Lesson 8 - Hygiene End of Unit SAQ

** Your brewery is experiencing positive results on early fermentation samples from a WLN agar medium. You wish to identify the potential contaminants as quickly as possible.

Discuss both the methodology and applicability of rapid biochemical or molecular tests that will identify the contaminants.

Response:

Traditional methods can mean that it takes up to a week or more to identify organisms, whereas rapid methods can produce results within hours.

Listed below are some of the more commonly used rapid biochemical and molecular tests that can identify specific strains:

API

ELISA

GC

Hybridisation

PCR

The API 20E test kit has been used to identify Gram-negative brewery bacteria for many years. It circumvents the need for complex multiple tests on specialised media and is simply performed using a disposable microtube array of 23 tests. The test does require a pure colony from agar plates grown aerobically and anaerobically, which is then suspended in saline for inoculation into the kit wells. After a 48 hour incubation, the results are read as colour changes and entered into an online database for identification. The Gram-negative non glucose fermenters Zymomonas and the acetic acid bacteria require further identification tests. However, the Enterobacteriaceae Obesumbacterium, Enterobacter, Klebsiella Citrobacter, and Escherichia are all differentiated and identified through the database.

ELISA tests are immunological tests that detect an antigen (a unique protein polypeptide) from specific bacteria. They do this through binding to an antibody-linked fluorescent probe that is specific for that antigen.  

Although some commercial antibody kits for the LAB and Pectinatus have been developed, the commercialisation of antibodies for beer spoilers has been a lesser area of priority than that of pathogenic bacteria detection.

Gas chromatography (GC) provides a novel possibility for rapid detection of microorganisms by measurement of their metabolites, for example acids, diacetyl, acetoin, and 2, 3-butanediol. We need to first plot a baseline of the beer volatile peaks; once this has been performed, we can trace the peaks of contaminated beer against that of pre-determined bacterial culture. GC allows us to detect microorganisms within a few hours. However, we need relatively high levels of contamination for it to be measurable.

Hybridisation is a molecular method, whereby a fluorescent labelled strand of a defined microorganism’s DNA combines with complementary DNA in an unknown sample. The resulting double strand of DNA is detected under a microscope as the target organism. FISH is an enhancement, where in situ hybridisation is possible on a glass microscope slide. Commercial measuring equipment and probes for all beer spoilage bacteria are available and can be combined with flow cytometry measurement.

Polymerise chain reaction, or PCR, is probably the most widely adopted rapid method of microbial detection in beer. There are many suppliers of desktop equipment and probes to choose from. DNA from a sample anneals to the organism probe DNA and is amplified up to a billion-fold through thermal cycling with DNA polymerase enzyme. During the amplification, DNA concentration is measured by fluorescence detection.

Importantly, brewery beer spoiler probes are readily available, meaning that this technology is easily accessible to most brewers.