Building a home QC lab... questions

[butterpants]

I'm always looking for a challenge and tend to get bored easily once my mind feels as if it has conqured something. Just a fact of life for me.... so sours and wild yeast brewing is next on my list to tackle. Problem is it takes so damm long to ferment out... so I need a brewing related project to keep my mind stimulated in the meantime.

I want to put together a microbiological quality control lab for my brewing. Currently in the process obtaing hardware and textbooks to teach it to myself. I've looked over what Jamil and Chris say in Yeast and seen some of the documentation the Brewing Science Institute (which is like 30 minutes from my house) puts out for equipment. Was wondering if any of you guys are doing this and had suggestions on....

1) Microscope models
2) Autoclave alternative
3) Microbiology Textbooks
4) Staining/testing kits n media
5) Source for lab odds n ends
6) Hemocytometer

Before someone jumps in and says "go back to school" or something similar.... I was in a molecular biology PhD program back in the 90ies and left to go take a job with more interesting prosspects. Most of the lab technology and techniques are familiar eventhough molecular and microbiology aren't much alike. I'd like to be able to test and identify all my produced beers for wild yeast and bacterial contamination and also be able to isolate and produce pure cultures on a small scale.

 

[helibrewer]

I just finished the Brewing Microbiology course at UC Davis and am considering the same thing. We used 1000X scopes with phase contrast and that was plenty even for Lacto and Pedio so I don't think you need 2000X. Seems they are available for about $600 (eBay).

I use a large pressure cooker as an autoclave, I can fit my plate chiller in it.

White Labs actually sells a lot of lab supplies including pre-filled media. You can order the dry media and mix it yourself but the order quantities are large so it may not be economical. Jamils book covers most of the media types. I found WLN to be quite good and WLD works great for bugs.

For counting, a quality hemocytometer runs about $60. You might also consider a 1-1000ul digital pipette for accurate dilutions for counting (ebay ~$40)

Then basic asceptic technique stuff: 70-75% ethanol (I use an Everclear knockoff for $15/750ml) in a spray bottle, small alcohol lamp, loop (wire or disposable). If you want to do sterile membrane filtration it gets a bit more involved but Nalgene has a setup.

 

[butterpants]

Oh sweet Heli thanks for your input. How was the course? I've thought about flying out to White Labs and taking a course but forking out 5k plus hotel plus a week or two off seems excessive.

2000x is a massive amount of magnification. Any specific models of microscope you care to endorse.

I was thinking about prepoured plates. I won't be doing a quantity anytime in the future that making my own will save me $$.

Did you guys buy the WL reagent and testing kits for your course?

Yea hemocytometer, forgot about that. Pipetteman for sure... gotta have it.

Not thinking membrane filtration right now cuz to do it right would destroy my budget.

How big of a pressure cooker did you get?

 

[trentm]

I'm always looking for a challenge and tend to get bored easily once my mind feels as if it has conqured something. Just a fact of life for me.... so sours and wild yeast brewing is next on my list to tackle. Problem is it takes so damm long to ferment out... so I need a brewing related project to keep my mind stimulated in the meantime.

I want to put together a microbiological quality control lab for my brewing. Currently in the process obtaing hardware and textbooks to teach it to myself. I've looked over what Jamil and Chris say in Yeast and seen some of the documentation the Brewing Science Institute (which is like 30 minutes from my house) puts out for equipment. Was wondering if any of you guys are doing this and had suggestions on....

1) Microscope models
2) Autoclave alternative
3) Microbiology Textbooks
4) Staining/testing kits n media
5) Source for lab odds n ends
6) Hemocytometer

Before someone jumps in and says "go back to school" or something similar.... I was in a molecular biology PhD program back in the 90ies and left to go take a job with more interesting prosspects. Most of the lab technology and techniques are familiar eventhough molecular and microbiology aren't much alike. I'd like to be able to test and identify all my produced beers for wild yeast and bacterial contamination and also be able to isolate and produce pure cultures on a small scale.

Cynmar.com is the source I use for most of my equipment and supplies. While they are a little slow (usually takes a week to 10 days to get my stuff in CA), they have great costumer service and good mid-range quality. Below is a list of stuff I purchased to get started counting yeast. For a microscope, I have a AM scope (from amscope.com). It's a cheap but usable microscope for yeast counting, probably not usable for observing bacteria. I would recommend a mechanical stage and binocular eyepiece with whatever scope you choose. For a pressure cooker: I have an All American Sterilizer 21 quart. This is a great sterilizer but expensive, look for a used one. The advantage to this type of sterilizer is you can do a dry cycle for things like pipette tips or slants. If you don't see a need for dry sterilization the cheaper pressure canners with the "jiggler" works fine. I have also converted an old style keg into a multi-purpose brewing vessel that includes pressure capability. It's great for sterilizing large quantities (canning wort) or large items (photo attached). Caution: pressure cooking can be dangerous especially with a homemade devise. That said, I've used mine hundreds of time with no issues. There are cheap hemocyotmeters out there for ~$20 but beware it is almost impossible to see the grid (at least with my cheap scope). There are also expensive ones, ~$200 but these may not be necessary as the ~$60 ones mentioned above work fine.

Another source worth mentioning are surplus lab supply stores. You can find them on ebay. I'm fortunate to have one nearby in Gilroy CA called Outback Equipment. I was able to get a nice laminar flow hood and orbital shaker for $200 each.

One final word of caution: Home microbiology is highly addictive. There seems to be no end to the possibilities. However, it is the best thing that has happened to my beer.

 

[helibrewer]

I am looking at the AmScopes, there are a lot of models so it should be easy to find what you like. I am thinking about this one: http://www.amscope.com/microscopes/...eterinary-trinocular-compound-microscope.html

Davis supplied the media and we plated Ale/Lager yeast, Brett, Lacto, and E. coli on each of the media to see which are most effective for each organism.

Here's the pressure cooker I use. I added the "T" and valve to the gauge:





Sent from my iPad using Home Brew

 

[trentm]

I am looking at the AmScopes, there are a lot of models so it should be easy to find what you like. I am thinking about this one: http://www.amscope.com/microscopes/...eterinary-trinocular-compound-microscope.html

Davis supplied the media and we plated Ale/Lager yeast, Brett, Lacto, and E. coli on each of the media to see which are most effective for each organism.

Here's the pressure cooker I use. I added the "T" and valve to the gauge:

View attachment 226730View attachment 226731



Sent from my iPad using Home Brew

Let me give another word of caution regarding the AM scopes. Somehow they are selling magnification that is not there, for mine at least. The 40X objective with the 10X eyepieces should give you 400X magnification. In reality, the 40X objective will give you about the same magnification as a 10X objective on a high dollar scope with really good optics. That said, the price of the really good optics is >40X.

 

[butterpants]

Thank you for the suggestions.

Back in the day for countertops we lysol'ed them, waited 5 min then flamed the entire work surface. Seems like my normal countertop will not be happy after that. Have you guys tried snagging some lab grade countertop material?

I'm not sure I'll go balls to the wall with a hood but down the road it could be interesting.

I will check out AmScopes, thank you for the link.

You guys pick up a centrifuge as well? I've never seen a lab that didn't have one and could think of a few uses. Not super high speed but enough to spin down large sample tubes.

What are you using the T handle for on the pressure cooker for? Just monitoring and release?

 

[trentm]

Thank you for the suggestions.

Back in the day for countertops we lysol'ed them, waited 5 min then flamed the entire work surface. Seems like my normal countertop will not be happy after that. Have you guys tried snagging some lab grade countertop material?

I'm not sure I'll go balls to the wall with a hood but down the road it could be interesting.

I will check out AmScopes, thank you for the link.

You guys pick up a centrifuge as well? I've never seen a lab that didn't have one and could think of a few uses. Not super high speed but enough to spin down large sample tubes.

What are you using the T handle for on the pressure cooker for? Just monitoring and release?

I worked successfully without a hood for several years, so it is more of a luxury than a necessity. Beer wort agar and media are pretty forgiving, however I like to use YPDA media for some applications and I had trouble with contamination before I was able to get the hood. I did have more problems working in a kitchen environment than in a garage. Also it seems early morning is a better time to work as there may be less organisms floating around on dust particles at that time of day. Regarding sterilizing a working surface, you can have good success by spraying down the surface with 70% alcohol (isopropyl works, EtOH may be better), let is set a minute and wipe with a paper towel. Here is a link to a blog where there is some good advise on creating a home lab: http://suigenerisbrewing.blogspot.ca/

I would love to have a lab workbench. I haven't really came across a need for a centrifuge as a finished yeast culture flocculates quickly. However, I bet if I had one, I would find uses. I want to know more about the T-handle as well.

 

[jbaysurfer]

I worked successfully without a hood for several years, so it is more of a luxury than a necessity. Beer wort agar and media are pretty forgiving, however I like to use YPDA media for some applications and I had trouble with contamination before I was able to get the hood. I did have more problems working in a kitchen environment than in a garage. Also it seems early morning is a better time to work as there may be less organisms floating around on dust particles at that time of day. Regarding sterilizing a working surface, you can have good success by spraying down the surface with 70% alcohol (isopropyl works, EtOH may be better), let is set a minute and wipe with a paper towel. Here is a link to a blog where there is some good advise on creating a home lab: http://suigenerisbrewing.blogspot.ca/

I would love to have a lab workbench. I haven't really came across a need for a centrifuge as a finished yeast culture flocculates quickly. However, I bet if I had one, I would find uses. I want to know more about the T-handle as well.

Me 3 on the T handle! I have the same pressure cooker fwiw.

 

[butterpants]

Let me give another word of caution regarding the AM scopes. Somehow they are selling magnification that is not there, for mine at least. The 40X objective with the 10X eyepieces should give you 400X magnification. In reality, the 40X objective will give you about the same magnification as a 10X objective on a high dollar scope with really good optics. That said, the price of the really good optics is >40X.

So in your experience you would or would not recommend AM? How is the build quality overall? Obviously there are several tiers or optical quality.... bargain barely working (****), functional ****, mid grade workhorse, high end and very stupid expensive NIH lab high end.

 

[biertourist]

You guys pick up a centrifuge as well? I've never seen a lab that didn't have one and could think of a few uses. Not super high speed but enough to spin down large sample tubes.

There's a guy on the interwebs who sells an adapter that you can affix to a dremel tool that will convert it into a very small capacity centerfuge; he's open sourced the design and you can even print it on a 3D printer.... Let me see if I can find it and I'll come back here and edit this post. -I'm almost certain he lives in Ireland or Northern Ireland...

It's created by Cathal Garvey and it's called the Dremelfuge. You can buy it or just download the 3D model and load it up in a 3D printer.
http://www.shapeways.com/model/77306/dremelfuge-classic.html

It supports a max size tube of 1.5ml, though.

Adam

 

[butterpants]

There are a few guys using AM Scopes stuff out there but man there are really some bad reviews out there on the interwebs.

Thoughts on a used or referb Olympus/Zeiss from ebay? Which models you've guys have experience with?

I'm a firm believer in buy once, cry once.... but not spending 2k on a microscope.

 

[ajdelange]

Let me give another word of caution regarding the AM scopes. Somehow they are selling magnification that is not there, for mine at least. The 40X objective with the 10X eyepieces should give you 400X magnification. In reality, the 40X objective will give you about the same magnification as a 10X objective on a high dollar scope with really good optics. That said, the price of the really good optics is >40X.

I think you are talking about 'empty resolution'. A yeast cell appears to be 10 times bigger at 400X than it does at 40X but is is a bigger blur in which you cannot see any more detail than you can in the 40X image. The is because the cheap objective doesn't have the required 'numerical aperture' to support 400X (or higher) magnifications. Selling, and heavily advertising, empty resolution is an old microscope and telescope maker's trick.

 

[ajdelange]

Which models you've guys have experience with?

B&L Galen III with phase kit. I bought it years and years ago at which time it was promoted as a medical student's microscope (hence the name).

Not cheap. You can get them reconditioned for around $700 (w/o the phase kit).

 

[butterpants]

AJ do you think phase contrast is a necessity?

 

[passedpawn]

AJ do you think phase contrast is a necessity?

I'd love the phase contrast for my scope, but for counting cells it's not necessary. If you are taking pics of microbes, it's fantastic.

Ebay has some decent hemocytometers for ~ $30. I bought one a while back and it seems fine to me.

I bought a used olympus scope on ebay as well. Again, it has been great. I wish it had the trinocular head though. I bought one of these SLR adapters for my camera. It fits perfectly in the eyetube of my Olympus, as well as the larger eyetube of my Meiji inspection scope.

Cynmar is great.

If you're interested, I posted pics throughout this discussion: Pics of Yeast under my new scope

 

[ajdelange]

For counting cells its definitely not required. 40 - 60X brightfield is plenty for that but if you want to see any of the internal structure of yeast it is necessary. For bacteria I don't know (I bought the phase kit for something other than brewing where I did want to see the interiors). Pedios are so tiny and lactos so thin I can't imagine you would see much internal structure even with high power (1000X) and phase. Between having to fiddle with phase and immersion oil I don't have much desire to examine those things in detail. If I can see that they are in my beer I know I've screwed up and that's usually enough data.

 

[trentm]

So in your experience you would or would not recommend AM? How is the build quality overall? Obviously there are several tiers or optical quality.... bargain barely working (****), functional ****, mid grade workhorse, high end and very stupid expensive NIH lab high end.

Yes. I have had my AM scope for about a year now and for my purpose it has served well. It works fine at 400X (40X objected and 10 eyepiece) for counting cells and you can even distinguish between Brettanomyces and Saccharomyces. The latter came in handy when I was able to separate the Sacc from the Brett in a culture from Orval dregs. So overall I am satisfied with the scope. Among your categories above I would rank it as "functional". When searching for a scope I was tempted by the mid grade workhorses found on ebay but I did not have the knowledge to know what I was getting. Ebay may be a good source if you know microscopes or have a good consultant. But even then you may get a good quality scope that is worn out. The "sold as is" on most of what I thought were good quality scopes scared me off. With the AM scope you get a new functional scope with a warranty and perhaps some level of customer service (I haven't needed any) at reasonable price. The overall build seems good, the stage movement is smooth and precise, the course and fine focus are tight and work great while the optics are good enough that counting cells is comfortable with little eye strain. If I had to do it over again, I would get the same scope.

On the other hand, if you have a need to observe bacteria, you won't have much luck with this scope. In my experience, even with the "high end" light microscopes, it is a real challenge to work with bacteria.

If you do decide on the AM scope, be sure to get the mid-range priced hemocytometer (it will have a mirrored surface where the counting grid is located). While the cheaper (no mirrored surface) may work fine with a higher quality scope, you won't be satisfied using one with the AM scope (my experience).

By the way, I recently found a free app that really makes cell counting and calculations easier. The "Hemocytap" app has a counter for live and dead cells and does the calculations, including; live cells, dead cells, and final volume density. The only issue for me is the dilution ratio which is critical to be done right. For some reason it is set up for dilutions that have been made by, for example, adding 1ml of the sample to 10ml of diluent and they call that a 1:10 dilution ratio. I make serial dilutions so a 1:10 dilution ratio to me is 1ml of the sample to 9ml of diluent. So you have set the app for a serial dilution to, for this example, 1:9.

 

[Warthaug]

Someone was kind enough to link to my blog - I've got some videos (and more in production) about some of the things you may be trying to do in your home lab. I'm a microbiologist by profession, so I may be able to help you out a little, especially on the microscope and media front.

Firstly, the autoclave. Don't waste your money - a quality pressure cooker is 1/10th the price and works just as well. So long as your pressurecooker gets to 15PSI or higher it'll do just as good of a job.

Microscopes are a much more difficult thing to address, there are a number of manufacturers out there who make good scopes - Nikon, Leica, Olympus and Zeiss lead the pack but there are many others. But defining exactly what scope features you need depends on what you wish to do with it. First (I think you know this already), the total magnification of a scope is determined by multiplying the magnification of the objective lens (the lens near your sample) by the magnification of the ocular (the part you look through). Most oculars are 10X, but 2X, 5X and 20X also exist. If you have a 40X lens and 10X ocular, your scope has a total magnification of 400X; you'd get the same with a 20X lens and 20X ocular, although for reason too complicated to go into here, this setup would give you a poorer resolving power. Any ways, what total magnification you need depends on what you want to do:

• Count yeast - you can get away with as little as 100X for this, although 200x - 400x is probably more practical
• Identify the presence of bacteria - minimum to do this reliably is 400x, although older eyes may appreciate 600x
• Species identification by morphology - you cannot ID species solely by microscopy, but you can separate brett from sacc, or lacto from pedio, based on morphology (assuming you know they are in there via other means). To do this you need 600x to 1000x. More on this below...
• Beyond 1000x - there isn't much of a purpose for this; beyond 1000x you are usually exceeding the resolution limit of your optics, meaning that while objects will appear larger, they will be no more detailed than at 600x or 1000x.

Hopefully, that gives you an idea of the magnification range you need. If you are thinking about the higher end there are a few other things to worry about. Lens quality matters a lot at the higher end - poor quality lenses will produce low-detail images and not collect a lot of light (making for dark images). At the higher end - especially if looking at a 100x objective (i.e. 1000x total mag(, you should be looking for a high numerical aperture lens - at least 0.8NA, ideally 1.0 or higher. The NA defines both the light collecting ability and the resolution of the microscope, meaning a high mag lens with low NA is almost useless. Unfortunately, these high-NA lenses are often oil-immersion or water-immersion. This isn't the end of the world, but it does complicate microscope use a little, and you have to be careful not to get the oil/water on your air lenses. The plus-side is that the oil lenses in particular really do a lot for image quality.

The other issue people have raised is phase contrast. Personally, I wouldn't consider a scope without it, but if all you want to do is counts you can get away without it. Microscopes rely on pigments (which yeast and brewing bacteria don't have) and diffraction to visualize stuff. Since yeast/bugs are mostly water they don't diffract much, meaning few details can be observed outside of general cell shape - and it can be hard to simply find the cells. Phase contrast "amplifies" the diffraction, making finding and observing subjects much eaiser. Again, adding phase adds some complexity (you need to align the phase optics), although that is a skill that is trivial to implement with a bit of practice.

For counting a hemocytometer is a must - a cheap one is more than enough though (just clean it well after each use). The purchase of a few basic dyes (i.e. a viability dye like trypan blue, plus a general staining dye like saffrin) can help a lot - especially if you don't have phase. Some basic micropipettes (ebay's loaded with them) can help with doing accurate dilutions. I'd recommend glass petri dishes & tubes; both because you can "cast" your gels right in the pressure cooker, and because they are cheaper in the long run than disposable plastics.

In terms of growing bugs on plates, a number of medias are available, but I've found the combination of beer-wort agar (DME @ 1.006SG + 1.5% agar) and potato-dextrose agar serve for 99%+ of what is practical at home.

Just my $0.02

Bryan

EDIT: shameless self-plug - you can link directly to my youtube channel here.

 

[WoodlandBrew]

I'm very happy with the Amscope microscope that I have:
http://www.woodlandbrew.com/2012/11/amscope-binocular-compound-microscope.html

And this is some information on hemocytometers:
http://www.woodlandbrew.com/2012/12/hemocytometers-side-by-side.html

 

[butterpants]

B&L Galen III with phase kit. I bought it years and years ago at which time it was promoted as a medical student's microscope (hence the name).

Not cheap. You can get them reconditioned for around $700 (w/o the phase kit).

Can you point me in the direction of exactly what kit/parts I'd need to convert one to a phase contrast?

I have a line on a Galen III in what seems like good condition for 300$.

 

[butterpants]

Someone was kind enough to link to my blog - I've got some videos (and more in production) about some of the things you may be trying to do in your home lab. I'm a microbiologist by profession, so I may be able to help you out a little, especially on the microscope and media front.

Firstly, the autoclave. Don't waste your money - a quality pressure cooker is 1/10th the price and works just as well. So long as your pressurecooker gets to 15PSI or higher it'll do just as good of a job.

Microscopes are a much more difficult thing to address, there are a number of manufacturers out there who make good scopes - Nikon, Leica, Olympus and Zeiss lead the pack but there are many others. But defining exactly what scope features you need depends on what you wish to do with it. First (I think you know this already), the total magnification of a scope is determined by multiplying the magnification of the objective lens (the lens near your sample) by the magnification of the ocular (the part you look through). Most oculars are 10X, but 2X, 5X and 20X also exist. If you have a 40X lens and 10X ocular, your scope has a total magnification of 400X; you'd get the same with a 20X lens and 20X ocular, although for reason too complicated to go into here, this setup would give you a poorer resolving power. Any ways, what total magnification you need depends on what you want to do:

• Count yeast - you can get away with as little as 100X for this, although 200x - 400x is probably more practical
• Identify the presence of bacteria - minimum to do this reliably is 400x, although older eyes may appreciate 600x
• Species identification by morphology - you cannot ID species solely by microscopy, but you can separate brett from sacc, or lacto from pedio, based on morphology (assuming you know they are in there via other means). To do this you need 600x to 1000x. More on this below...
• Beyond 1000x - there isn't much of a purpose for this; beyond 1000x you are usually exceeding the resolution limit of your optics, meaning that while objects will appear larger, they will be no more detailed than at 600x or 1000x.

Hopefully, that gives you an idea of the magnification range you need. If you are thinking about the higher end there are a few other things to worry about. Lens quality matters a lot at the higher end - poor quality lenses will produce low-detail images and not collect a lot of light (making for dark images). At the higher end - especially if looking at a 100x objective (i.e. 1000x total mag(, you should be looking for a high numerical aperture lens - at least 0.8NA, ideally 1.0 or higher. The NA defines both the light collecting ability and the resolution of the microscope, meaning a high mag lens with low NA is almost useless. Unfortunately, these high-NA lenses are often oil-immersion or water-immersion. This isn't the end of the world, but it does complicate microscope use a little, and you have to be careful not to get the oil/water on your air lenses. The plus-side is that the oil lenses in particular really do a lot for image quality.

The other issue people have raised is phase contrast. Personally, I wouldn't consider a scope without it, but if all you want to do is counts you can get away without it. Microscopes rely on pigments (which yeast and brewing bacteria don't have) and diffraction to visualize stuff. Since yeast/bugs are mostly water they don't diffract much, meaning few details can be observed outside of general cell shape - and it can be hard to simply find the cells. Phase contrast "amplifies" the diffraction, making finding and observing subjects much eaiser. Again, adding phase adds some complexity (you need to align the phase optics), although that is a skill that is trivial to implement with a bit of practice.

For counting a hemocytometer is a must - a cheap one is more than enough though (just clean it well after each use). The purchase of a few basic dyes (i.e. a viability dye like trypan blue, plus a general staining dye like saffrin) can help a lot - especially if you don't have phase. Some basic micropipettes (ebay's loaded with them) can help with doing accurate dilutions. I'd recommend glass petri dishes & tubes; both because you can "cast" your gels right in the pressure cooker, and because they are cheaper in the long run than disposable plastics.

In terms of growing bugs on plates, a number of medias are available, but I've found the combination of beer-wort agar (DME @ 1.006SG + 1.5% agar) and potato-dextrose agar serve for 99%+ of what is practical at home.

Just my $0.02

Bryan

EDIT: shameless self-plug - you can link directly to my youtube channel here.

Oh man thanks Bryan for the truly detailed response. I need to ruminate on some of those points a bit before I can reply.... plus I'm about to get on a plane.

 

[butterpants]

I'm very happy with the Amscope microscope that I have:
http://www.woodlandbrew.com/2012/11/amscope-binocular-compound-microscope.html

And this is some information on hemocytometers:
http://www.woodlandbrew.com/2012/12/hemocytometers-side-by-side.html

Thanks for posting that.... I do have your book and counted your talk about AM Scopes as a positive endorsement!

 

[ajdelange]

Can you point me in the direction of exactly what kit/parts I'd need to convert one to a phase contrast?

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.

 

[trentm]

And this is some information on hemocytometers:
http://www.woodlandbrew.com/2012/12/hemocytometers-side-by-side.html

Thanks for link WoodlandBrew. I have visited your site before but somehow missed your write up on hemocytometers. So you inspired me to try using my iPhone to take photos using my scope (same model as yours) and found with a steady hand, success could be achieved. When I purchased my scope I also chose an economy hemocytometer and was very disappointed. I first thought the economy microscope was to blame but like you, I had access to a lab grade hemocytometer, borrowed it and found good results. However, paying more for a hemocytometer than a microscope seemed ridiculous. Then I took a chance on the brightline hemocytometer offered by Cynmar for $65. It seems the quality of image provided by this hemocytometer is comparable to the >$200 ones sold through other suppliers. For me the price was defiantly worth it.

The first image shows the economy hemocytometer on the left and the brightline on the right (notice the mirrored surface of the counting chamber). The second image was taken using the economy hemocytometer and the third image is with brightline hemocytometer (both at 400X). The fourth image just shows the view I get at 100X (10X objective with 10X eyepiece).

How do get the micron key in your images?

By the way, for those our there looking for hemocytometers, most scientific suppliers use the spelling "hemacytometer"

 

[ajdelange]

There are some notes in use of the hemocytometer at http://www.wetnewf.org/pdfs/hemocytometer.html. Emphasis is on counting (which square a cell should be assigned to) and on how to interpret counts (how many cells/squares need to be counted for a given level of accuracy).

 

[trentm]

There are some notes in use of the hemocytometer at http://www.wetnewf.org/pdfs/hemocytometer.html. Emphasis is on counting (which square a cell should be assigned to) and on how to interpret counts (how many cells/squares need to be counted for a given level of accuracy).

Wow! What yeast nutrient are you using? All my sources indicate Saccharomyces cerevisiae cells measure from 5 - 10 microns in diameter. Perhaps the measurement of the square you indicated is actually 0.25 mm.

 

[ajdelange]

Something does seem fishy here. I think the line spacing is probably 1 mm as I believe that's pretty much a standard. I'd normally run up to the lab and check the measurements but that's 600 mi away so I can't do that. That leaves the yeast. It is not unusual for certain strains to be appreciably larger than 10u but I have no idea at this point which strain these were nor whether any strain I actually use is a 'giant'. What I suspect is that these cells, which are not by any means young viable cells but rather old dead or near dead cells from the sediment in a beer bottle, may be swelling prior to bursting (releasing all that smelly stuff we associate with autolysis) but I don't disallow the possibility that I made an error in measuring the haemocytometer.
'

 

[passedpawn]

Something does seem fishy here. I think the line spacing is probably 1 mm as I believe that's pretty much a standard. I'd normally run up to the lab and check the measurements but that's 600 mi away so I can't do that. That leaves the yeast. It is not unusual for certain strains to be appreciably larger than 10u but I have no idea at this point which strain these were nor whether any strain I actually use is a 'giant'. What I suspect is that these cells, which are not by any means young viable cells but rather old dead or near dead cells from the sediment in a beer bottle, may be swelling prior to bursting (releasing all that smelly stuff we associate with autolysis) but I don't disallow the possibility that I made an error in measuring the haemocytometer.
'

200um

On mine, the minor graduations are 50um, and triple lines 200um apart

The full field is 1mm square

 

[trentm]

200um

on mine, the minor graduations are 50um, and triple lines 200um apart

the full field is 1mm square

This thing has stuck in my mind until I think I now have it figured out. Someone please correct me if this seems wrong:

The full grid of the counting chamber is 3mm square and contains 9 large squares that measure 1mm square (highlighted red in the picture below). Because the cover slip of the hemocytometer is mounted 0.1mm above the grid the volume of the area defined by 1 large square is 0.1 microliter or 1/10,000 of a ml. If one were counting large objects, say >20 microns, 5 of the largest squares (typically the 4 corner squares and the center square) would be counted and for calculation the sum would be divided by 5 to get the average number in one large square and the number would be multiplied by 10,000 to estimate the number of cells in 1ml. This could be done with yeast cells and high quality microscope but it is more practical to to count yeast cells using the center square. The center square also measures 1mm square if you measure the center line (I think) of the triple lines. So when counting the center square one would count 5 of smaller squares (yellow in the picture) using the center line of the triple line for boundaries. A.J's write-up describes this well as well as how to count cells that are on the line, http://www.wetnewf.org/pdfs/hemocytometer.html. Now instead of taking the average of the 5 squares as we did with the large squares, we will multiply the count of 5 small squares by 5 to get an estimate of the total number of cells in the center square which we know the volume is 0.1 microliters and multiply by 10,000 to get total number of cells in 1mm.

I have been puzzled as to why the triple lines on the center square. I think the answer is: By using the inside line of the triple lines a new boundary in defined within the small squares that measures a smaller volume (0.004 microliters), notice there are 25 small squares in the center square compared to 16 small squares in the 4 corner squares, even though the the total volume of the center square is the same as each of the 4 corner squares. Now the tiny squares (blue in the picture) within the small squares of the center square have a volume of 0.00025. So at this level, even smaller cells could be counted.

 

[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.