

[helibrewer]

Here is the WLN media:

And here is the WLD (selective) media:

You can see that the selective media does not grow S. cerevisiae so it is good if you are only looking for bugs.

We cleaned objective lenses with Windex and lens paper

 

[Warthaug]

1) Are you guys using an incubator for plate growth? If not room temp is ok?

I don't use an incubator. They can accelerate growth, but they can also alter gene expression in undesired ways.

3) For staining/fixing are their certain slides that are preferred? What thickness of coverslips to get?

Other have mentioned this already, but fixation is rarely needed. The only time I do anything like it is if I am trying to get a good count on yeast vs. bacterial numbers, or am trying to get a really good look at morphology. In these cases I rely on heat fixation, which is dead-simple:

1. Place a few drops of a liquid culture on a slide. If working from a plate, place a few drops of water on a slide, then mix in a colony of yeast using a sterile loop
2. Spread out the suspension until the middle 3rd or so over the slide is covered
3. Let sit until completely dry; don't rush it - letting it sit too long is harmless, not letting it sit long enough will ruin it
4. Once dry, very quickly pass the slide - yeast/bugs side down - through the flame of your burner. When I say quick I mean quick - less than a second. This direct exposure to flame will bind your sample to the slide; any longer and the sample gets cooked.
5. Stain/image/etc as you require

Obviously, you don't do that for viability staining

4) How are you cleaning your optics? Alcohol and lens paper?

I would recommend isopropyl and lens paper. If using an oil immersion lens, first dab off the oil before cleaning with alcohol. In either case, lightly whet the lens paper with alcohol and wipe with that - never pour/spray alcohol on the lens.

5) Which housing tubes should I use for slants? How long as you guys getting away with freezing them at household fridge temps?

I freeze my yeast, but not in a lab situation so the methods are not really comparable. When I used to slant I preferred the 10-15ml screw-top glass tubes; reusable, you can prep your slants by autoclaving the media right in the tube, and they are big enough to give you enough media volume to allow for long-term storage.

Yeast can be kept stably on slants for several years using a simple protocol:

1. Grow yeast on slant until there is a good layer of yeast
2. Seal the tube and place in the fridge for 2-3 days. This will induce the formation of trehalose, which will aid the yeast's long-term survival
3. Fill the tube with sterile mineral oil, and re-seal the top *(e.g. with vinyl electrical tape). There are reports of yeasts stored like this being recovered after 60+ years - although most people would recommend re-culturing every 3-5 years.

Obviously, if you're using the yeast a lot the above is not a preferred method. You can also overlay a slant with sterile water; yeast last about a year like that, or you can tightly seal the caps - good for 3-6 months. There are lots of other yeast-storage options other than slants as well.

6) Glassware.....between borosilicate bottles for keeping sterile wort, beakers for mixing agar, flasks for propagation and graduated cyls what count and sizes are mandatory or need multiple of? Stoppers as well or only tinfoil? The biggest batches I ferment are 12 gallons.

For 12 gal batches you'll probably want a 1, 2 and 4L flask for starters. Sterile wort can be kept in plain-ol' mason jars (can in a pressure cooker like any other preserve). Mason jars also make great beakers for mixing media - no need to buy the expensive lab stuff. I use foil, but there are foam stoppers that are a little more convenient.

7) Thinking of a stirplate with a heating element. Anyone doing this?

Seems risky, in terms of overshooting temps and what not. In the lab we certainly would not do this.

8) Do they make Bunsen burners that will run off compressed camp fuel bottles or even larger propane tanks? I really don't like alcohol lamps (if you've ever had a crazy ******* throw a Molotov cocktail at you, you'll know why).

I have seen propane models that run off of the little compressed tanks, but they seem to be few and far between.

9) Methylene Blue and gram stain kit....any other stains needed?

I've found viability dyes to not be overly useful; because they rely on enzymatic action dead cells can be missed to to residual activity. I've had a few cases where cultures that stained with many viable cells were unable to grow.

10) For the guys pouring their own plates.....into plastic or glass? I was initally thinking of just buying prepoured but the shelf life worries me. Think i'd rather have a bag of WLN/WLD and just be able to make them as I go since I have the autoclave. I'm assuming (hoping) the dry media has an extended shelf life.

I use both, but prefer glass. Glass can be reused, making it cheaper, and you can prepare media right in the plate (no need for sterile pours). Dry media will last years on the shelf; especially if you seal the containers tight enough to limit water entry.

Bryan

 

[orangehero]

How about one of these?

http://www.amazon.com/dp/B004U8CP88/?tag=skimlinks_replacement-20

The piezo is junk but it's actually a really nice burner for less than $6.

 

[butterpants]

^^Totally forgot about a light camp stove. I own a jetboil too....Good call, it's perfect.

Oh wow lots of good information here. Thanks guys for tracking with my questions! I was interested in fixing slides only for showing others (like my kid). For the stirplate/hotplate combo my thoughts were on heating mixtures of things while actively stirring not in any shape or form of incubation.

Ordered a pile of stuff. I just made ebay some fat cash this week. In a few weeks I should have my basement workspace converted and culturing. Exciting. I have more questions....

1) When you're doing cell counts for density and vitality what dilution range do they have to be in for quick counts to be accurate as possible? For instance lets say I have a 1.8L starter and I decant it down to 200mL. I take 1mL of the starter slurry and add it to 99mL of sterile water to get a 1:100 dilution. Will this be sufficient density of cells to count or in your practice would another, or multiple sets of serial dilutions be made? When is best to add viability dye during this procedure? How are you accounting for the added volume of dye?

2) Is there an inexpensive yet feasible (don't want to burn down the house) alternative to anaerobic "packs" for fermenting your plates without the presence of oxygen?

3) I've searched yet haven't been able to find a good deal on a small laminar flow hood, so I got to thinking what if I construct a work space out of laminated countertop with a partial plexiglass shield that contains 2 sources of light...a fluorescent bulb AND a UV bulb? I could work within the enclosed space with the UV lamp on and a small flame keeping the wild bug population very low. It could be left on for extended periods to sterilize all interior surfaces and no dealing with filters/air flow calculations. The downside would be personal exposure to the radiation so gloves and long sleeves would need to be worn but the plexiglass (Lexan, Home Depot...cheep) would filter out almost all of the UV escaping the pseudo fume hood. I'm thinking you might need some sort of swan neck exhaust port just for the hot gasses from the bunsen burner.... thoughts on any of this? I'll attach a couple of pictures I found on google...just imagine them without any air filtration, only UV sterilization.

4) So really all I need to test, select and isolate for lacto, pedio, brett + sacro would be WLN vs WLD (+ ana vs aerobic) while observing colony morphology and then a growth media such as UBA to propagate once isolated? How many plates (100mmx15) do you get from a 500gr bottle of dry media?

5) If you have large glassware and need to sterilize it in your oven BUT it's borosilicate glass, can you crank up the temp and do it faster than the 250F/2 hours? I'm thinking about a 5L flask or odd shaped things. Just wondering

6) Any recommended apps/spreadsheets for making cell counts and the calculations after for pitch rate easier? Found a few in the Play store. Just looking for an endorsement or warning to avoid. Couldn't find the one mentioned earlier in this thread.

7) Can you autoclave Bell mason jars with their lids on and loose? If not do you just take the lids completely off?

8) So if I'm mixing up lets say WLD and WLD agar. I put it in a mixing vessel then transfer the liquid to media bottles. Lightly cap and autoclave. I can wait for it to cool a bit then pour plates or I can just park the bottle on the shelf a few weeks....but what is the preferred method of reheating the mixture for pouring? Is there a limited number of times it should be heated and cooled before performance degrades?

9) For DI water are you guys just using distilled bottled water from a grocery store in some kind of lab friendly dispenser or sterilizing it first?

 

[helibrewer]

I found 1:1000 dilutions worked best for counting. I don't do viability on a hemacytometer, the stain will eventually effect your hemacytometer.

One easy way to do anerobic growth is to use slants and stab into the media, anerobic stuff will grow in the "stabbed" area within the media.

Do not work under a powered UV light, use the light before you start work then turn it off and use standard aseptic techniques.

For the autoclaved media you want to pour your plates when the media is "can touch your cheek" warm...just don't actually touch your check, that WLD is toxic.

For Mason jars, place the lid on them then the ring, but tighten the ring only enough to hold the lid in place. A well sealed lid after cooling indicates that you have a good sterile sample.

Sterile water and distilled water are different. For DI, just use store bought.

You don't have to be too fancy with your flame source, just something that can flame your loop and create a slight air current away from your working area.

Let your prepared plates sit for several days (media side up) before using them. It's much better to know if they are contaminated before you spend all the time streaking them.

 

[Warthaug]

1) When you're doing cell counts for density and vitality what dilution range do they have to be in for quick counts to be accurate as possible?

The answer is "it depends" - the dilution you need is proportional to the yeast density, which can be quite variable. 1:100 often works for fermenting wort (e.g. in a starter or a fermenter), 1:1000 to 1:10,000 is more typical for slurry or anything with a lot of bacteria in it.

As a "rule", for your yeast counts to be accurate you need to be counting 100-300 cells on the hemocytomoeter, at your selected dilution. Less than that and counts can be off due to sample variability, more than that is hard to count.

2) Is there an inexpensive yet feasible (don't want to burn down the house) alternative to anaerobic "packs" for fermenting your plates without the presence of oxygen?

Yep, an overlay plate. Its pretty simple:

1. Prepare a plate as you normally would, but with ~1/2 the normal amount of media
2. Sterilize more medium, and cool to just above its gelling point (40C or so). You want an excess amount of this
3. Streak out your sample on the first plate, than immediately overlay with the cooled (but not yet solidified) agar. Fill the plate completely & cap while still liquid (a bit of gel should leak around the edges)
4. The gel should set quickly, once set seal the edges of the plate with vinyl tape (electricians tape)

This is not completely impervious to air, but is sufficient for all but the strictest of anerobes - anything that would grow in anaerobic-phase beer would do fine in this plate.

Harvesting bugs from these plates is a bit more challenging; generally you recover colonies by using a stab or punch.

3) I've searched yet haven't been able to find a good deal on a small laminar flow hood

Don't waste your money. Laminar flow hoods are generally not used for microbiology applications - standard aseptic techniques are more than sufficient. In essence, they are a crutch for poor technique (or, in the case of pharmaceutical applications, a guard against mistakes). The only other thing to use them for is to protect yourself from pathogens, and the design you've picked wouldn't do that - it would blow them in your face.

And, FWIW, UV sanitation is pretty weak; a few seconds exposure to starsan or bleach has a grater effect than several minutes of UV.

4) So really all I need to test, select and isolate for lacto, pedio, brett + sacro would be WLN vs WLD (+ ana vs aerobic) while observing colony morphology and then a growth media such as UBA to propagate once isolated? How many plates (100mmx15) do you get from a 500gr bottle of dry media?

You don't need anaerobic for any of those - both pedio and lacto are areotolerant anaerobes, which means they don't need oxygen but also don't give a s**t if its around. Depending on the species, the impact of oxygen on their growth will range from a mild growth impediment to a mild growth enhancement. I culture lacto and pedio all the time without any anaerobic media or chambers without issue. The standard for commercial preparation of both of those organisms (e.g. for yoghurt, sausage, probtiotic pills, etc) is non-aerated, but otherwise air-exposed fermenters.

The medium you choose is upto you. Commercial media are expensive, but can simplify work-flow through the inclusion of selective agents. Beer-wort and potato-dextrose agar are cheap, but either require colony identification or the addition of selective or differential agents such as antibiotics and pH indicators to achieve the same thing as commercial media.

In terms of volumes, the answer is "it depends". A typical 100 mm plate should contain ~30 ml of media. How far a bottle goes depends on the amount of medium required to make the needed volume of gel, and how much you waste (some waste is inevitable).

5) If you have large glassware and need to sterilize it in your oven BUT it's borosilicate glass, can you crank up the temp and do it faster than the 250F/2 hours? I'm thinking about a 5L flask or odd shaped things. Just wondering

Yep - sterilization time is proportional to temperature:
http://www.cdc.gov/hicpac/disinfection_sterilization/13_10othersterilizationmethods.html

6) Any recommended apps/spreadsheets for making cell counts and the calculations after for pitch rate easier? Found a few in the Play store. Just looking for an endorsement or warning to avoid. Couldn't find the one mentioned earlier in this thread.

I like yeastcalc.co - free, and is accurate. Its a re-write of the former yeastcalculator page.

Cell counts don't really need an app - (# cells counted per quadrant) * dilution * hemocytometer conversion factor (usually 10,000). With a bit of use you'll probably just do the math in your head.

7) Can you autoclave Bell mason jars with their lids on and loose? If not do you just take the lids completely off?

Lids on & loose work just fine.

8) So if I'm mixing up lets say WLD and WLD agar. I put it in a mixing vessel then transfer the liquid to media bottles. Lightly cap and autoclave.

Don't bother with the mixing vessel; weigh out the medium and put into the flask/jar; measure out the water and pour in. Swirl flask/bottle to mix, cap lightly (or with foil if using a flask) and autoclave. No need to make extra dishes

I can wait for it to cool a bit then pour plates or I can just park the bottle on the shelf a few weeks....but what is the preferred method of reheating the mixture for pouring? Is there a limited number of times it should be heated and cooled before performance degrades?

Repeated re-heats can degrade some of the medias (e.g. those containing antibiotics or dyes), but two or three reheats should be OK as you're sticking to basic medias. Simply place the bottle of media into a pot of water, and heat on your stove until melted.

9) For DI water are you guys just using distilled bottled water from a grocery store in some kind of lab friendly dispenser or sterilizing it first?

Don't bother with it for media - tap water is fine. Heating will drive off any chlorine, and the trace minerals in the water should not otherwise impact your media. Even in a research lab, we use tap water for most of our bacterial culture media. DI is for more sensitive cells - like them wussie mammalian cells.

Bryan

 

[ajdelange]

1) When you're doing cell counts for density and vitality what dilution range do they have to be in for quick counts to be accurate as possible?

The accuracy depends on the square root of the number of cells you count. The coefficient of variation is its reciprocal. Thus if you count 100 cells (total in all squares) your Cv = 1/sqrt(100) = 0.1 and the standard error in your count is Cv times what you counted or, in this example (0.1)(100) = 10 cells. Or just looking at the Cv expressed as a percentage 10%. If you count 200 cells your Cv= 1/sqrt(200) = 0.0707 or 7%. I would think either of those levels acceptable but if you want tighter accuracy you will have to count more cells - lots more. For 1000 counted Cv = 3.2%.

 

[butterpants]

Thread keeps getting better and better, thanks again guys.

100-200 cells counted and an accuracy range from 7-10%. AJ I can live with that, thanks. 1k no way. Diminishing returns on the loss of my mental sanity. Heli, if you're not using a hemocytometer to count stained cells, how are you determining viability....or is that just not a concern for you? Bryan I checked on the FDA site and it looks like 340F@60min will get my big pyrex items sterilized.

Some more questions/discussion points:

1) Watched a White Labs YouTube video not too long ago on making yeast starters (which I have been doing since I began brewing but I'd consider them an authority on yeast and was looking for refinement of technique)....it appeared to be around a 1.5-2L starter in a 4L Erlenmeyer flask with stirbar. Nice amount of room for gas exchange....BUT the presenter then took the aluminum foil cover on top of the flask and cranked it down sealing it all around....something I've never done, infact just the opposite. I leave it loose for gas exchange. Thoughts on oxygen/stirplate starters/oversized flasks/lid sealing? You guys are the yeast whisperers right? What say you

2) Do you recommend actually mixing up my own differential media from its raw individual components (WLD/WLN i'm thinking) instead of buying it in a dry premixed form? Significant cost savings? My worry is that some of the trace products (bromocresol green, cyclohexamide) I'll have to purchase in quantities I'll never use making it no longer financial advantageous.

3) Why would one use "beer" agar over "wort" agar....from a plating or growing perspective? If making wort agar is it important to obtain a hot break and rupture starch granules before adding the wort constituent or can you just weigh out DME?


Packages have started arriving

 

[ajdelange]

100-200 cells counted and an accuracy range from 7-10%. AJ I can live with that, thanks. 1k no way.

That reminds me. A very important part of the kit that hasn't been mentioned is one of those counter thingies that theater ticket takers use. Its a VR counter that increments once each time you push the little lever. You can count like the wind with one of those, never have to write anything down and don't have to remember anything except which squares you have counted.

 

[trentm]

I don't use an incubator. They can accelerate growth, but they can also alter gene expression in undesired ways.

I haven't heard of this, could you elaborate. Seems odd to me, isn't an incubator just a way of keeping a consistent temperature? How would the environment in a incubator at 75F be different from a room temperature environment at 75F?

 

[trentm]

Don't waste your money. Laminar flow hoods are generally not used for microbiology applications - standard aseptic techniques are more than sufficient. In essence, they are a crutch for poor technique (or, in the case of pharmaceutical applications, a guard against mistakes). The only other thing to use them for is to protect yourself from pathogens, and the design you've picked wouldn't do that - it would blow them in your face.

And, FWIW, UV sanitation is pretty weak; a few seconds exposure to starsan or bleach has a grater effect than several minutes of UV.

Bryan, I have great admiration for your microbiological knowledge but I am surprised by this statement. I am going to give an alternate view with all due respect.

My profession is Plant Pathology and as I am sure you know it is the study of plant disease. My area is very applied and I do a lot of work in the field and greenhouse, however, lab work is required in order to isolate and produce inoculum to make plants sick and to look at the effects of the disease. Over my 15 years in the field, I have worked in 7 different labs and collaborated with dozens of other labs. Often these labs are fairly primitive, base in field locations, others are at major Universities and USDA/ARS research stations. Without exception all Plant Pathology labs I have worked in or visited are equipped a laminar flow hood and use these hoods for all microbiological work when there is the possibility of contamination. Within my experience, in a lab situation, working with open cultures or media outside a hood is a thing of the past.

I do agree that basic work required for the advanced homebrewer can be achieved without a hood. I have had great success for over 5 years in my home (garage) lab without one. As for a crutch, nothing, in my opinion will forgive poor sterile technique including a hood. In Plant Path we work with highly contaminated plant material including roots and soil from field conditions. Isolating fungal or bacterial pathogens from these samples can be very challenging, and just as isolating yeast from a field sample a laminar flow hood is a great tool. Personally, I find many other uses for my hood, none of which include guarding against mistakes. I believe the $200 I spent on my hood was a great addition to my lab. On the other hand, spending $2000 - $10,000, the price of a new hood, would be extravagant.

+1 on the UV sterilization. Well equipped hoods do use UV light, but the idea is to use the light when the hood is not in use to maintain sterility within the hood environment between uses.

 

[butterpants]

That reminds me. A very important part of the kit that hasn't been mentioned is one of those counter thingies that theater ticket takers use. Its a VR counter that increments once each time you push the little lever. You can count like the wind with one of those, never have to write anything down and don't have to remember anything except which squares you have counted.

Yep I snagged one of those... just forgot to list it

 

[trentm]

^^Totally forgot about a light camp stove. I own a jetboil too....Good call, it's perfect.

Oh wow lots of good information here. Thanks guys for tracking with my questions! I was interested in fixing slides only for showing others (like my kid). For the stirplate/hotplate combo my thoughts were on heating mixtures of things while actively stirring not in any shape or form of incubation.

Ordered a pile of stuff. I just made ebay some fat cash this week. In a few weeks I should have my basement workspace converted and culturing. Exciting. I have more questions....

1) When you're doing cell counts for density and vitality what dilution range do they have to be in for quick counts to be accurate as possible? For instance lets say I have a 1.8L starter and I decant it down to 200mL. I take 1mL of the starter slurry and add it to 99mL of sterile water to get a 1:100 dilution. Will this be sufficient density of cells to count or in your practice would another, or multiple sets of serial dilutions be made? When is best to add viability dye during this procedure? How are you accounting for the added volume of dye?

2) Is there an inexpensive yet feasible (don't want to burn down the house) alternative to anaerobic "packs" for fermenting your plates without the presence of oxygen?

3) I've searched yet haven't been able to find a good deal on a small laminar flow hood, so I got to thinking what if I construct a work space out of laminated countertop with a partial plexiglass shield that contains 2 sources of light...a fluorescent bulb AND a UV bulb? I could work within the enclosed space with the UV lamp on and a small flame keeping the wild bug population very low. It could be left on for extended periods to sterilize all interior surfaces and no dealing with filters/air flow calculations. The downside would be personal exposure to the radiation so gloves and long sleeves would need to be worn but the plexiglass (Lexan, Home Depot...cheep) would filter out almost all of the UV escaping the pseudo fume hood. I'm thinking you might need some sort of swan neck exhaust port just for the hot gasses from the bunsen burner.... thoughts on any of this? I'll attach a couple of pictures I found on google...just imagine them without any air filtration, only UV sterilization.

4) So really all I need to test, select and isolate for lacto, pedio, brett + sacro would be WLN vs WLD (+ ana vs aerobic) while observing colony morphology and then a growth media such as UBA to propagate once isolated? How many plates (100mmx15) do you get from a 500gr bottle of dry media?

5) If you have large glassware and need to sterilize it in your oven BUT it's borosilicate glass, can you crank up the temp and do it faster than the 250F/2 hours? I'm thinking about a 5L flask or odd shaped things. Just wondering

6) Any recommended apps/spreadsheets for making cell counts and the calculations after for pitch rate easier? Found a few in the Play store. Just looking for an endorsement or warning to avoid. Couldn't find the one mentioned earlier in this thread.

7) Can you autoclave Bell mason jars with their lids on and loose? If not do you just take the lids completely off?

8) So if I'm mixing up lets say WLD and WLD agar. I put it in a mixing vessel then transfer the liquid to media bottles. Lightly cap and autoclave. I can wait for it to cool a bit then pour plates or I can just park the bottle on the shelf a few weeks....but what is the preferred method of reheating the mixture for pouring? Is there a limited number of times it should be heated and cooled before performance degrades?

9) For DI water are you guys just using distilled bottled water from a grocery store in some kind of lab friendly dispenser or sterilizing it first?

Sorry for the delayed reply. I'll comment on what has not been covered or where I think I have something to add.

1) You may find counting before you allow the yeast to settle and just after you remove the culture from the stir plate to be the most accurate. After the yeast have settled, the cells have clumped together and it is sometimes hard to get a homogeneous suspension. I make sure to keep up with the size of the culture by measuring and tallying each step. If you find you need to count the slurry, you may have to do serial dilutions until you find an appropriate concentration. It may be however somewhere between 1:100 and 1:1000.

For an un-concentrated culture containing ~300 million cell/ml a 20:1 dilution works well for me. At that concentration and dilution you will be counting ~60 cells/small square if you are counting the center square, for a total of ~300 cells.

You can you use the viability dye as your last diluent. I make my last dilution using 0.5 mL of 0.01% methylene blue to 0.5 mL of the sample. Remember to calculate the final dilution.

3) For me, a laminar flow hood is a great tool but not a requirement. The advantage to working in a hood is you have a sterile environment that allows you to work with open cultures without the concern of environmental contamination. Unless your environment is completely sterile, you will still need to work around a flame and pray you are in the updraft.

4) I have no experience with selective media. Of course the number of plates you can pour will depend on how much media is required. Some have said ~30 mL per plate. I shoot for 20 mL.

6) The “Hemocytap” app is only available from Apple. I mentioned earlier it was free but it is actually $1.99.

7) As mentioned, leave the rings loose but tighten them after you remove them from your sterilizer. The rings need to be tight to ensure the lids seal properly.

 

[trentm]

Thread keeps getting better and better, thanks again guys.

100-200 cells counted and an accuracy range from 7-10%. AJ I can live with that, thanks. 1k no way. Diminishing returns on the loss of my mental sanity. Heli, if you're not using a hemocytometer to count stained cells, how are you determining viability....or is that just not a concern for you? Bryan I checked on the FDA site and it looks like 340F@60min will get my big pyrex items sterilized.

Some more questions/discussion points:

1) Watched a White Labs YouTube video not too long ago on making yeast starters (which I have been doing since I began brewing but I'd consider them an authority on yeast and was looking for refinement of technique)....it appeared to be around a 1.5-2L starter in a 4L Erlenmeyer flask with stirbar. Nice amount of room for gas exchange....BUT the presenter then took the aluminum foil cover on top of the flask and cranked it down sealing it all around....something I've never done, infact just the opposite. I leave it loose for gas exchange. Thoughts on oxygen/stirplate starters/oversized flasks/lid sealing? You guys are the yeast whisperers right? What say you

2) Do you recommend actually mixing up my own differential media from its raw individual components (WLD/WLN i'm thinking) instead of buying it in a dry premixed form? Significant cost savings? My worry is that some of the trace products (bromocresol green, cyclohexamide) I'll have to purchase in quantities I'll never use making it no longer financial advantageous.

3) Why would one use "beer" agar over "wort" agar....from a plating or growing perspective? If making wort agar is it important to obtain a hot break and rupture starch granules before adding the wort constituent or can you just weigh out DME?


Packages have started arriving

1) I like the idea of a large headspace because it allows the stir plate to do an adequate job of stirring which allows for gas exchange and creates a large surface area. In my opinion the large head space is not so much for the air present in the space before the yeast start to grow, as the space will quickly become saturated with CO2 if sealed, but to allow more surface contact with the air you that can coax into the flask throughout the fermentation. Due to the conical shape of an E. flask, the surface area is diminished the fuller the flask.

A loose fitting foil cover is the best in my opinion because, as you know, microbe laden dust does not fall up but air can move in and CO2 out, due to action of the vortex. Foam or cotton plugs are another option and can keep out crawling insects but you may have more of a blow off issue if your system creates a large krausen.

2) Sorry, again no experience with differential media. My guess is buy the premixed form.

3) No knowledge on “Universal Beer Agar”. Here is a link to a fact sheet: http://www.neogen.com/Acumedia/pdf/ProdInfo/7574_PI.pdf. For culturing with the intention to brew, wort agar is said to be best because the yeast get used to the environment they are in. I noticed in the recipe that Glucose is the sugar source and yeast can get lazy on Glucose. For wort agar, I brew up a batch of wort for starters and media, remove as much break material as possible and can it. I like to make my own because I pitch the entire starter. Even though I have removed much of the break, after canning more break appears. When making solid media I decant the wort from the break because I like clear plates and slants. When using it for a starter, I add at least some of the break because I have heard that yeast benefit from small amounts of break material. I don't know about “rupturing starch granules”.

 

[Warthaug]

Bryan, I have great admiration for your microbiological knowledge but I am surprised by this statement. I am going to give an alternate view with all due respect.

My profession is Plant Pathology and as I am sure you know it is the study of plant disease. My area is very applied and I do a lot of work in the field and greenhouse, however, lab work is required in order to isolate and produce inoculum to make plants sick and to look at the effects of the disease. Over my 15 years in the field, I have worked in 7 different labs and collaborated with dozens of other labs. Often these labs are fairly primitive, base in field locations, others are at major Universities and USDA/ARS research stations. Without exception all Plant Pathology labs I have worked in or visited are equipped a laminar flow hood and use these hoods for all microbiological work when there is the possibility of contamination.

Outside of non-bacterial cell culture, in the health sciences side of things we don't use hoods. In fact, they are counter-indicated for BSL 1 and BSL 2 organisms (i.e. our permits explicitly recommend against using hoods for those organisms). Its not until we hit BSL 3 organisms where we use a hood - to protect ourselves, not our samples - and those hoods are dedicated strictly to that work.

Within my experience, in a lab situation, working with open cultures or media outside a hood is a thing of the past.

Than you plant biologists are weird :cross: I work in a large microbiology department, have visited microbiology departments across the world, been part owner of 3 of biotech startups, worked briefly in industry, and in none of those environments I have never seen a hood used strictly for bacterial/fungal work, outside of BLS3/4 organisms.

Bryan

 

[Warthaug]

I haven't heard of this, could you elaborate. Seems odd to me, isn't an incubator just a way of keeping a consistent temperature? How would the environment in a incubator at 75F be different from a room temperature environment at 75F?

Sorry, by incubator I thought you meant growing at 30-37C. Regardless, many genes are regulated by environmental factors such as temperature, osmolarity, pH, etc. Meaning that yeast cultured under conditions different from than of beer may express different genes, and thus produce different fermentation profiles, flavour compounds, viability, etc. I've done some experimentation with higher-temp starters to accelerate growth, and found (in general) that going more than 3-4 degrees above the recommended fermentation range produces yeast which ferment more slowly and throw more off-flavours than do yeast grown within the recommended fermentation range, despite the beer itself being in the recommended range.

Bryan

 

[Warthaug]

1) Watched a White Labs YouTube video not too long ago on making yeast starters (which I have been doing since I began brewing but I'd consider them an authority on yeast and was looking for refinement of technique)....it appeared to be around a 1.5-2L starter in a 4L Erlenmeyer flask with stirbar. Nice amount of room for gas exchange....BUT the presenter then took the aluminum foil cover on top of the flask and cranked it down sealing it all around....something I've never done, infact just the opposite. I leave it loose for gas exchange. Thoughts on oxygen/stirplate starters/oversized flasks/lid sealing? You guys are the yeast whisperers right? What say you

It doesn't matter all that much - the amount of O2 actually entering the starter is rather small (I think Kai did some experiments on this end). The main benefit of stirring is removal of CO2, which will have no trouble getting past a tightly "sealed" foil cap.

2) Do you recommend actually mixing up my own differential media from its raw individual components (WLD/WLN i'm thinking) instead of buying it in a dry premixed form? Significant cost savings? My worry is that some of the trace products (bromocresol green, cyclohexamide) I'll have to purchase in quantities I'll never use making it no longer financial advantageous.

If you're making enough on your own than it is far cheaper to make it from scratch, but if only using small amounts it may be cheaper to buy a small bottle of pre-mixed.

3) Why would one use "beer" agar over "wort" agar....from a plating or growing perspective? If making wort agar is it important to obtain a hot break and rupture starch granules before adding the wort constituent or can you just weigh out DME?

I don't think there is a difference; commercially it is usually sold as wort-agar, but I treat the terms as interchangeable. That said, beer-agar may indicate a hopped product, which would have a weak selectivity against gram positive bacteria. DME + water + nutrient is more than enough - conversion, hydration, etc, are already complete with DME (there is no starch in DME; its all converted). I take mine through the hot-break, simply because that is inevitable if boiling long enough to sanitize the wort.

Bryan