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European Biopharmaceutical Review

Detection Validation

One of the most important aspects of a prevention strategy involves monitoring the presence of bacteriophages in fermentation and the facility environment. Is that an easy task which can be done in the correct way by a microbiologist, or do you need a set of superpowers in order to detect phage? The answer is that being a microbiologist is not usually enough, unless they are extensively trained in phage detection techniques. Superpowers are not necessary, however. Deep knowledge and a few years of practice in work with bacteriophages should suffice in most cases.

Problems in Phage Detection

The majority of virulent phages are relatively easy to detect. However, even phages which produce the most evident contaminations – often found in fermentation T1-like phages – can sometimes be difficult to detect. Detection of phages from other groups of bacteriophages – which are less frequent in fermentation, yet when taken together still attack bioprocesses with similar frequency – may in some situations prove challenging even for those with extensive phage detection experience.

The problems become even more pronounced in the case of temperate phage detection. One particular issue is the inability to efficiently induce some prophages using the most common methods, such as by triggering SOS regulon. This method is effective in the induction of prophages belonging to the lambdoid group, for example, but it fails when used with P2 prophages groups, which are much more dangerous. The problem is that, in most cases, SOS regulon-based methods are used as a sole prophage induction test, but this approach cannot guarantee accurate results.

Some phages, even if properly induced, cannot be detected using standard plating methods (1). The use of innovative new methods and customisation is becoming necessary.

One of the best indicators that use such methods is the observation of liquid culture behaviour and appearance, plus its comparison to usual parameters. Each deviation, such as cell aggregation, slow growth, foaming and extensive biofilm-like structures formation, may be considered a warning signal and will require the use of more sensitive methods if standard ones produce negative results. However, microbiologists must be aware that liquid culture observation may be misleading if a strain is used, which can have certain mutations or express products that change cell parameters. This could take the form of, say, mutation or growth conditions, which change the amount of Ag43 on the surface of E. coli, and which in turn may increase their tendency to form aggregates.

There are several possible problems which may cause false negative or inconclusive results. The most frequent are technical errors made during sample handling and test execution. It is very common for people who are not experienced in phage detection techniques to try to reproduce them from textbooks and scientific publications, and will not produce them in the correct way. When one considers how few microbiology courses contain any phage handling and detection practice, it becomes easier to understand how even skilled microbiologists sometimes find phage detection difficult. Paradoxically, the situation can be worse in facilities which do not suffer frequently from phage contamination, due to the fact that staff will not have not worked enough with phage-contaminated samples and lack the necessary skills needed to perform the task correctly.

Another frequent mistake is the wrong choice of indicator strain. An incorrect selection will affect phage host range patterns, which is a great concern to those who wish to research phage therapy. Anyone who is employed to try to detect phages should understand that the majority of phages have narrow host ranges, and many of them are specific to a single bacterial strain. To make the situation worse, some strains can gain resistance (partial or full) to individual or, occasionally, groups of phages. Our laboratory found that some bacterial strains, theoretically identical but obtained from different sources, possess widely varying phage resistance patterns. The reason for this is the inconsistent histories of sister strains between each lab. Some may even contain prophages which have been introduced accidentally, causing additional confusion and, in some cases, false negative results (2).

To Validate or Not to Validate?

Validated methods are useful because they have been proven to work in the situation used in the test, but nonvalidated methods also work well because of their flexibility. At first glance, validated tests look more convincing.

They demonstrate the ability to detect phage and standardise the method used, so it is very reproducible and makes it easier for anyone to perform this test, even if they are not familiar with the method. On the other hand, validated methods do not allow for too much flexibility, which can have a great impact on phage detection. An improper choice of indicator strain means that one may generate false negative results for the entire test, as we have seen. Additionally, it is important to choose phages which will validate the methods used, which can hugely influence test performance. If it proves too easy to detect phage – for example if T1 is chosen – the test may not be sensitive enough to detect slowly growing phages with poor plaque formation kinetics. Thus, the tests should be optimally constructed in a way that allows for detection of problematic bacteriophages.

With non-validated methods, the probability of phage detection is higher when two conditions are fulfilled: firstly, when the test is performed by a person who is experienced in detecting various types of phages; and secondly, when a test is constructed it has been dedicated to particular bioprocess and situations. Where customised tests are concerned, the probability of making an error rises dramatically for unskilled personnel; however, in the correct hands the probability of phage detection also dramatically increases.

So the question remains: which method to choose? The answer depends on the situation. If one wants to perform in-house phage testing, then the validated method is definitely the best. The ideal solution is to construct and validate separate tests for each fermentation process conducted in the facility, even if only one bacterial species is used. The only exception may be when different processes utilise the same bacterial strain and the only variant is plasmid insert. In this situation, one universal method can be developed for all processes.

Of course, when any authority requires validated tests, there is a very strong argument to choose validated procedures, even if their performance is sub-optimal. If a facility outsources its phage testing, it can be a good idea to ask the service provider how much the construction and the validation of the method will cost in order to perfectly fit the process utilised. It may be quite affordable when the long-term business relationship with the service provider is considered, and the costs may prove lower than the possible impact of undetected phage contamination; thus, this relatively small investment may prevent a more expensive phage outbreak recovery.

Customised methods are highly recommended in a few specific situations. One example is when tests are to be provided for facilities utilising large amounts of different strains, such as contract manufacturing organisations. In such cases, the idea that ‘one test fits all’ will not work, and our recommendation is to look for a service provider with a good record of customised phage testing. As such tests are performed to protect your fermentation – and the business in general – the right choice of laboratory testing is crucial.

Another situation where customised tests are recommended are in emergency scenarios when phage tests are important in identifying the source of a process failure, as well as defining the nature of the situation within the facility. As test results are the basis for further, sometimes expensive, action to correct any errors, the option of customised tests should be chosen as they provide greater accuracy, especially in the detection of low-level phage contamination.


Phage detection tests are not easy to perform in the correct manner. The risk of generating false negatives, and sometimes even false positives, is ever present. False negatives can allow for phage to spread within a facility and cause an outbreak, which in turn can paralyse productivity for an extended period. Another possibility is for undetected phage to contaminate the final product, which may cause the need for product recall. At first glance false positives do not seem so dangerous; however, they could trigger unnecessary and quite expensive decontamination procedures, which usually causes the temporary stop of production. Choosing the type of test to be performed, whether outsourced or completed in-house, should be strictly dependent on the process samples from which they originated. It is important to avoid tests which are not optimised for particular processes, as the use of such tests may easily produce false negative results.


1. Los JM, Golec P, Wegrzyn G, Wegrzyn A and Los M, Simple method for plating Escherichia coli bacteriophages forming very small plaques or no plaques under standard conditions, Appl Environ Microbiol 74: pp5,113-5,120, 2008

2. Rotman E, Amado L and Kuzminov A, Unauthorized horizontal spread in the laboratory environment: the tactics of lula, a temperate lambdoid bacteriophage of Escherichia coli, PLoS ONE 5(6): e11106, 2010

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Marcin Los is CEO of Phage Consultants. He has an MSc from the University of Bradford. Marcin spent one year at the Fraunhofer Institute for Silicon Technology in Itzehoe, Germany, as a researcher involved in the construction of novel virus detection methods, before obtaining a PhD in 2004 and a DSc in molecular biology from the University of Gdansk, Poland in 2011. He was Secretary of the Main Board of Polish Genetic Society and is currently an Associate Professor at both the University of Gdansk and the Institute of Physical Chemistry in Warsaw.

Marcin Los
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