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People in Cleanrooms: Understanding and Monitoring the Personnel Factor | IVT

Peer Reviewed: Cleanroom Contamination


ABSTRACT

Cleanroom contamination can arise from a number of sources. Most contamination within the pharmaceutical facility can be traced to humans working in cleanrooms. The paper discusses staff gowning and personnel behavior in pharmaceutical cleanrooms, and how cleanroom risk can be minimized. The human skin ecosystem is discussed. The Human Microbiome Project (HMP) from the US NIH characterized microorganisms found in association with both healthy and diseased humans. Information from this project has great impact on cleanroom activities including gowning practices. Topics associated with cleanroom garments are discussed including fabric types, garment lifespan, recycling, laundering, human changing procedures, training, behavior, hand sanitization, ongoing assessments, and associated topics.

 

INTRODUCTION

Cleanroom contamination can arise from a number of sources. These may vary depending upon the type of cleanroom, its geographic location, the types of products processed, and so on. Nevertheless, these sources can generally be divided into the following groups1:

  • People
  • Water
  • Air and ventilation
  • Surfaces
  • Transport of items in and out of clean areas 

Most contamination within the pharmaceutical facility can be traced to humans working in cleanrooms2. This is, in some way, evidenced from the association of microorganisms transient or residential to skin being the primary isolates from environmental monitoring in controlled environments3. Human personnel shed high numbers of skin cells mostly as skin flakes. The cleanroom garments worn by personnel cannot contain all human detritus.

The paper discusses staff gowning and personnel behavior in pharmaceutical cleanrooms. Further, it considers how cleanroom risk can be minimized. Basic training for all cleanroom staff including activities such as cleanroom entry and gowning practices is examined.

 

HUMAN SKIN

Before proceeding to look at gowning, it is worthwhile to consider the human skin ecosystem. The human body is an intricate system that hosts trillions of microbial cells across the epithelial surface and within the mouth and gut. These microorganisms play a role in human physiology and organ function including digestion and immunity. The microorganisms also affect the outside environment as they are shed from the skin or deposited through different orifices. This latter issue has important implications for cleanrooms. The outer layer of human skin can host up to one million microorganisms per square centimeter4. The population, as well as the diversity, varies according to anatomical locale.

 

TABLE 1. HUMAN BODY SITES AND TYPICAL NUMBERS OF MICROORGANISMS WITHIN AREA

AREA

NUMBER OF MICROORGANISMS/cm2

Scalp

1 million

Saliva and nasal fluid

10 million/gram

Back

100

Groin

1 – 20 million

Forehead

100 – 1000

Hand

10,000 – 100,000

Armpit

1 – 10 million

Feet

1 million

 

Research suggests that a typical person sheds 1,000,000,000 skin cells per day of a size 33 µm x 44 µm x 4 µm -- equivalent to a rate of 30,000 to 40,000 dead skin cells shed from the surface of the skin every minute. Of these, Whyte indicates that approximately 10% of particles carry microorganisms5. There are, on average, four microorganisms per skin cell. A term commonly used to describe skin flakes with adhered microorganisms is “microbial carrying particles.”

The significance of this is that people are not only a source of contamination, but also are an agent for transferring contamination possibly to locations that could pose a product risk. Microorganisms are spread from sneezing, coughing, and touching. While microorganisms suspended in the air are less of a concern, should such organisms gravitate towards a product or critical location, they may present a significant risk.

Human Microbiome Project

Our understanding of the risk the people pose to cleanrooms has been advanced by the findings from the Human Microbiome Project. The Human Microbiome Project (HMP) is a United States National Institutes of Health initiative. The project goal is to identify and characterize the microorganisms found in association with both healthy and diseased humans (the human microbiome). The human microbiome describes the aggregate of microorganisms and their genetic interactions that reside on the surface and in deep layers of skin, in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal tract.

Some of the most illuminating HMP research has been with the human skin. The skin is a complex ecosystem, supporting a range of microbial communities that live in distinct niches. These niches are affected by available nutrients as well as by several non-nutritional factors such as pH, humidity, and temperature. With the skin, however, as epithelial cells are continually being shed, many microbial communities on the external surface are rarely stable6.

The outcomes of the HMP research have shown that there is a high population on, and a considerable diversity of microbial species across, the outer layer of the skin. There are approximately 1000 species of bacteria from 19 phyla on human skin. Of these, most bacteria can be categorized into four phyla: 

  • Actinobacteria (51.8%). Actinobacteria are a group of Gram-positive bacteria with high guanine and cytosine content, such as Micrococcus, Corynebacteria and Propionibacteria.
  • Firmicutes (24.4%). This includes the genera Clostridia and Bacillus.
  • Proteobacteria (16.5%). This is a major phylum of bacteria that includes a wide variety of pathogens such as Escherichia, Salmonella, Vibrio, Helicobacter, and many other notable genera.
  • Bacteroidetes (6.3%). The phylum Bacteroidetes is composed of three large classes of Gram-negative, nonspore-forming, anaerobic, and rod-shaped bacteria7.

Reasons for the topographical variations relate to the physicochemical properties of the skin. This is especially so for temperature, pH, amounts of oil, and moisture8. From this, there are three main ecological areas of the skin: sebaceous, moist, and dry. Examples of microbial divergence include Propionibacteria and Staphylococci species dominating the sebaceous areas (with a high oil content). Dry, calloused areas (arms and legs), Gram-positive cocci (primarily the Micrococcaceae) are found on the arms and legs and Gram-positive rods are found in high numbers on the torso. Staphylococci and Corynebacteria are found together with some Gram-negative bacteria in moist areas9. These types of microorganisms generally reflect the types recovered from cleanrooms10.

The reason that Gram-positive bacteria predominate across the skin is because the skin is generally a dry environment, and any fluids present on the surface generally have a high osmotic pressure11. Thus Gram-positive bacteria (especially the Staphylococci and Micrococci) are better adapted for such environments, not least to being resistant to desiccation. Where other species occur this is due to variations in temperature and with areas of higher sweat production12. For example, this can lead to higher levels of fungi on the feet13. In relation to pharmaceutical manufacturing, the presence of any such organisms remains problematic.

A further observation is that the ratio of the microorganisms recovered from the skin is relatively evenly divided between the aerobic and the anaerobic. The aerobic microorganisms tend to live on the outermost layers of the skin and the anaerobic microorganisms live in the deeper layers of the skin and hair follicles14.

The information from the human microbiome project about the rich depth of variety of microorganisms on the skin introduces several implications for cleanroom environmental monitoring. The most important question of whether gowning practices are adequate to exclude all microorganisms from the richest areas of the skin microbiome. This is a pertinent point given that most bacteria free-floating in cleanroom air current are not free-living but are instead the result of direct particle shedding of desquamated skin cells and subsequent re-suspension of skin detritus in the air stream.

The answer to this question should lead to a consideration of:

  1. The types of cleanroom undergarments used and an examination as to whether these provide an effective barrier, especially for the more moist parts of the body.
  2. The importance of the outer gown covering all parts of the body, including the forehead.
  3. The quality of cleanroom certified undergarments.
  4. The level of training required for operators in relation to gowning and the way that gowning qualification as conducted.
  5. How long a cleanroom suit should be worn for in relation to material integrity against operator perspiration.
  6. The environment in which gowns are donned, where higher air-change rates might prove effective.
  7. How often gowns should be recycled which involves washing and irradiation. At some point the material fibers will weaken, thereby reducing the bacteria filter efficiency of the gown. The wearer of the gown should know what types of testing is conducted on recycled gowns and what the procedures are for rejecting gowns where a loss of integrity is detected.

Cleanroom microbiologists may wish to consider how concerned they are with each of the items listed in excluding microorganisms found on all regions of the skin. There must be good understanding of the environmental monitoring methods used to assess cleanrooms. These may not show how good or bad gown changing and gown wearing is. These concerns are best addressed through good gowning practices.

 

PROTECTING PRODUCTS FROM PEOPLE

Despite some advances with automation and robotics, in most situations people cannot be eliminated from cleanrooms. Control of contamination from people in cleanrooms is achieved by application of two principles: 

  • We “wrap” the people to minimize the amount of “shedding” of microorganisms.
  • We put localized protection around the product to minimize the amount of contact with the people. 

The localized protection issue is achieved through local air protection, such as unidirectional airflow cabinets and isolators. With clothing, personnel working in cleanrooms are required to wear special clothing designed for the clean environments. Such clothing is necessary, as indicated above, because the human body creates its own micro-environment of potentially damaging particulate contamination.

To be effective, cleanroom clothing must:

  • Form a particulate barrier for the human micro-environment.
  • Allow freedom of movement and be comfortable.
  • Address any specialist requirement, e.g. static dissipation.
  • Avoid being a significant particulate contributor in itself.

Cleanroom garments must meet specific protection criteria. Not all cleanroom garments are of the same quality. This involves manufacturing the garments from special materials, following particular construction methods, and then tailored for individual styling. The gowns must be comfortable, easy to apply and practical in use. Some gowns are disposable and others are made to be re-laundered and sterilized depending on the cleanroom grade.

 

CLEANROOM GARMENTS AND FABRICS

Fabrics used in the manufacture of cleanroom garments must have the following features15:

  • Be low in particulate shedding.
  • Permit the body to breathe while trapping particles within the garment. The contaminant should be retained within the garment and not released into the surrounding atmosphere.
  • Be flexible enough for comfortable wearing.
  • Withstand repeated cleaning and sterilization cycles.
  • Meet any specific requirements such as control of static.
  • Meet opacity requirements.
  • Look and feel as good as possible.
  • Be cost-effective.

Fabric Categories

There are three broad categories of fabric used in the construction of cleanroom garments:

  • Woven fabrics. Woven or re-usable fabrics are the most commonly used fabrics in cleanroom environments. Such garments are woven on sophisticated looms from yarns of continuous filaments of polyester. The thickness of the yarn and filaments is important -- the finer the yarn, the tighter the weave can be made, and the better the filtration. The pattern and tightness of the weave is important to reduce the pore size to a minimum. The use of continuous filament polyester means that there are few loose ends from which particles may be shed.
  • Laminated or membrane fabrics. Laminated fabrics are favored for some high-grade microelectronic environments. These types of garments are not commonly used in the pharmaceutical sector.
  • Disposable or limited life materials. The most common of these non-woven fabrics are from spun bonded olefin and polypropylene. Comprising a densely interlinked matt of fibers, these fabrics can provide good results for a limited period. Garments from such materials need to be processed and decontaminated before use in the cleanroom. Disposable or limited use garments are mainly used in those environments where protection of the wearer against potentially hazardous products is required. 

Garment Considerations

Garments are designed to provide protection for the head, body, hands and feet. In establishing a system for garment selection, it is important to consider the broader aspects of cleanroom use: suitability of fabric, garment style, layers, the nature of the tasks involved, costs, regulatory requirements, and any specific customer requirements. The classification of the cleanroom will inevitably be the major factor in determining the degree of personnel protection required and the fundamental choice of garments.

One important issue with gowns is the maximum length of time that a gown can be worn. As people perspire, the integrity of the gown will weaken. Complicating factors are the temperature and humidity of the cleanroom and the variations between people. The length of time will also depend upon the grade of the cleanroom. In aseptic areas, such as ISO 14644 class 7 / EU GMP Grade B areas, gowns are typically worn only for the length of the shift (normally four hour periods to enable operators to take breaks). In lower grade cleanrooms, a gown might be worn for several sessions during the course of the working day.

Other factors affecting the lifespan of the gowns that are subject to recycling are repairs and the number of permitted washing cycles. With repairs, it is prudent to have a repair policy. This will vary across facilities, and again, it will be affected by the cleanroom class. With aseptic areas, if a gown becomes torn it is normally discarded. In other grades of cleanroom, a gown can be repaired depending upon the size of the hole and the impact on the material. Some organizations set a maximum size for any hole or tear and for the number of times a gown can be repaired.

Gowns that are recycled are subject to laundering. Gowns are washed by special washing machines with suitable detergents, dried, folded, and then wrapped in cleanroom packaging. For gowns that are to be used in aseptic areas, such gowns are irradiated. A policy should be in place outlining how often a gown can be processed -- typical times range between 20 and 40 times. To make the tracking task easier, many gowns sterilized by irradiation or gassing are fitted with barcodes and scanned. It is further important to establish the extent that the sterilization process affects the integrity of the gown material.

In order to assess the contamination risks from re-laundering, gowns are subject to particle counting. There are different ways to do this, although the most common means is the Helmke Drum particle emission test. With this, the test method simulates particle shedding of clothing under movement. The garment under test is tumbled in a rotating drum (approximately 10 revolutions per minute) to release particles from the surface of the cleanroom garment in a controlled manner. An automatic particle counter is used to sample the air within the drum to determine the average particle concentration of the air during the initial ten minutes of the test. The common standard is the IEST "Recommended Practice RP-CC003.3: Garment System Considerations for Cleanrooms and Other Controlled Environments".

An alternative measure is the Body Box test. This method simulates particle filtration and release under real wear conditions. As a consequence it measures the contamination of the cleanroom by the clothing/wearer. For this, particle counters determine the quantity of particles generated by the wearer/garment that are emitted into the chamber.

 

CHANGE PROCEDURE

Cosmetics, such as powder, rouge, eye liner, mascara, and lipstick must be banned in cleanroom environments. Jewelry, such as rings, watches, necklaces, bracelets, earrings and other items, together with all forms of visible piercing, are commonly not allowed in cleanrooms.

The best method of changing into cleanroom garments is one that minimizes contamination getting onto the outside of the garments. Change areas can vary in design, but it is common to find them divided into three zones:

  1. Pre-change zone. Outside of changing rooms 'tacky mats' or polymeric flooring can be positioned to help reduce the level of particles carried on footwear16.
  2. Changing zone. The changing room design contributes to the assurance of appropriate personnel access and microbial contamination control. The changing room should be provided with filtered air. Intermediate (bag) filters will typically be suitable for this purpose, though High Efficiency Particulate Air (HEPA) filtration may be used. The air pressure should be negative with regards to the manufacturing area corridor, but positive relative to external adjacent areas.
  3. Cleanroom entrance zone. This must be of the same grade or class as the main cleanroom into which the area leads.

Ideally there should be separate routes through airlocks for material required in cleanrooms. Taking items through personnel change areas should be discouraged.

 

TRAINING

Personnel training in gowning is an important function. Gowning practices must be assessed periodically and monitored frequently. Training programs should ideally include visual assessment and microbiological assessment. The microbiological assessment varies, but can include the exposure of settle plates during the change process and the assessment of gown cleanliness through post-use suit contact plates. The results of the cleanroom sampling should not exceed those of the room class. If results are exceeded, the individual may be an unusually high shedder of skin particles.

Training required for staff who work in cleanrooms should include:

  • Introduction to micro-organisms and microbiological contamination control.
  • Entry and exit of production facilities (including gowning).
  • Personal hygiene training.
  • Microbiological risks associated with specific production tasks.

Training must be documented and regularly reviewed. Training must be effective. Actual performance of personnel competency in gowning should be demonstrated on a regular basis.

 

CLEANROOM PERSONNEL BEHAVIOR

Working in clean environments demands knowledge, discipline, motivation as well as a thorough understanding of contamination risks among all personnel involved. Each individual cleanroom should have its own documented rules and procedures.

Training includes reminding personnel that they must not be allowed to touch critical products and equipment with their naked hands. All critical work must be undertaken wearing gloves. Critical activities requiring personnel contact such as aseptic processing or sampling must be done through the use of clean utensils such as tweezers, forceps, and the equivalent. All devices and gloves used must fully comply with the cleanliness demands of the cleanroom and work undertaken in the cleanroom. They must be cleaned, disinfected, or sterilized as appropriate for the criticality or activity and risk of contamination.

Another aspect of best practice is in instructing personnel in the appropriate behaviors within the cleanroom. The generation of contamination is proportional to activity conducted. A person with head, arms, and body moving can generate about 1,000,000 particles ≥ 0.5 µm/min. A person who is walking can generate about 5,000,000 particles ≥ 0.5 µm/min. However a person in motionless position can generate only 100,000 particles ≥ 0.5 µm/min. In addition, personnel should reduce activities like talking, singing, whistling, coughing, sneezing etc., especially when being close to the handled products and production equipment.

People working in cleanrooms and other forms of controlled environments must be physically healthy. Diseases in the upper respiratory tract as well as stomach disorders can create problem in hygienic applications. 

Another factor that can impact upon the environment is the number of people in the cleanroom. Only necessary and limited number of persons should be allowed in a cleanroom at the same time. The more persons simultaneously present in a cleanroom, then the higher the contamination level will be, i.e., the higher concentration of particles in the air). This is particularly important in relation to changing rooms.

 

HAND SANITIZATION

Good personal hygiene is a requirement of all pharmaceutical cleanroom activities. However, studies show poor compliance is common in relation to basic hand washing technique. Hand hygiene and glove hygiene are important given the high numbers of microorganisms found on the human body including the hands and the risks posed by hands as a means of contamination transfer. Microorganisms associated with hands are found mainly on the surface of the skin and under the superficial cells of the stratum corneum. The dominant species is Staphylococcus epidermidis that is found on almost every hand, together with other species of Staphylococcus and species of the genera Micrococcus17.

Hands must be washed with soap and water prior to entry to the cleanroom. Hand washing facilities should not be located in an actual cleanroom, but rather in an area leading to the cleanroom changing room. As an alternative, a hygienic handrub can be used.

Where gloves are required these should be put on using a method designed to prevent the ungloved hand from touching the clean or sterile outer part of the glove. Once in the cleanroom, gloved hands should be subject to periodic hand sanitization18.

 

 

 

FIGURE 1: HAND SANITIZATION (IMAGE: TIM SANDLE)

 

When decontaminating hands with an alcohol-based antiseptic hand rub, apply product to palm of one hand and rub hands together, covering all surfaces of hands, fingers and wrists, until hands are dry (alcohol-based hand rubs are not to be used with water). The process typically takes between thirty seconds and one minute. Follow the manufacturer’s recommendations regarding the volume of product to use.

The technique for applying alcohol to gloved hands is similar to applying a handrub to skin. It is important to ensure that all surfaces are covered. With glove sanitization, there are two alcohols of choice: ethyl alcohol (ethanol) and isopropyl alcohol (IPA). Other alcohols, such as methyl alcohol (methanol) are unsuitable19. Of the two alcohol forms, IPA is slightly more bactericidal than ethanol, although ethanol has better viricidal properties20. Another factor is application to the skin, and here IPA can be quite harsh. Thus, ethanol is more often applied to bare skin (often in a denatured form) whereas IPA is more often applied to gloves.

These sanitizers have bactericidal action against vegetative cells but not spores. The concentration of alcohol to water varies, although the optimal range is 60 to 90% (volume/volume). Below 60%, bactericidal action drops, and above 90% there is insufficient water for the bacterial cell to absorb water. The alcohol does not enter the cell and is unable to denature the bacterial proteins21. Most preparatory concentrations are 70%. 

While most bacteria are killed after ten seconds of contact with alcohol22, contact times in practice are longer due to the variability of hand rubbing. Typical contact times are thirty seconds. 

It is important that the selection of a hand sanitizer is qualified. There are different approaches that can be taken for qualification. Most of these require individuals to wear gloves and to place their hands into broth containing a high concentration of a non-pathogenic microorganism. Disinfectant is then applied, and the bacterial reduction is assessed through placing the treated hands into broth and performing dilutions.

 

ON-GOING ASSESSMENT

In higher-grade cleanrooms such as those used for aseptic processing, it is common practice to assess the risk of personnel to the process by taking suit contact plates of the gown as worn by the person as they leave the aseptic area. The gown must be discarded after the plates have been taken due to the potential effect on the gown integrity when an agar plate contacts the gown. 

It is good practice to begin suit sampling with a higher number of samples. These can then be reduced over time. Some facilities perform more samples from the gown during gowning test qualifications compared with routine sampling. Sites considered for selection include the top of the head, the face mask, both arms, middle torso, and both legs. In terms of limits, for EU GMP Grade B/ISO class 7 areas, the aim is often to adopt the same limits as per the limits applied to finger plates. The action level for gowns is ordinarily 5 CU/25cm2.

Experience has shown that higher counts are obtained from the top of the head, perhaps because this is the warmest region of the body. Care must be undertaken when sampling as so not to break the integrity of the gown.

In addition to gowning control, a procedure should be in place for the notification of health conditions by staff. Staff who are ill (coughs, colds, and so on) should not enter cleanrooms. This is because the illness may affect product quality. It is important to control the potential risks from personnel carrying

  • Infectious disease.
  • Open lesions on any exposed part of body.
  • Shedding skin conditions, such as eczema or psoriasis, dermatitis, and dandruff (skin scales may harbor objectionable micro-organisms that may impact pharmaceutical products and patients).
  • Gastric upsets.

Personnel with any of the above conditions must be excluded from working within cleanrooms for the duration of their illness.

 

CONCLUSION

This paper has considered the personnel factor and the relationship between people and cleanrooms. It addressed why people are a risk in relation to the skin microbiome, and how good gowning practices can help to minimize that risk. The paper has also considered other factors that can affect contamination risks from people, including the importance of good behaviors and the necessity of hand sanitization. Capturing these various issues through procedures and imparting the key concepts through training are a necessary part of cleanroom management.

 

REFERENCES

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  2. Hyde, W. (1998). Origin of bacteria in the clean room and their growth requirements. PDA J Sci Technol; 52:154–164
  3.  Sandle, T. (2011). A Review of Cleanroom Microflora: Types, Trends, and Patterns, PDA Journal of Pharmaceutical Science and Technology, 65 (4): 392-403
  4. Proksch E.; Brandner J.M.; Jensen J.M. (2008) The Skin: An Indispensable Barrier, Exp. Dermatol. 17 (12): 1063–72
  5. Whyte, W. (1981) Setting and impaction of particles into containers in manufacturing pharmacies, J. Paren. Sci. Technol., 36: 255-268
  6. Roth, R.R. and James, W.D. (1988): Microbial Ecology of the Skin. Annu. Rev. Microbiol. Vol. 42, pp. 441–64
  7. Grice, E.A., Kong, H.H., Renaud, G., Young, A.C. (2008). A diversity profile of the human skin microbiota. Genome Research.18:1043-50. (PMID:18502944)
  8. Costello, E.K., Lauber, C. L., Hamady, M., Fierer, N., Gordon, J.I., Knight, R. (2009). Bacterial community variation in human body habitats across space and time, Science, 326: 1694–1697
  9. Chen YE, Tsao H. (2013). The skin microbiome: current perspectives and future challenges, J Am Acad Dermatol. 69(1):143-55
  10. Grice, E.A., Kong, H.H., Conlan, S. et al (2009). Topographical and Temporal Diversity of the Human Skin Microbiome, Science, 324: 1190 – 1192
  11. Gao, Z., Tseng, C.H., Pei, Z., and Blaser, M.J. (2007). Molecular analysis of human forearm superficial skin bacterial biota. Proc. Natl. Acad. Sci. 104: 2927–2932
  12. Kong, H.H. and Segre, J.A. (2012). Skin Microbiome: Looking Back to Move Forward, Journal of Investigative Dermatology, 132: 933–939
  13. Findley, K.,  Oh, J. Yang, J., Conlan, S. et al (2013). Topographic diversity of fungal and bacterial communities in human skin, Nature, 498(7454):367-70. doi:10.1038/nature12171
  14. Cogen A.L, Nizet, V. and Gallo, R.L. (2008): Skin Microbiota: A Source of Disease or Defence?, Br. J. Dermatol., 158 (3): 442-55
  15. Ramstorp, M. (2011) Microbial Contamination Control in Pharmaceutical Manufacturing. In Saghee, M.R., Sandle, T. and Tidswell, E. (Eds.) Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices, Business Horizons: New Delhi, pp615-701
  16. Sandle, T. (2006) The use of polymeric flooring to reduce contamination in a cleanroom changing area, European Journal of Parenteral and Pharmaceutical Sciences, 11 (3): 7
  17. Kampf, G., Kramer, A. (2004). Epidemiologic Background of Hand Hygiene and Evaluation of the Most Important Agents for Scrubs and Rubs, Clinical Microbiology Review, p. 863–893
  18. Sutton. S., (2009). Hand Washing-A Critical Aspect of Personal Hygiene in Pharma, Journal of Validation Technology, 15 (4): 50-55
  19. Spaudling, E.H. (1964) Alcohol as a surgical disinfectant, AORN J., 2: 67-71
  20. Klein, M. and DeForest, A. (1963) The inactivation of viruses by germicides, Chem. Specialist Manufact. Assoc. proc., 49: 116-118
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  22. Morton, H.E. (1983) Alcohols. In Block, S.S. (Ed.) Disinfection, Sterilisation and Preservation, Philadelphia: Lea and Febiger, pp225-239



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