- tamsinmarshall4
- Apr 3
- 7 min read
Updated: Apr 4
Opinion Review Article | Open Access | Published 2nd April 2025
A spore in every pore? Cardboard and cleanrooms don’t mix
Tim Sandle, Ph.D., CBiol, FIScT | EJPPS | 293 (2024) | Click to download pdf
A standard part of the contamination control measures for transferring materials into a facility is that cardboard has no place in a cleanroom (or indeed any ‘controlled-not-classified’ area), with cardboard generally removed in airlocks prior to the transfer of consumables (or, as Kanegsberg and Kanegsberg recommend, ideally removed in an area outside of but proximal to the transfer area, with items then immediately moved into the transfer area)¹.
What is cardboard?
Cardboard is a very generic term for any heavy paper-based product. The construction ranges from fairly thick paper termed ‘paperboard’ through to ‘corrugated fibreboard’ (formed of multiple plies of material). Some forms are laminated, wax coated or treated for wet strength². Most cardboards are paper-based (formed from fibrous materials turned into pulp and bleached), although some can be wood-based. The main polymers are cellulose, hemicellulose and lignin, and it is cellulose that many microorganisms are able to biodegrade³.
Various technical specifications are applied to cardboard, including moisture content (which is typically between 6.5 to 9.5%) and selected as means of assessing material strength, with low moisture a means to reduce the compression strength; edge crush based on force per unit width; box compression strength; burst strength; flat crush rigidity; bending resistance; impact resistance; cushioning, shock absorption; tear resistance; and overall grammage (weight per meter square).
Microbial load
While the papermaking process drastically reduces the total microbial counts, it does not reduce the bioburden down to a significantly low level for a controlled environment. One study showed that untreated paper pulp contains between 10^8 and 10^9 CFU (per gram dry weight, of which 10^2 to 10^4 were spore forming bacteria); after cardboard has been manufactured, this reduces down to between 10^3 to 10^6 per gram (dry weight). In pulp, the predominant organisms were mesophilic and thermophilic actinomycetes⁴.
Different studies into the microbial load of cardboard packaging show that there is some variation based on the different chemical/physical characteristics of the material. Other variables include storage, including environmental conditions.
With microbial loads, there are differences between new and reusable packaging. With reusable cardboard, this generally presents aerobic mesophilic loads between 10^3 to 10^6 CFU/cm^2⁵, while virgin fibre packaging presents cell loads ranging between 10^2 and 10^5 CFU/cm^2⁶. Of the vegetative (albeit some spore forming) bacteria of concern, the extracellular enzymes of actinomycetes are perhaps most adept for degrading cardboard⁷.
In terms of more problematic species, several microorganisms detected on cardboard packaging surfaces include spore forming bacteria such as Bacillus spp. and Clostridium spp., together with moulds such as Aspergillus spp. and Cladosporium spp⁸,⁹. Of endospore bacteria, organisms in the Bacillus cereus group appear to be far the most common¹⁰.
In the most serious instances of fungal contamination a colour change will occur due to pigment diffusion (dye migration from the mycelium to the paper), for example Penicillium will create yellow spots; Fusarium or Rhodotorula, pink; and Alternaria or Cladosporium, grey or green). However, Aspergillus species are seemingly the most destructive damagers of paper and cardboard¹¹.
pH is an internal factor that plays an important role in the types of microorganisms that might be found. Bacteria prefer alkaline conditions (pH 6.8-8.0), whereas fungi grow better under acidic conditions (pH 4.0-6.0).
As well as cardboard itself, additional contamination will arise from:
Personnel.
Contact with other objects.
Atmospheric air.
Building air (including organisms common to the built environment:
Aureobacterium, Bacillus, Cellulomonas, Micrococcus, Sarcina, Staphylococcus).
Dust (such as grains of sand, street dust, black carbon particles, plant origin fragments, heating systems, wall degradation, carpets, and human skin).
Time
Over time, as a result of microbial metabolism discolouration, decomposition and reduction of the strength of paper. Time is highly variable and dependent upon the cardboard and the microorganisms; however, inevitably, progressive biodeterioration will occur.
Susceptibility to attack by microbial agents also depends on the excipients added to the cellulosic material in the paper making process e.g. glues, dyes, and fillers; these can provide additional food sources and accelerate the process.
Surface porosity
Surface porosity is an influencing factor. Many microorganisms are like golf balls rolling into a field of holes and here they more easily sink into porous materials – such as cardboard, wood or cloth.
What happens then will vary, depending on the material and the microorganism. Many vegetative organisms, sucked into a porous surface might not be able to transfer to anything that comes into contact with the surface. In addition, moisture tends to be drawn away from the surface, which makes it less friendly to vegetative cells. However, spore-forming bacteria will survive in such conditions which is one reason why cardboard is a concern in relation to bacterial endospores.
In addition, non-porous surfaces are easier to disinfect and will hold less liquid.
Corrugation
Another factor is corrugation. A piece of research evaluated both temperature and time to determine if typical corrugated manufacturing processes, which combine a fluted or arched layer of paper sandwiched between two smooth layers, were sufficient for decontamination. The study employed a temperature and time profile representative of manufacturing practices where linerboard reaches temperatures of 80 to 95^C for approximately nine seconds. That profile was attained in the laboratory by placing corrugated material between two 2.5 centimetre thick, preheated aluminium plates for the specified time. Under these conditions, linerboard contaminated with a mix of various thermotolerant vegetative organisms reached the specified temperature for the identified time resulting in a five-log reduction in the number of organisms present on the liner surface. This met the US EPA’s defined requirement for sanitization.
Such temperatures would not be effective in the case of most endospores, but they do suggest that as a starting material, corrugated cardboard will be of a lower bioburden than non-corrugated material.
Storage
Another key factor is storage, especially in terms of increasing the bioburden through microbial growth or additional contamination. Temperature, relative humidity and light play a crucial role in terms of increasing or decreasing microbial growth.
Paper and cardboard can readily absorb water from air. The water content in cardboard is one of the main factors determining the rate of colonization of the paper surface by microorganisms.
Based on this, one study demonstrated the importance of storage and supply chain conditions (hygiene and low humidity) to prevent the mould growth and the increase of their role in the cross-contamination of materials contained inside¹². Reducing air humidity can be achieved by raising the temperature (these parameters are closely related). However, prolonged exposure to high temperatures leads to a reduction in paper strength and a decrease in pH (factors which can leave the material more susceptible to biodeterioration).
The most important humidity factor is that nothing that is wet should enter the facility. Ideally materials coming in from a delivery lorry or other transportation from a warehouse should be completely protected from the elements. Such materials should remain in entry airlocks, with circulating air, until they are dry.
What is ideal? One area where there has been extensive research is with archives. For instance, German researchers determined that archives have a recommended air temperature of 15°C (13°C -18°C up to 25°C) and a relative air humidity of 45% (40-50%), creating optimal conditions that minimize microbial growth¹³.
Disinfection
Paper and cardboard can be disinfected but the methods are complex and expensive. Certain forms of paper can be autoclaved (a common way of having paper in cleanrooms) and cardboard can be subjected to irradiation or a gas or vapor decontamination process. These processes are difficult to verify and complicated to establish. For a standard cleanroom, this begs the question of why go to this trouble? Simply don’t have the material entering the facility in the first place.
Other materials
As indicated above, the introduction of cardboard into cleanroom facilities should be avoided. In contrast, laminates, metal foils, and blister-pack materials all have smooth impervious surfaces with a high-temperature stage employed in their manufacture and, therefore, have low surface microbial counts¹⁴.
What can we learn?
In some ways this article is simply cementing good practices:
Do not deliver items to facilities when it is raining or where they are not properly protected.
Incoming items should ideally have the first layer or packaging removed.
Items should be placed into an airlock, with circulating air, to ensure they are dry.
Cardboard should be removed and discarded prior to transfer to a controlled-not-classified area.
Cardboard must not be presented to a cleanroom.
Sprodicial transfer disinfection should be established and in place to move items into the cleanroom.
For all stages, temperature and humidity should be controlled, with low humidity conditions ideal.
It may also be the case that if you have fungi and or actinomycetes in your cleanroom, or perhaps Bacillus species, then it is worth verifying if cardboard is entering and the potential for cross-contamination from the cardboard is being controlled. These simple steps can help reduce the level of external bioburden entering the facility.
The revised and updated second edition of Tim Sandle’s book ‘Biocontamination Control for Pharmaceuticals and Healthcare’ is now available.
References
01. Kanegsberg, B., Kanegsberg, E., and K. O’Donoghue, K. Keeping Product Clean In and Out of the Cleanroom, Part 1: The Interface, Controlled Environments Magazine, Feb. 2009
02. ASTM D996-16 Standard Terminology of Packaging and Distribution Environments, 2023
03. Mitchell R., McNamara C.J. (2010). Cultural heritage microbiology. Fundamental studies in conservation science. ASM Press, Washington
04. Suihko, M-L, Skyttä, E. A study of the microflora of some recycled fibre pulps, boards and kitchen rolls, Journal of Applied Microbiology,1997, 83 (2): 199–207
05. Binderup, M., Pedersen, G. A., Vinggaard, A. M., Rasmussen, H., Rosenquist, H., and Cederberg, T. (2002). Toxicity testing and chemical analyses of recycledfibre-based paper for food contact. Food Addit. Contam. 19, 13–28
06. Suominen, I., Suihko, M. L., and Salkinoja-Salonen, M. (1997). Microscopic studyof migration of microbes in food-packaging paper and board. J. Ind. Microbiol.Biotechnol. 19, 104–113
07. Stobińska H. (1976). Microbial decomposition of raw materials and paper products. Polish Paper Review 37:422-424
08. Patrignani, F., Siroli, L., Gardini, F., and Lanciotti, R. (2016). Contribution of two different packaging material to microbial contamination of peaches: implications in their microbiological quality. Front. Microbiol. 7:938
09. Turtoi, M., and Nicolau, A. (2007). Intense light pulse treatment as alternative method for mould spores destruction on paper–polyethylenepackaging material. J. Food Eng. 83, 47–53
10. Outi Priha, Katri Hallamaa, Maria Saarela, Laura Raaska, Detection of Bacillus cereus group bacteria from cardboard and paper with real-time PCR, Journal of Industrial Microbiology and Biotechnology, 2004, 31 (4): 161–169
11. Nyuksha Yu P. (1994). The biodeterioration of paper and books. [In:] Garg K.L., Garg N., Mukerji K.G. (Eds.) Recent advances in biodeterioration and biodegradation. Naya Prokash, Calcutta, pp. 1-88
12. Siroli, Lorenzo & Patrignani, Francesca & Serrazanetti, Diana & Chiavari, Cristiana & Benevelli, Marzia & Grazia, Luigi & Lanciotti, Rosalba. (2017). Survival of Spoilage and Pathogenic Microorganisms on Cardboard and Plastic Packaging Materials. Frontiers in Microbiology. 8. 10.3389/fmicb.2017.02606.
13. Zyska B, Żakowska Z. (2005). Materials microbiology. Lodz University of Technology, Łódź. (in Polish).
14. Payne, D.N. Microbial Ecology of the Production Process. In Denyer, S. and Baird, R.M. (Eds.) Microbiological Control in Pharmaceuticals and Medical Devices, CRC Press, Boca Raton, 2007: 60-61
Author Information
Corresponding Author: Tim Sandle, Head of Microbiology
Bio Products Laboratory ,
UK Operations, England
Email: timsandle@btinternet.com
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