Characterize Your Contaminant: Gram staining, Catalase testing

Not to spook you or anything, but there are about a trillion different bacteria out there. The good news is that the subset that commonly affects beer is very small. Nevertheless, it can run the gamut from the good, to the bad, to the ugly. If bad bacteria are those that could harm us, then the ugly are beer ruiners that spoil the profile in other ways. Lactic acid bacteria, for example, can both be good when used intentionally, and ugly since some of them are more resilient in beer than we’d like. They can be a big problem if they get in uninvited. 

In typical beer, hygienic brewing processes along with beer’s inherent barriers help to protect it. ABV, low pH, hop bittering compounds, low oxygen content, carbonation, even the absence of nutrients that were already metabolized by yeast during fermentation — all of this is what makes beer successfully unwelcoming to bacteria. That’s by design. Our ancestors in beer were no dum-dums. So, when we’re spot checking our suds for bugs, we’re not really doing it to make sure it’s healthy for us (though we also are), it’s primarily about ensuring that the stability, flavor and look of our beer will remain for the long haul.

High threat, low threat

When beer spoiling bacteria is detected, understanding more about what kind of bacteria it is will tell us more about what level of threat there is to our beer. If, for example, characterization methods tell us it’s a type that can’t survive long in beer’s conditions, then it’s a relatively low spoilage threat. A high level threat would be one that isn’t stopped by beer’s defenses. This kind is free to persist and become a big problem for the taste, look and overall stability of our beer. The majority of contaminants that craft breweries come across are low-risk hygiene indicators, like organisms that come up with a faulty pump seal, improper CIP or a small biofilm that is beginning to form. A result that suggests a high-risk beer spoiler and you’ll need to start triaging a much bigger problem before any bad beer is out in a consumer’s hand. The biggest beer spoiling threats are Lactobacillus, Pediococcus, Megasphaera and Pectinatus.

Genetic testing vs. other lab methods

Generally speaking, the more precisely we can characterize our contaminant, the better. Genetic testing is a fast and precise way of getting an ID, but that’s not always practical for the run-of-the-mill contaminants you run across in the brewery. Other methods, even methods that are less precise, can still give us good information. They give us more of a threat level than a specific ID. Gram staining and catalase testing are two of these. Together, they help us eliminate or include what type of contaminant we could have according to two traits that differentiate types of organisms into potentially high risk or potentially low risk. These kinds of tests might fit more accessibly into a brewery’s QC protocols than genetic testing does, which can be really important for early detection. The more information the better.

Gram staining involves staining cells in a certain way to see if they appear purple or pink. Directly, the test reveals a bacteria’s cell wall structure, and as a result, whether the bacteria we’re looking at is likely to be hop resistant or hop sensitive. Indirectly, knowing this trait just helps us identify whether our contaminant is consistent with the known traits of a certain high risk organism or not. 

• Gram-Positive: If a bacterial colony ends up stained purple at the end of the test, its cell walls are the kind that let antibacterial hop compounds in. Theoretically, that would make Gram positive contaminants consistent with hop sensitive bacteria, and therefore low risk — and most are; most bacteria that let bitter hop compounds in are eventually killed off by hops’ antibacterial properties., essentially messing with a bacterial cell’s pH, preventing it from benefiting from nutrients. But there’s some bacteria with a cell wall structure that lets hop compounds in that can also usher them back out again, which makes them more resilient to hop compounds. These kinds of bacteria also happen to be resilient to some of beer’s other protections, too, like alcohol and acidity. That makes them a big threat if they get where they aren’t wanted. Hop resistant lactic acid bacteriacan persist in beer’s conditions, adding sour, buttery and sulfurous flavors as well as causing ropiness and/or sliminess. In conjunction with the catalase test next, you can see if a gram-positive result can be sorted as a higher or more moderate risk for spoiling your beer.
• Gram-Negative: If a bacterial colony ends up stained pink at the end of the Gram test, it’s because its cell walls have an extra membrane layer. That layer keeps hop compounds out. Though hops don’t have an antibacterial effect on Gram negative bacteria, they’re also a group of bacteria that are the unlikeliest to survive in the finished beer anyway because they are mostly all susceptible to beer’s other defenses. It’s a good thing, too, because bacteria in the Gram-negative characterization can be things like coliforms, enterobacter and other hygiene organisms. If you have any of these, it’s a good indication that you have a breach in your CIP effectiveness, like a faulty gasket or a small biofilm starting to form. This is a great heads up to get to the root of the problem before any higher risk beer spoilers show up to the party. In conjunction with the catalase test next, you can see if a gram-negative result can be sorted as a moderate or lower threat.

Directly, catalase testing looks for how resilient an organism is likely to be to oxygen environments via catalase enzyme activity. Most bacteria that can use oxygen for respiration produce the catalase enzyme, and most bacteria that can’t tolerate oxygen environments don’t. So, the test involves smearing a sample of a contaminant on hydrogen peroxide and watching for bubbles (in the cell, bacteria’s catalase enzyme breaks up hydrogen peroxide, a byproduct of the bacterial cell’s metabolism, in order to protect the cell from what’s called oxidative stress). Indirectly, when catalase testing results are read with Gram staining results, we can characterize our contaminant more accurately, and determine what level of threat we’re looking at.

• Catalase-Negative: Beer is largely an oxygen-free environment, so bacteria that can live no matter what the oxygen environment is like and bacteria that only survive in oxygen-free environments can survive in beer and spoil it. No bubbling on a catalase test indicates no catalase enzyme activity, which is characteristic of organisms that don’t tolerate oxygen well, so they could be a potential spoiler in that sense, if this information were simply taken on its own. However, we use this information in conjunction with Gram staining results to deduce whether we’re dealing with certain bacteria we know to be higher or lower risk. Continue to the next section to see how we read these test’s results together.

• Catalase-Positive: If rapid bubbling happens, there is catalase enzyme activity, which means the organism you have is likely aerobic, a.k.a. an oxygen user. This rules out obligate anaerobes, which are bacteria that can’t tolerate oxygen. But it doesn’t rule out facultative anaerobes, which can survive in either aerobic or anaerobic conditions. Some lactic acid bacteria are facultative anaerobes. They may also be catalase-positive, though they would show weak bubbling. Since they can tolerate aerobic environments and thrive in anaerobic ones, too, then you still have a moderate risk for beer spoilage with this result.

Gram and Catalase Together

We don’t use these tests only to detect traits in our contaminant, meaning our use of these tests doesn’t end at assessing our contaminant’s likely hop tolerance via its cell wall structure (Gram staining), or its aerobic, non-aerobic or micro-aerobic behavior (catalase testing). We’re using a contaminant’s traits in these two instances to identify what the contaminant could or couldn’t be according to what we know about certain bacteria’s traits. If our results indicate a combination of traits that make it possible that our contaminant could be an especially pernicious spoilage organism for beer, then we should initiate our relevant processes for a high level of alert. If its traits eliminate the liklihood that it’s one of these, we have a much less stressful situation on our hands — although one that still needs to be investigated. When we use these tests in combination with other methods, we’re mitigating risk really effectively. 

HIGH THREAT

• Gram-positive, catalase-negative could be either Lactobacillus or Pediococcus. It is a result that’s often linked to beer spoilage. If these lactic acid bacteria have HorA/HorC genes (genes related to hop resistance), it can lead to an even bigger threat to hoppy beer styles. If you see this result, take note of the morphology of the cells to help differentiate: Rods indicate Lactobacillus, cocci indicate Pediococcus.

MODERATE THREAT

• Gram-positive, catalase-positive is less likely to be Lactobacillus or Pediococcus. So, it’s considered slightly less of a threat than the above. Also, because it is an easier contaminant to grow, it is less likely to be missed. 
• Gram-negative, catalase-negative is probably the one we’d least like to see. We can rule out lactic acid bacteria, but we still have concerns for Megasphaera and Pectinatus. Note: Because these bacteria are so sensitive to oxygen — and higher alcohol, they are only threats to beers that are lower than 5% ABV and when dissolved oxygen levels are kept very low.

LOW THREAT

• Gram-negative, catalase-positive is potentially less alarming. Gram-negative rules out the possibility of Lacto. Catalase-positive suggests it isn’t as likely to thrive in beer. That would limit its impact. Acetic acid bacteria can be considered a threat, but only in situations where oxygen is present, like barrel-aged beers and mixed fermentations.
  

Catalase testing and Gram staining, especially when understood together, can provide useful information to better categorize the organism in question. At the end of the day, protocols of your QC program will have to guide the decision on what to do next, but this sort of testing can provide helpful, useful information that you can make that decision a more informed one.

If you keep a log of contaminants you see in the brewery, you will have a better idea of whether you have seen something before and where the potential source of the micro is. Keep pictures, if possible, and indicate the size and morphology of the organisms. If you do send something for genetic identification, make sure to log that, too. In the future, you can use this information to assess whether what you’re seeing now is the same hygiene indicator you’ve seen and solved for handily in the past, or if it is something more serious. 

The more confidently you identify the risk-level of your contamination, and its source, the more it will help you find an easier way to the solution next time, which could save you time and money. Improving your process and getting better at root cause analysis will make each contaminant seem like less of a problem.