My Lab

Still alive

It’s been a while. Quite a while.

A lot has happened. Having a total knee replacement and soon to have Rotator Cuff surgery, I’ve been literally out of commission. For several weeks, I didn’t even have a beer! After the knee replacement, I had absolutely no desire to have a beer. Must have been the medication. Don’t worry, I’m enjoying a Poet by New Holland Brewing Co. as I type this post.

I haven’t been able to brew due to surgery and other developments. The doctor gave me a two-thumbs up last week, but now I won’t be able to lift anything for the next month or so. Today I began working on my new brewery. During my convalescence I decided to go electric on my brewery. So, last Friday I decided to do as much as I could to get the process started. Additionally, I’ll be making a clean-room for my yeast. I began working on a home-made laminar hood and decided to take over a room in the basement for the brewery. Walls came down and carpet left the room today with hopes of painting the floor with a two-part epoxy on Tuesday. I’ll be out of commission for about a month, but at least I’m on my way.

The next time you see a post, I should have the ability to continue working on the brewery. Can’t wait.

 

UPDATE 04/17/2015

What I was able to accomplish prior to surgery.

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Quality Control Experiments – Part 2

Results are in. All types of mold, bacteria, and wild yeast are flying around my basement with immunity. Yes I clean, sanitize, and sterilize. When I transfer liquids, I make sure windows are closed, a flame is around the containers, and all precautions are taken.

This experiment almost made me quit brewing.

I sat back and wondered what I was doing wrong. If I was doing everything I could humanly do to keep everything clean, why were there so many bug ending up in my cultures?

My beers turn out great. I always keep several tubes of wort and monitor them for bacteria. I have some tubes from six months ago that still show no sign of bacteria. This has, however, pointed out many flaws in my lab that will be corrected. Below are the results.

Plate 1
Air sample using UBA. Using Gram Stain, under microscope at 40x (below).

Plate #1b     Plate #1 UBA      Slide1b.Gram Stain.Plate 1.40x

Plate 2
Air sample using agar plate. Gram Stain using 10x, 40x, and the last two are 100x oil immersion. (below)

Plate #2 - Agar     Plate #2 after 24 hours     Slide2.Gram Stain.Plate 2.10x

Slide2a.Gram Stain.Plate 2.40x.4     Slide2.Gram Stain.Plate 2.oil.3     Slide2a.Gram Stain.Plate 2.oil.4

Plate 3
Swab samples. Gram Stain at 40x and oil immersion 100x. (below)

Swab Plate  Plate #3     Plate #4a     Slide6.Gram Stain.Swab.40x.1

Slide3.Gram Stain.Swab3.oil.2

Perhaps one of the most startling results came from the swabbing on the incubator. Although samples were taken from five different areas, absolutely no bacteria or fungi were detected.

Swabbed area in red ovals

Swabbed area in red ovals

I have begun building my new lab. I’ve transferred all yeast, DME, and hops from the fridge and placed in another fridge, storing at the lowest possible temperature (-20F in the freezer, 37F in refrigerator). Additionally, I am using a stainless steel table as my work area and constructing a Laminar air flow booth for culturing.

New lab

Current Project – part 2 (just the beginning)

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My rude awaking began with this culture. Actually, I let this grow for quite some time, long past my observations. However, it confirmed what I theoretically knew; mold, bacteria, and yeast are all around. Visually confirming something you cannot observe without the use of a microscope is powerful. The carpet in the petri dish I produced can be replicated in any but the most sterile lab conditions. Really – there are no visible molds growing ANYWHERE in my house. My lab is in the basement and you cannot smell anything that resembles mold or mildew. The filter on my furnace is 5″ thick and can filter to 3μm, and is equipped with a UV lamp.

So what to do?

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1. Assume everything you handle is contaminated with mold, bacteria, or wild yeast. Let your guard down and you’ll be sorry. Very sorry.

2. If you think you’ve cleaned your lab and equipment well enough, you haven’t. Read #1 again.

3. Yes, air moves. Yes, all of those bad bugs catch the wave and ride it until they reach something worth feeding on – like your wort, agar plate, and yeast culture. I read several books and blogs that suggested closing the vents or turning off the furnace / air conditioner. That is one of the best suggestions.

4. Finally, when in doubt, remember this – there is mold, bacteria, and/or wild yeast everywhere you believe is clean. “Indeed, studies of human exposure to air pollutants by EPA indicate that indoor levels of pollutants may be 2 to 5 times – and occasionally more than 100 times – higher than outdoor pollutant levels.” (EPA, 2014). You WILL have contaminated yeast cultures if you believe otherwise.

After seeing the culture, I almost gave up on being a yeast rancher. If this is what I have to look forward to, and have to deal with this every time I culture, then why even try? I did think those thoughts, for about 30 seconds. Then I remembered something – just one of my brain cells can kick any bug’s ass, blindfolded.

Besides, I love the smell of fermentation in the morning – it smells like victory.

EPA. (09/13/2013). Questions About Your Community: Indoor Air.  Retrieve from http://www.epa.gov/region1/communities/indoorair.html

Quality Control Experiments

Yes, I need to finish the Current Project blog, however, I am beginning several experiments at the same time. Having found wild yeast and Gram-positive cocci, I am concerned that my seemingly clean lab / brewing area is really a hot-bed for bacteria. It helps that my Universal Beer Agar has about two weeks of shelf life, so why not begin the experiments now. I’ll also be culturing some English Ale yeast to test several storage techniques – I’ll cover that in another blog.

To begin this experiment, I decided to place two open plates on my cabinets. Last night I placed an open UBA plate on my peninsula cabinet and an open agar plate near my test equipment. Believing that both the wild yeast and cocci came from a test tube sitting open in the rack, I hope the air samples can substantiate my hypothesis.

DSC00562                   DSC00563

Tonight I swabbed four areas; the first being the counter where the agar plate was sitting. Although I am judicious and clean my work areas prior to doing anything, for this experiment I did not clean the area with disinfectant prior to swabbing. The second area swabbed was the stove. Circles represent areas that were swabbed.

cabinet                   Stove

The test tube holders are another area as a possible bacteria cesspool, along with the various pieces of lab equipment. The other area, although not used while brewing, is the incubator.

lab equipment                    Incubator

I placed all inoculated plates into the incubator at 80° F and will check them in 72-hours. Assuming each plate grows a culture, I will post the results after they are stained and examined.

Current project – part 1

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I began this current project with a few simple questions – how many bugs are flying around my lab? Is my work area really as “clean” as it looks? And, how likely is it that any batch of beer is going to be infected by a bug (bacteria, wild yeast, etc.)?

FIRST THINGS FIRST

As most projects go, I started by researching the subject.  I read and reread Yeast, watched several YouTube videos on how to culture yeast, and read many blogs and articles on the process of yeast ranching. Taking some of the yeast (Lallemand Nottingham Ale) from a current fermenting beer, I began my journey as a yeast rancher.

I have yet to read anyone who says culturing dry yeast is worth it, but I think losing $3.99 is better than $6.99. So, this was my test batch to see whether I could keep the area clean and sanitary while culturing the yeast. I began by putting everything that could fit into my stove-top autoclave. Spend some money and get a good autoclave rather than a pressure cooker. If you are going to herd yeast, this is one piece of equipment that you must have.  Be sure to follow the instructions. You’ll want to purge the autoclave of air and allow pressure to build up to around 17 -20 psi (about 235-240 F) to sterilize your glassware and agar.

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Purging air from autoclave

The next step requires that you pay attention to your surroundings. There are many formulas for agar, all sharing the same ingredients: agar, DME, and yeast nutrients. Boil the DME and nutrients, then add the agar. Pour into either a culture tube or petri dish. The amount of agar in the petri dish need not be very thick. The culture tube should have a little less than half the total volume available (if the tube is 30ml, then you need a little less than 15ml). Place everything into the autoclave and sterilize for at least 35 minutes.

After the glassware is touchable, remove everything from the autoclave. While the dish is warm, but the agar is set, turn the dish upside down and let cool for several days, or until the condensed water is gone. Take some Parafilm and wrap the petri dish if you do not intend to inoculate immediately. The culture tubes are a bit different. Remove them while the agar is still liquid and place them on a 30 degree angle (thus the name slants). Be sure the screw cap is semi-loose while the agar is setting up. As with the plate, allow enough time for the condensation to dry. Parafilm when ready.

20140112_134909

My lab / brew area

WORDS OF CAUTION

If you take away anything from this blog, remember to make sure that everything you touch – that will in any way come into contact with anything from this point on – is sanitized and/or sterilized. Remember, you want your laboratory to be as clean as possible; free from draft, dirt, and anything that will contaminate your yeast. If you think your area is clean, it isn’t. If you are of the mindset that there is absolutely no way mold or bacteria can get into your culture, you are imagining things. Unless you have a cleanroom (as in a commercial laboratory), you’ll have to take every precaution you have heard or read about to even have the slightest hope of culturing yeast that carry zero contaminants.

Getting back to inoculating your dishes and slants.

Make sure you have a Bunsen burner, inoculating loop, nitrite gloves, and alcohol (look below for a complete list of necessary equipment). Before you attempt to inoculate your plate or slant, you should practice holding two tubes with one hand, know where you will put the cap, and practice taking a sample from one tube and placing the inoculating loop into the other. There are many great articles written by experts showing how this should be done.

 bunsen burner  Take the loop and place above the flame.

20131230_204152 This will kill all bacteria. 

I used to pride myself on keeping my lab clean, making sure no bugs would enter my beer. Oh, was I ever in for a rude awaking.