Simple Laboratory Methods for Microbrewers
By Frank Commanday (Brewing Techniques)
Quick Results for Quality Assurance
The time normally associated with microbiological testing causes many brewers to view such work as tedious drudgery. A couple of relatively easy methods can make light work and produce quick results, saving brewers both time and money.
For many microbrewers, lab work is the last thing on their minds. After all, production has first priority, and any task unrelated to cellar or brewhouse operations is often done during spare moments (that is, rarely). Brewers who are accustomed to routine lab work are tempted to fall behind on their microbiological plating in the face of production contingencies. For years, plates showed negative results, so why worry? Why stay an extra hour, make media, and hassle with the petri dishes and pipettes? Similarly, brewers who lack lab facilities max not want to bother with the extra expense and complication. Good sanitation (or what is considered good sanitation) is enough to ensure clean beer, right? Wrong.
In both cases, all the hard work of the brewing, business management, and marketing staff is for naught if an infection develops and surfaces in the finished product. Although this fear alone may be sufficient to motivate the busy microbrewer to get a little busier, making the lab methods easier and faster certainly makes the responsibility less daunting.
This article describes methods that do just that. The methods are simple and inexpensive yet reveal a wide variety of common contaminating bacteria quickly. This last point is important, because it is useful to know whether a given pitch of yeast is clean before you pitch it. Even with American Society of Brewing Chemists–approved plating methods using UBA (universal beer agar) incubated aerobically and in anaerobic jars, complete results may be unavailable for 6– 7 days, which is too long to wait if you are testing your yeast before each stage of propagation or if you are repitching ale yeast.
Two media available from J.E. Siebel Sons’ Co. (Chicago, Illinois), when used together, give preliminary results in 16–48 hours. Further, neither requires incubation under anaerobic conditions.
HRM (Hsu’s rapid medium) is used in an aerobically incubated petri dish (plate), with the sample mixed into the molten medium. When it contains ~6 ppm cycloheximide, also known as actidione (available through large chemical manufacturers such as Sigma Chemical [St. Louis, Missouri] and local chemical or laboratory supply stores), it detects a wide range of brewery bacteria, including those that are aerobic, facultative (able to grow aerobically or anaerobically), and aerotolerant (anaerobes that are able to survive or marginally grow in the presence of air). Used without cycloheximide, HRM is a good medium for yeast culture.
HLP (Hsu’s Lactobacillus-Pediococcus medium) is specific for the two normally slow-growing anaerobic beer pests that give it its name. Instead of being used in a pour plate, HLP is a semigel (soft agar) used in a screw-cap tube.
Materials and Preparation
The only equipment you need is the following:
• 1 small laboratory incubator
• 1 laboratory hot plate
• 1 pressure cooker/canner over a stove or gas burner
• 2 1-L Pyrex beakers
• 1 125-mL Pyrex beaker
• 1 100-mL Pyrex bottle with stopper
• 12 2-mL Eppendorf tubes
• 1 1-L Pyrex conical flask
• 15 16 X 150 mm Pyrex screw-cap tubes
• 1 box disposable pipetter tips
• 1 roll nonabsorbent cotton
The key to making plating quick and easy is mixing, dispensing, and sterilizing media ahead of time. One 200-mL batch of HRM in 32 tubes, for example, is sufficient for 16 plates.
Preparing a stock solution: Before mixing the media, prepare a stock solution of 1.1 mg/mL cycloheximide. This task can be made easier and safer by weighing out several 110-mg portions in 2-mL plastic Eppendorf tubes ahead of time. You will need an analytical balance to do this, and it helps to know someone who has access to a lab (beer is usually an effective barter …). Cycloheximide is toxic (it shuts down cellular protein synthesis) and should be handled with latex gloves and a dust mask. It is a good idea to sterilize 100 mL of water in the Pyrex bottle before adding a 110-mg cycloheximide portion. Store this stock solution in the lab refrigerator. Adding 2 mL of this stock solution to 200 mL of HRM medium yields 11 ppm of cycloheximide. This concentration is significantly more than the 4 ppm recommended if the stock solution were added after sterilizing the medium. The method described here calls for dispensing the medium into tubes before sterilization; the high concentration of cycloheximide stock solution added to the HRM compensates for the fraction of cycloheximide destroyed during sterilization. Even if some of the cycloheximide is not destroyed, Pedioccocus growth will proceed uninhibited at levels <20 ppm (3). An alternative method involves preparing a 0.6 mg/mL stock solution, running it through a 0.45-µm membrane filter disk (for example, on the end of a syringe) into a sterile container, and dispensing it aseptically into each of the test tubes.
Preparing the media: To prepare HRM, add 14 g of the powdered medium to 200 mL of distilled water in a 1-L conical flask. To dissolve the powder, bring the water to a boil over low heat for 1 min while stirring (you can also steam in without pressure in the pressure cooker). Add 2 mL of cycloheximide stock solution. Dispense 6 mL of hot medium into each of 32 capped test tubes and sterilize at 250 °F (121 °C) for 10 min. Store in the lab refrigerator.
To prepare HLP, sterilize 13 screw-cap tubes (with caps partially screwed on) and a 125-mL beaker covered with aluminum foil for 15 min at 250 °F (121 °C). Mix 14 g of HLP powder with 200 mL of distilled water in a 1-L conical flask. Dissolve the powder by swirling the flask during a gentle boil and boil for 2–3 min. Do not boil longer than 2–3 min, because this medium contains heat-sensitive cycloheximide (4 ppm). Fill each tube in the 110-mm mark using the sterilized beaker and tightly cap. Store in the lab refrigerator.
About 15 plastic pipette tips should also be sterilized ahead of time. Stuff a small wad of cotton into each, just below the end, and place into a capped test tube. The cotton filters out contaminants from the air during sample dispensing and protects the pipetter mechanism from contact with liquid. Sterilize 15 min at 250 °F (121 °C). Note that sterile tips can also be purchased.
You are now ready for about two weeks of hassle-free QC!
First, remove a sterilized sample of actively fermenting wort. For an enclosed fermentor, flame the sample valve with a propane torch to prevent contaminants growing in the spout from being introduced into your sample. To do this, rinse the valve with water from a spray bottle, then flame all around and inside the spout until you see steam. Bleed out some wort to cool and flush the valve. Fill a sterile test tube slowly (without overflow — this stuff is foamy).
Back at the lab, put an HLP tube and two HRM tubes (with caps loosened) in a beaker half-filled with water and boil until the media are melted. Close the HLP cap and transfer the tubes to a beaker of 100 °F water. With the 0.5-mL pipetter and sterile tip, add 0.5 mL of the wort sample to an empty, labeled 60 X 15 mm petri dish, and 0.5 mL to the attemperated HLP tube and invert twice to mix. Add a tube of attemperated HRM to the dish and gently swirl in a figure-eight pattern to disperse sample. Add the second tube to the dish. Place the HLP tube and HRM dish in the incubator set at 86 °F (30 °C).
All transfers of media and samples must be done using sterile technique. Although a covered inoculating cabinet or glove box isn’t absolutely necessary, make sure that you haven’t just handled a load of dusty malt and that your lab surfaces are clean and wiped down with dilute bleach or rubbing alcohol. It is also necessary to briefly flame the ends of sample and media tubes just after removing and before replacing caps and to work in the proximity of the propane torch’s flame (the plume of hot air is sterile and dust-free).
Examine the HRM plates and HLP tube over the next two days and count and record the number of colonies, if any appear. Although most colonies will appear within that time, slow-growing colonies may appear during the third day, which is the day to record final results. The results give you a rough indication of your level of sanitization.
Although counts > 100/mL of fermenting wort indicate serious contamination, counts of ~10/mL are cause for concern. Wort pitched with yeast from fermentors showing any bacterial growth should be tested, and, if the contamination level remains the same or increases, the yeast from that batch should be either repitched or acidwashed.
Know Your Enemy
Although this method allows you to avoid repitching contaminated yeast, how can you identify the source of contamination? Identifying the culprits can yield clues.
Small white colonies on your HRM plates and comet-shaped colonies embedded in the HLP tubes, for example, indicate lactic (Pediococcus and/or Lactobacillus) contamination, which could be caused by a poorly sanitized fermentor or a contaminated pitch. Large beige colonies on HRM plates often correspond to aerobic-wort bacteria (for example, Enterobacter, Hafnia, Klebsiella, and others), which stain Gram-negative. Poor sanitation of wort lines or the heat-exchanger or contamination of the rinse water may have caused contamination by these acid-intolerant rods. Examination of colony cells under a microscope may confirm your suspicions.
Hot-side contamination (in the whirlpool, for example) is evidenced by the presence of Bacillus. These large Grampositive rods form large colonies on HRM with a granular surface caused by production of heat-resistant spores (endospores).
Supplemental Tests To Finished Beer
Incubating samples of unpitched wort in finished beer to “force” the growth of contaminants provides a good supplement to plating. This simple method can detect levels of contaminants too low for plating to detect. The test is limited to serving as a warning flag, however, because it cannot reveal the extent of contamination or (directly) the identity of the offending bugs.
To start, you need a supply of sterile, cotton-plugged 125-mL or 250-mL conical flasks. Simply flame the sample valve on your bright-beer tank (as described above) and fill the flask about a quarter full. Incubate at 86 °F (30 °C) and examine over 7 days. Bottled beer can be incubated in the same manner. The beer should remain clear and be free of sourness or off-aromas (such as solvent, fruit, diacetyl, vegetable, phenol, and others). A similar method can be applied to unpitched wort to reveal possible contamination by wort bacteria or Bacillus.
Because contaminated fresh bright beer and packaged beer often have counts < 1/mL, the plating methods outlined in this article are inappropriate. Instead, it is wise to first filter 12 oz of the product through a 0.45-µm membrane filter disk and lay the disk on HRM agar in a petri dish (see instructions enclosed with the medium). This quantitative method should be used to complement regular force tests.
With the exception of the force tests, none of the methods outlined in this article can reveal contamination by wild yeasts. If samples consistently show little or no bacterial growth, however, the probability of yeast contamination is low because of evidently good sanitization. This is consolation, but not proof! I recommend periodic (every 6 months) yeast culture testing by a good lab, using a range of selective media (for example, lysine and Lin’s and Siebel’s WL) for wild yeast detection, along with tests for viability, respiratory deficiency, and genetic variability (that is, variation in colony size and morphology). With the resulting information, you may decide to repropagate more often, purify your culture, or even obtain a new culture.
Peace of Mind
Using simple force tests and preparing HRM and HLP rapid media ahead of time results in easy routine lab work. You will be rewarded with peace of mind when samples run clean and clear, and you will be given sufficient warning to allow you to act quickly and preemptively if contamination becomes evident.
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