Biological indicators, random growth. Is your decontamination cycle really at fault?

It is not uncommon, during performance qualifications of a decontamination system, to observe growth following incubation of biological indicators. The investigations conducted following these nonconformities are often long, and generally result in the cycle that has been developed being called into question.

Have you considered “Rogue BIs”?

Rogues : Illustration

1. What is a “Rogue BI”?

A so-called “Rogue” indicator is one that shows inexplicable and inconsistent signs of growth after incubation, during phases of performance qualification or of cycle development. This growth does not generally have a rational explanation, is not consistent over the cycles that are carried out and, above all, will sometimes be entirely contrary to the result that would logically be expected. These inconsistent results cause time to be spent on investigations to explain them, and delay the start-up or restarting of production equipment.

Here are a few approaches to be considered in the event that biological indicator results appear strange and difficult to explain.


2. Consequences of rogues during cycle development.

These can be very extensive. Without a valid decontamination cycle, production cannot be launched or relaunched after re-qualifications, involving significant cost to the manufacturers, without forgetting problems that may be raised by the regulatory authorities during audits.

Some indicator manufacturers recommend the use of triplicate indicators during qualifications (3 indicators in the same position), suggesting that if 1 indicator out of 3 grows in this position, this is de facto validated. Bioquell does not entirely agree with this procedure. A positive control cannot simply be ignored. Bioquell’s policy on this subject is to repeat different cycles in order to ensure that this growth can be imputed either to the cycle that has been developed, or to the quality of the biological indicator used.


3. What is the cause of rogues?

Bioquell has conducted a study involving several manufacturers that market these indicators, observing carriers with an SEM (scanning electron microscope). It is in fact difficult to study the causes that lead to rogue indicators, since that involves destroying them (by incubation). It is therefore necessary to be able to observe them before incubation. The results obtained suggest that these rogues may be caused by several specific problems.


3.1 Medium present in the inoculum (Image 1)


Generally caused by poor cleaning of the bacterial suspension before inoculation onto the stainless steel disc. The culture medium will then protect the spores from all contact with the biocidal product during the decontamination cycle, as the product cannot cross this protective barrier. The spores will therefore survive and grow when they are incubated.

Rogues : Image 1
Image 1. Spores imprisoned in the solid culture medium.


3.2 Damaged stainless steel disc (Image 2)


The carrier is a crucial constituent component of the indicator, and damage to the disc can help the spores to survive. This damage often takes the form of scratches, or recesses on the surface of the disc, where spores can find refuge, and so be protected during the cycle.

Rogues : Image 2
Image 2. Damage to the surface of the stainless steel disc.


3.3 Conglomeration or multiple layers (Image 3)


Most indicator manufacturers will insist on the importance of a single layer and uniform distribution of the microbial load on the disc. In fact, the stacking of layers of spores causes significant resistance by the indicator, since the biocide cannot traverse the multiple microbial layers, so leaving the deepest layers of microorganisms unexposed. (~biofilm).

Rogues : Image 3
Image 3. Multilayers of microorganisms.


4. What are the typical symptoms of rogue BIs?

4.1 Inconsistent results across cycles

The most significant and troubling effect of rogue indicators in the study, is without doubt the inconsistent results obtained across the cycles that are carried out, especially during a PQ.

Reminder: We will suppose here that a single batch of biological indicators is used. If several biological indicator batches are used, especially during performance qualification, or any other seasonal requalification, the results are not usable.

Case 1: Inconsistent results for several identical cycles with controlled, monitored parameters:

It may happen that on 3 consecutive runs, with the same reference cycle, and in identical environmental conditions, the results show marked inconsistencies.


For example: Growth in cycle 2 in a position which does not show signs of growth in cycles 1 and 3

  • Another position shows growth solely in cycle 1
  • A final position develops growth in cycles 1 & 3


After checking that the cycles were indeed identical in all respects, and that the environmental parameters were fully complied with, this variation in results may be explained by the presence of rogue BIs.


Case 2: Positive indicators with more powerful, supposedly more effective cycles:

After an unsuccessful series of tests leading to invalid results during qualifications, the instinct is very often to implement a more powerful decontamination cycle. What a disaster when new positions are perceived to be positive. A more powerful cycle is normally supposed to eliminate non-compliant positions observed during weaker cycles, and not to introduce new uncertainties. It is therefore not logical that new positions are positive when they were not positive with previous, weaker cycles.

4.2 Pousses non immédiates

Usually, a positive indicator shows signs of growth fairly rapidly (d+1 or d+2) after incubation. It is rarer to see indicators showing signs of growth at d+5 or d+6. This may be a sign of rogue indicators. In fact, some of the causes of rogues noted above show that they can sometimes be brought about by the superimposition of layers of spores, or by crevices on the carrier, allowing the spores to be protected. It is therefore possible that the phenomenon of growth at d+5 or d+6, demonstrates this artificial protection effect of spores that are not reached by the decontamination. The appearance of positives at the end of the incubation period can be explained by the fact that the upper layers of destroyed spores take time to pass into suspension in the broth, before being replaced by spores in the protected sublayers that are not destroyed. The culture medium therefore takes more time to reach these spores which are still active, hence the significant latency observed during incubation.

4.3 How should an indicator seen under the microscope be presented in order to reduce the risk of rogues? (Image 4)


The Bioquell approach to the prevention of rogues begins with the care given to preparation of the bacterial inoculum. Several cleaning and rinsing methods are practiced, to ensure complete removal of the culture medium and of vegetative cells before inoculation onto the stainless steel disc. Photos of the inoculum are also taken in order to be able to compare batches.

Rogues : Image 4
Image 4. Neat, regular, homogeneous distribution of spores spread across the stainless steel disc.


A new procedure has also been adopted to test Bioquell indicators. The KM100 test examines batch resistance, so helping to identify batches that are more resistant than the others. The aim here is to identify batches with a reference resistance (D-value) as close as possible to that recommended by Bioquell for use of its decontamination systems.


Bioquell also takes SEM images of a sample of indicators, for all marketed batches, in order to check the distribution and quality of the indicators released. These images are available to Bioquell customers on request, to help them with their quality procedures. This test is not a criterion for batch release, since just a small part of production is tested, but contributes to Bioquell quality criteria.

Nevertheless, despite all efforts made during the manufacture of indicators, rogues will unfortunately always exist. Indeed, indicators are manufactured from living biological material which is difficult to control completely. Bioquell’s aim is to reduce the proportion of rogues in marketed batches to the greatest extent possible.

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Jules Boulicot – Bioquell

Jules Boulicot, ingénieur commercial au sein de l’entreprise de Bioquell, est expert en en Désinfection de Surfaces par Voie Aérienne (DSVA). Il occupe un rôle majeur en gestion de projets et en intégration de solutions techniques dans les process critiques clients, pour de multiples acteurs majeurs de la production/R&D pharmaceutique et biotech (équipements et services).


HBEL: Health-Based Exposure Limits
PDE: Permitted Daily Exposure


1963 (US GMP regulation): Some cleaning consideration “Equipment shall be maintained in a clean and orderly manner”
1978 (US CFR): Section 211.67 added describing equipment cleaning and maintenance
1993 (US FDA guideline): Guide to inspections for cleaning validation2000 (ICH Q7): Guide for active pharmaceutical ingredients
2004 (US FDA guide): Guide to inspections validation of cleaning processes
2010 (PDA): TR 49 – Points to consider for Biotechnology Cleaning Validation
2010 (ISPE): Risk-Based Manufacture of Pharmaceutical Products
2012 (PDA): TR 29 – Points to consider for Cleaning Validation
2015 (EMA): Guideline on setting health based exposure limits for use in risk identification in the manufacture of different medicinal products in shared facilities
2015 (Eudralex Vol. 4 – GMP Guidelines Annex 15): Qualification and Validation