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:
- Prepare a plate as you normally would, but with ~1/2 the normal amount of media
- Sterilize more medium, and cool to just above its gelling point (40C or so). You want an excess amount of this
- 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)
- 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