quarta-feira, 5 de setembro de 2012
Basics of Isolator Cleaning
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Basics of Isolator Cleaning
By Dr. Thomas H. Treutler
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.Isolators are increasingly installed in pharmaceutical production laboratories due to the increased handling of hazardous drug ingredients as well as the need for smaller batches and more flexible production environments. Isolators can potentially lower the installation and maintenance costs compared to large scale cleanroom environments. While manufacturing facilities have established SOPs for isolators, this article focuses on the importance of proper cleaning and wiping procedures.
ISOLATORS AND DECONTAMINATION
Decontamination is the reduction or removal of biological or chemical agents, including non-active particles to non-hazardous levels to products, processes, or the environment by means of physical or chemical procedures.
Specifically in pharmaceutical manufacturing environments, research laboratories, and hospital pharmacies, the effective decontamination of biological agents like bacteria, viruses, fungi, protozoa, prions, and spores is essential.
Isolators like fume hoods, biosafety cabinets, and gloveboxes are used to create environments with low levels of environmental pollutants such as biological agents, aerosol particles, and dust. These separative devices have a controlled level of contamination, specified by the number of particles with a defined size per cubic meter, providing controlled environments that are specifically tailored to the needs of its operator. This classification of cleanrooms and isolators, however, is not taking into account specific requirements regarding biological contamination. In order to maintain the low levels of environmental pollutants, isolators have to be decontaminated on a regular basis.
ISOLATOR CLEANLINESS
Isolator cleanliness levels are defined by different classifications, shown in Table 1 and Table 2. These classifications are evaluating the environmental pollution by particles, however, not taking into account specific requirements regarding biological contamination. In order to maintain the low levels of environmental pollutants, isolators have to be decontaminated on a regular basis.
Quality supervisors in facilities using isolators have to determine the acceptable level of biological agents in their respective environment and decide on the method to achieve these levels. Several factors influence the choice of method and materials.
POTENTIAL CONTAMINANTS
Isolators are used in a variety of industries working with different material and under different requirements. Potential contaminants in isolators can therefore range from biological contaminants (e.g. pharmaceutical industry, hospital pharmacies), radionuclides (e.g. pharmaceutical industry, research laboratories) to general particulate contaminants (e.g. semiconductor industry).
CHEMICAL AGENTS: INACTIVATION
Spills of hazardous chemical agents in isolators or potential reaction products immobilized on isolator surfaces have to be inactivated or diluted to non-hazardous levels. The chemicals and chemical processes used for inactivation depend on the contaminant.
BIOLOGICAL AGENTS: DISINFECTION AND STERILIZATION
To reduce the level of biological agents in an environment, disinfectants/sanitizers and sterilants can be used. Sanitizers and disinfectants are terms used in different industries for the same kind of product. Whereas the food and foodprocessing industry uses the term sanitizers, the pharmaceutical industry, laboratories, and hospitals are predominantly using the term disinfectant.
Disinfection describes a process that eliminates many or all pathogenic microorganisms on inanimate objects, except bacterial spores.1 On the other hand, sterilization describes a process that destroys or eliminates all forms of microbial life and is carried out by physical or chemical methods.1 Depending on the biological agent and the material or media holding it, sterilization can be achieved through the application of heat, chemicals, irradiation, high pressure, or filtration. It is essential to understand the difference between both processes to assure that contamination level requirements of work environments are met. Whereas some commercial and technical literature is confusing readers by using both terms interchangeably, it should be noted that disinfection and sterilization describe two processes with very different requirements in outcome. It is not appropriate to talk about partial sterilization or even replace the word disinfection with sterilization.
The efficacy of sterilization depends on a number of factors like:
•prior physical cleaning (effective surface and biofilm reduction)
•presence of organic and inorganic load-level and type of microbial contaminants
•concentration of sterilant
•exposure time of sterilant
•pH, temperature, and humidity of environment
•geometry of objects and spaces
•physical properties of objects
Frequent application of sterilization processes is facing two major challenges; the potential build-up of resistance against the used sterilization agent as well as disadvantageous interactions with humans and surfaces that get in direct contact with these agents. The applied processes have to be well understood in order to avoid these detrimental effects.
The efficacy of different sterilization methods has been evaluated and reported by a number of publications. Tested microbial agents include bacteria, spores, and viruses.3,4,5 As discussed in these articles, microbiological agents may show a significant difference in resistance to the discussed sterilization methods. Therefore previously mentioned factors (the efficacy of sterilization depends on a number of factors such as in list one as well as the specific resistance of microbiological agents) play a vital role in the selection of the appropriate sterilization method.
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CLEANING OF ISOLATORS
Decontamination or cleaning, the reduction or removal of biological or chemical agents, including non-active particles is a multi-step process that depends on the contaminant and the required cleanliness level.
In isolators with processes using chemical agents the successful inactivation of these agents precedes any removal attempt in order to avoid further contamination of the environment or reaction with the isolator surfaces and cleaning materials. After successfully inactivating hazardous chemicals, high absorbency wipes are used to physically remove the reaction products.
When choosing isolator cleaning tools and materials, it is recommended that operators introduce the least amount of particle and fiber generating materials into the isolator. Typically a cleanroom laundered 100% continuous filament polyester knit material with sealed edges is recommended for use to clean surfaces inside the isolator. Additionally isolator cleaning tools with replacement covers that have been tested for particle and fiber release are appropriate to extend the reach of the cleaning area as well as providing ergonomic benefits to the operator.
One can also use cleanroom wipes with specific surface treatments to allow the wiper to capture and retain particulate contamination, resulting in more efficient cleaning and reduced likelihood of recontamination of critical surfaces.
The recommended steps to be performed when cleaning a contaminated surface do not change and are the same for all kinds of contaminants.
1.Always clean from the cleanest to the dirtiest surface.
2.Clean with overlapping strokes and change wiper surface with each stroke.
3.If using an isolator cleaning tool or mop, change out cover material with each surface side of the isolator.
In the case of isolators with biological contaminants, like bacteria, spores, and viruses, regular sterilization might seem to be sufficient in killing the microbial agents. However, it is extremely important that prior to sterilization, a physical removal of these contaminants is done in order to avoid a subsequent buildup of biofilms that would increase the resistance to sterilization attempts in the future. Biofilm is composed of polysaccharides that consist of carbon, hydrogen, and oxygen. Hydrogen and oxygen are most likely to be found in most isolators with natural atmosphere, leaving killed microbial agents behind would provide the required carbon for bacteria to reproduce and form new biofilms.
CLEANING PROCESS SOP
Developing a Standard Operating Procedure (SOP) for your isolators is a difficult task and depends on the very specific requirements of a facility’s processes and regulation in its industry. As a rule of thumb, Table 3 can serve as a general guideline to develop your own SOP.6 Questions you should ask yourself are:
•What contaminants am I concerned about?
•Would they contaminate my processes (inside) or the environment (outside)?
•Are these contaminants inert, chemically-, biologically-, or radio-active?
•What contamination limits have to be considered?
The use of an isolator cleaning tool should also be considered to allow efficient cleaning6 of hard-toreach areas and guarantee an equal pressure distribution of your cleaning material (wipes/pads) on the isolator surface. The applied pressure is a determining factor in the physical removal of contaminants from a surface.
SUMMARY
Proper decontamination and cleaning of isolators is critical to the long term success of materials produced in these environments. Reducing the risk of cross contamination starts with a full understanding of the type of potential contaminants introduced before, during, and after the production process. Sterilization and spraying with disinfectants alone are not enough to remove residual particles that could result in the buildup of biofilms. Proper wiping and rinsing protocols are needed to ensure the total removal of contaminants and the cleanliness of the isolator.
References
1.Healthcare Infection Control Practices Advisory Committee (HICPAC), “Guideline for Disinfection and Sterilization in Healthcare Facilities,” 2008
2.McDonnell, G.; Russell, A.D.; “Antiseptics and Disinfectants: Activity, Action, and Resistance” Clinical Microbiological Reviews, Jan. 1999, p. 147-179
3.Mehmi, M.; Marshall, L.J.; Lambert, P.A.; Smith J.C.; “Evaluation of Disinfecting Procedures for Aseptic Transfer in Hospital Pharmacy Departments” PDA Journal of Pharmaceutical Science and Technology, Vol. 63, No. 2, p. 123-138
4.Block, S.S. Disinfection, Sterilization, and Preservation. Philadelphia: Lea & Febiger 1991
5.Siegerman, H. “Wiping Surfaces Clean” A2C2 Magazine, April 2003
6.“Isolator Cleaning Guide” 01 Aug 2010 Berkshire Corporation Dr. Thomas H. Treutler is CTO for Berkshire Corporation. He has years of experience in nanotechnology and applications in contained environments. Berkshire Corporation, 21 River Street, Great Barrington, MA 01230; (413) 931-3468 begin_of_the_skype_highlighting (413) 931-3468 end_of_the_skype_highlighting; ttreutler@berkshire.com; www.berkshire.com
REF: http://www.cemag.us/print/5090 Acessado em 05/09/12
segunda-feira, 11 de junho de 2012
Verticillium sp.
Verticillium sp.
Colonies are fast growing, suede-like to downy, white to pale yellow in colour, becoming pinkish brown, red, green or yellow with a colourless, yellow or reddish brown reverse. Conidiophores are usually well differentiated and erect, verticillately branched over most of their length, bearing whorls of slender awl-shaped divergent phialides. Conidia are hyaline or brightly coloured, mostly one-celled, and are usually borne in slimy heads (glioconidia).
Conidiophores, phialides and conidia of Verticillium sp.
Clinical significance:
Members of this genus are often isolated from the environment. It has been reported as a rare agent of mycotic keratitis.
Mycosis: Hyalohyphomycosis
Further reading:
Domsch, K.H., W. Gams, and T.H. Anderson. 1980. Compendium of soil fungi. Volume 1. Academic Press, London, UK.
Rippon, J.W. 1988. Medical Mycology. 3rd Edition. W.B. Saunders Co., Philadelphia, USA.
REF: http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Hyphomycetes_(hyaline)/Verticillium/ Acessado em 12/06/12
Colonies are fast growing, suede-like to downy, white to pale yellow in colour, becoming pinkish brown, red, green or yellow with a colourless, yellow or reddish brown reverse. Conidiophores are usually well differentiated and erect, verticillately branched over most of their length, bearing whorls of slender awl-shaped divergent phialides. Conidia are hyaline or brightly coloured, mostly one-celled, and are usually borne in slimy heads (glioconidia).
Conidiophores, phialides and conidia of Verticillium sp.
Clinical significance:
Members of this genus are often isolated from the environment. It has been reported as a rare agent of mycotic keratitis.
Mycosis: Hyalohyphomycosis
Further reading:
Domsch, K.H., W. Gams, and T.H. Anderson. 1980. Compendium of soil fungi. Volume 1. Academic Press, London, UK.
Rippon, J.W. 1988. Medical Mycology. 3rd Edition. W.B. Saunders Co., Philadelphia, USA.
REF: http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Hyphomycetes_(hyaline)/Verticillium/ Acessado em 12/06/12
Staphylococcus sciuri
We previously characterized over 100 Staphylococcus sciuri isolates, mainly of animal origin, and found that they all carried a genetic element (S. sciuri mecA) closely related to the mecA gene of methicillin-resistantStaphylococcus aureus (MRSA) strains. We also found a few isolates that carried a second copy of the gene, identical to MRSAmecA. In this work, we analyzed a collection of 28 S. sciuri strains isolated from both healthy and hospitalized individuals.
REF: http://jcm.asm.org/content/38/3/1136.full Acessado em 12/06/12
REF: http://jcm.asm.org/content/38/3/1136.full Acessado em 12/06/12
Staphylococcus hominis
Staphylococcus hominis is a coagulase-negative member of the bacterial genus Staphylococcus, consisting of Gram-positive, spherical cells in clusters. It occurs very commonly as a harmless commensal on human and animal skin. However, like many other coagulase-negative staphylococci, S. hominis may occasionally cause infection in patients whose immune systems are compromised, for example by chemotherapy or predisposing illness.
DescriptionColonies of S. hominis are small, usually 1–2 mm in diameter after 24 hours' incubation at 35 °C, and white or tan in colour. Occasional strains are resistant to novobiocin and may be confused with other resistant species (e.g. S. saprophyticus.)
It is one of only two species of Staphylococcus that display sensitivity to desferrioxamine, the other being S. epidermidis. Unlike S. epidermidis, S. hominis produces acid from trehalose, so the two tests together serve to identify the species.
[edit] BiologyNumerous coagulase-negative staphylococci appear commonly on the skin of human. Of these species, Staphylococcus epidermidis and S. hominis are the most abundant. While S. epidermidis tends to colonize the upper part of the body, S. hominis tends to colonize in areas with numerous apocrine glands, such as axillae and the pubic region. In a certain study, S. hominis was calculated to account for 22% of the total staphylococci species recovered from individuals, second to S. epidermidis at 46%. S. hominis is the predominant species on the head, axillae, arms, and legs. S. hominis, as well as most other staphylococci species common on the human skin, is able to produce acid aerobically from glucose, fructose, sucrose, trehalose, and glycerol. Some strains were also able to produce acid from turanose, lactose, and galactose, melezitose, mannitol, and mannose. Most strains colonize on the skin for relatively short periods of time compared to other Staphylococcus species. They, on average, stay on the skin for only several weeks or months. The cell wall contains low amounts of teichoic acid and glutamic acid. The cell wall teichoic acid contains glycerol and glucosamine. S. hominis cells are Gram-positive cocci, usually 1.2 to 1.4 micrometers in diameter. They appear normally in tetrads and sometimes in pairs.[1]
[edit] ResistanceBased on a total of 240 strains, all were resistant to lysozyme, some were slightly resistant to lysostaphin, 77% were susceptible to penicillin G, 97% to streptomycin, 93% to erythromycin, 64% to tetracycline, and 99% to novobiocin.[2]
[edit] CulturingWhen grown in agar cultures, colonies are usually circular, 4.0 to 4.5 micrometers in diameter. Agar colonies usually have wide edges and an elevated center. They are commonly smooth with dull surfaces, and are yellow-orange pigmented in the center of the opaque colonies. They grow both in aerobic and anaerobic conditions, but tend to grow significantly less in the latter. Optimal NaCl concentrations of the agar culture for the growth of S. hominis seems to be around 7.5%, and a salt concentration of 15% yielded poor growth to no growth at all. The optimal growth temperature range was around 28 to 40 °C, but good growth is still observed at 45 °C, while no growth is observed at 15 °C. S. hominis can be differentiated from staphylococci by its colony morphology and pigmentation patterns, predominant tetrad cell arrangement, poor growth in thioglycolate, low tolerance of NaCl, and carbohydrate reaction pattern. Each species is also significantly different in cell wall composition, lactic acid configuration, temperature extremes of growth, coagulase activity, hemolysis acetylmethylcarbinol production, nitrate reduction, and phosphatase, DNase, and bacteriolytic activities. Similarities in these properties between S. hominis and several other species suggest there is a close relationship between S. hominis and S. epidermidis, S. haemolyticus, and S. warneri.[3]
[edit] Antibiotic-resistant subspeciesS. hominis is normally found on human skin and is usually harmless, but can sometimes cause infections in people with abnormally weak immune systems. Most, if not all, strains are susceptible to penicillin, erythromycin, and novobiocin, but a divergent strain, S. hominis subsp. novobiosepticus (SHN) was found recently. This strain was named so because of its unique resistance to novobiocin and its failure to produce acid aerobically from trehalose and glucosamine. In addition, the 26 isolated strains of this new subspecies are resistant to nalidixic acid, penicillin G, oxacillin, kanamycin and streptomycin. They were also somewhat resistant to methicillin and gentamicin, and most strains were resistant to erythromycin, clindamycin, chloramphenicol, trimethoprim/sulfamethoxazole and ciprofloxacin, as well. In addition, S. hominis subsp. hominis is commonly found isolated from human skin, but there are no reports of the isolation of SHN from the human skin.[4]
The SHN is so similar to the original S. hominis, now called S. hominis subsp. hominis, that a MicroScan system that clinical microbiology laboratories use identified seven of 31 S. hominis subsp. novobiosepticus cultures as S. hominis subsp. hominis. The relationship between the two is currently unknown, but antibiotic-resistant isolates of S. hominis belong only to SHN. [5]
SHN strains seems to have thickened cell walls, and this tendency may be the result of a genetic background that also allows for vancomycin resistance. The thickened cell walls exist in subspecies with and without vancomycin resistance which suggests this subspecies did not originate from the acquiring of resistance genes. [6]
[edit] OriginThe combined resistance to novobiocin and oxacillin is hypothesized to have originated from a simultaneous introduction of genes controlling the resistance to the two. These genes were believed to have been acquired originally through heterologous DNA from a methicillin-resistant strain of one of the novobiocin-resistant species belonging to the S. sciuri or the S. saprophyticus groups. The larger genome size of the SHN compared to that of S. hominis subsp. hominis may be the result of the acquiring of heterologous DNA. This new, divergent strain was first described in 1998, and this microbe was first implicated in causing bactermia in 2002. Another hypothesis is the insertion of the mec A gene and its flanking sequence into the chromosome of SHN might have affected the expression of a closely linked gene, which converted the host to become novobiocin-resistant.[7]
REF: http://en.wikipedia.org/wiki/Staphylococcus_hominis Acessado em 12/06/12
DescriptionColonies of S. hominis are small, usually 1–2 mm in diameter after 24 hours' incubation at 35 °C, and white or tan in colour. Occasional strains are resistant to novobiocin and may be confused with other resistant species (e.g. S. saprophyticus.)
It is one of only two species of Staphylococcus that display sensitivity to desferrioxamine, the other being S. epidermidis. Unlike S. epidermidis, S. hominis produces acid from trehalose, so the two tests together serve to identify the species.
[edit] BiologyNumerous coagulase-negative staphylococci appear commonly on the skin of human. Of these species, Staphylococcus epidermidis and S. hominis are the most abundant. While S. epidermidis tends to colonize the upper part of the body, S. hominis tends to colonize in areas with numerous apocrine glands, such as axillae and the pubic region. In a certain study, S. hominis was calculated to account for 22% of the total staphylococci species recovered from individuals, second to S. epidermidis at 46%. S. hominis is the predominant species on the head, axillae, arms, and legs. S. hominis, as well as most other staphylococci species common on the human skin, is able to produce acid aerobically from glucose, fructose, sucrose, trehalose, and glycerol. Some strains were also able to produce acid from turanose, lactose, and galactose, melezitose, mannitol, and mannose. Most strains colonize on the skin for relatively short periods of time compared to other Staphylococcus species. They, on average, stay on the skin for only several weeks or months. The cell wall contains low amounts of teichoic acid and glutamic acid. The cell wall teichoic acid contains glycerol and glucosamine. S. hominis cells are Gram-positive cocci, usually 1.2 to 1.4 micrometers in diameter. They appear normally in tetrads and sometimes in pairs.[1]
[edit] ResistanceBased on a total of 240 strains, all were resistant to lysozyme, some were slightly resistant to lysostaphin, 77% were susceptible to penicillin G, 97% to streptomycin, 93% to erythromycin, 64% to tetracycline, and 99% to novobiocin.[2]
[edit] CulturingWhen grown in agar cultures, colonies are usually circular, 4.0 to 4.5 micrometers in diameter. Agar colonies usually have wide edges and an elevated center. They are commonly smooth with dull surfaces, and are yellow-orange pigmented in the center of the opaque colonies. They grow both in aerobic and anaerobic conditions, but tend to grow significantly less in the latter. Optimal NaCl concentrations of the agar culture for the growth of S. hominis seems to be around 7.5%, and a salt concentration of 15% yielded poor growth to no growth at all. The optimal growth temperature range was around 28 to 40 °C, but good growth is still observed at 45 °C, while no growth is observed at 15 °C. S. hominis can be differentiated from staphylococci by its colony morphology and pigmentation patterns, predominant tetrad cell arrangement, poor growth in thioglycolate, low tolerance of NaCl, and carbohydrate reaction pattern. Each species is also significantly different in cell wall composition, lactic acid configuration, temperature extremes of growth, coagulase activity, hemolysis acetylmethylcarbinol production, nitrate reduction, and phosphatase, DNase, and bacteriolytic activities. Similarities in these properties between S. hominis and several other species suggest there is a close relationship between S. hominis and S. epidermidis, S. haemolyticus, and S. warneri.[3]
[edit] Antibiotic-resistant subspeciesS. hominis is normally found on human skin and is usually harmless, but can sometimes cause infections in people with abnormally weak immune systems. Most, if not all, strains are susceptible to penicillin, erythromycin, and novobiocin, but a divergent strain, S. hominis subsp. novobiosepticus (SHN) was found recently. This strain was named so because of its unique resistance to novobiocin and its failure to produce acid aerobically from trehalose and glucosamine. In addition, the 26 isolated strains of this new subspecies are resistant to nalidixic acid, penicillin G, oxacillin, kanamycin and streptomycin. They were also somewhat resistant to methicillin and gentamicin, and most strains were resistant to erythromycin, clindamycin, chloramphenicol, trimethoprim/sulfamethoxazole and ciprofloxacin, as well. In addition, S. hominis subsp. hominis is commonly found isolated from human skin, but there are no reports of the isolation of SHN from the human skin.[4]
The SHN is so similar to the original S. hominis, now called S. hominis subsp. hominis, that a MicroScan system that clinical microbiology laboratories use identified seven of 31 S. hominis subsp. novobiosepticus cultures as S. hominis subsp. hominis. The relationship between the two is currently unknown, but antibiotic-resistant isolates of S. hominis belong only to SHN. [5]
SHN strains seems to have thickened cell walls, and this tendency may be the result of a genetic background that also allows for vancomycin resistance. The thickened cell walls exist in subspecies with and without vancomycin resistance which suggests this subspecies did not originate from the acquiring of resistance genes. [6]
[edit] OriginThe combined resistance to novobiocin and oxacillin is hypothesized to have originated from a simultaneous introduction of genes controlling the resistance to the two. These genes were believed to have been acquired originally through heterologous DNA from a methicillin-resistant strain of one of the novobiocin-resistant species belonging to the S. sciuri or the S. saprophyticus groups. The larger genome size of the SHN compared to that of S. hominis subsp. hominis may be the result of the acquiring of heterologous DNA. This new, divergent strain was first described in 1998, and this microbe was first implicated in causing bactermia in 2002. Another hypothesis is the insertion of the mec A gene and its flanking sequence into the chromosome of SHN might have affected the expression of a closely linked gene, which converted the host to become novobiocin-resistant.[7]
REF: http://en.wikipedia.org/wiki/Staphylococcus_hominis Acessado em 12/06/12
Penicillium sp
O Penicillium (lat. penicillus= pincel) é um género de fungos, o comum bolor do pão, que cresce em matéria orgânica biodegradável, especialmente no solo e outros ambientes húmidos e escuros. Por contágio, contaminam frutas e sementes e chegam a invadir habitações, sendo responsáveis pelos bolores que se instalam em alimentos para consumo humano.
Natural Habitats Soil • Seed • Cereal crops
Suitable Substrates in the Indoor Environment Foods (blue mold on cereals, fruits,
vegetables, dried foods) • House dust • Fabrics • Leather • Wallpaper • Wallpaper glue
Water Activity Aw=0.78-0.86
Mode of Dissemination Wind • Insects
Allergenic Potential Type I (hay fever, asthma) • Type III (hypersensitivity)
Potential Opportunist or Pathogen Penicilliosis
Industrial Uses P. chrysogenum for the antibiotic penicillin • P. griseofulvum for the antibiotic
griseofulvin a • P. roquefortii for Roquefort cheese • P. camemberti for Camembert cheese
• Brie, Gorgonzola, and Danish Blue cheese are also the products of Penicillium • Used to cure
ham and salami • Production of organic acids such as fumaric, oxalic, gluconic, and gallic
Potential Toxins Produced Citrinin • Citreoviridin • Cyclopiazonic acid • Fumitremorgen B
• Grisiofulvin • Janthitrems • Mycophenolic acid • Paxilline • Penitrem A • Penicillic acid
• Ochratoxins • Roquefortine C • Secalonic acid D • Verruculogen • Verrucosidin
• Viomellein • Viridicatumtoxin • Xanthomegnin
Other Comments Penicillium is one of the most common genera of fungi
ref: http://www.nordichomeinspection.com/uploads/Penicillium.pdf Acesso: 11/06/12
Natural Habitats Soil • Seed • Cereal crops
Suitable Substrates in the Indoor Environment Foods (blue mold on cereals, fruits,
vegetables, dried foods) • House dust • Fabrics • Leather • Wallpaper • Wallpaper glue
Water Activity Aw=0.78-0.86
Mode of Dissemination Wind • Insects
Allergenic Potential Type I (hay fever, asthma) • Type III (hypersensitivity)
Potential Opportunist or Pathogen Penicilliosis
Industrial Uses P. chrysogenum for the antibiotic penicillin • P. griseofulvum for the antibiotic
griseofulvin a • P. roquefortii for Roquefort cheese • P. camemberti for Camembert cheese
• Brie, Gorgonzola, and Danish Blue cheese are also the products of Penicillium • Used to cure
ham and salami • Production of organic acids such as fumaric, oxalic, gluconic, and gallic
Potential Toxins Produced Citrinin • Citreoviridin • Cyclopiazonic acid • Fumitremorgen B
• Grisiofulvin • Janthitrems • Mycophenolic acid • Paxilline • Penitrem A • Penicillic acid
• Ochratoxins • Roquefortine C • Secalonic acid D • Verruculogen • Verrucosidin
• Viomellein • Viridicatumtoxin • Xanthomegnin
Other Comments Penicillium is one of the most common genera of fungi
ref: http://www.nordichomeinspection.com/uploads/Penicillium.pdf Acesso: 11/06/12
Nigrospora spp
Nigrospora spp.
(described by Zimmerman in 1902)
Taxonomic classification
Kingdom: Fungi
Phylum: Ascomycota
Order: Trichosphaeriales
Family: Trichosphaeriaceae
Genus: Khuskia (teleomorph)
Description and Natural Habitats
Nigrospora is a filamentous dematiaceous fungus widely distributed in soil, decaying plants, and seeds. It is a common laboratory contaminant. Although it has been isolated from a few clinical samples, its pathogenicity in man remains uncertain [531, 1295, 2144, 2202].
Species
Nigrospora sphaerica is the best-known species of the genus Nigrospora.
Synonyms
See the summary of synonyms and teleomorph-anamorph relations for Nigrospora spp.
Pathogenicity and Clinical Significance
Nigrospora has been isolated from cutaneous lesions of a leukemic patient and from a case with keratitis. However, its pathogenic role as a causative agent is not well-known [1847, 2218].
Macroscopic Features
Nigrospora grows rapidly and produces woolly colonies on potato dextrose agar at 25°C. The colonies mature within 4 days. Color of the colony is white initially and then becomes gray with black areas and turns to black eventually from both front and reverse. Sporulation may take more than 3 weeks for some isolates [531, 1295, 2144, 2202].
Microscopic Features
Septate hyaline hyphae, hyaline or slightly pigmented conidiophores, and conidia are visualized. The conidiogenous cells on the conidiophores are inflated, swollen, and ampulliform in shape. They bear a single conidium (14-20 µm in diameter) at their apex. Conidia are black, solitary, unicellular, slightly flattened horizontally, and have a thin equatorial germ slit [531, 1295, 2144, 2202].
Compare to
Humicola
Nigrospora is differentiated from Humicola by its very black conidia that originate from hyaline, inflated conidiophores.
Laboratory Precautions
No special precautions other than general laboratory precautions are required.
Susceptibility
No data are available
ref: http://www.doctorfungus.org/thefungi/Nigrospora.php Acessado em 12/06/12
(described by Zimmerman in 1902)
Taxonomic classification
Kingdom: Fungi
Phylum: Ascomycota
Order: Trichosphaeriales
Family: Trichosphaeriaceae
Genus: Khuskia (teleomorph)
Description and Natural Habitats
Nigrospora is a filamentous dematiaceous fungus widely distributed in soil, decaying plants, and seeds. It is a common laboratory contaminant. Although it has been isolated from a few clinical samples, its pathogenicity in man remains uncertain [531, 1295, 2144, 2202].
Species
Nigrospora sphaerica is the best-known species of the genus Nigrospora.
Synonyms
See the summary of synonyms and teleomorph-anamorph relations for Nigrospora spp.
Pathogenicity and Clinical Significance
Nigrospora has been isolated from cutaneous lesions of a leukemic patient and from a case with keratitis. However, its pathogenic role as a causative agent is not well-known [1847, 2218].
Macroscopic Features
Nigrospora grows rapidly and produces woolly colonies on potato dextrose agar at 25°C. The colonies mature within 4 days. Color of the colony is white initially and then becomes gray with black areas and turns to black eventually from both front and reverse. Sporulation may take more than 3 weeks for some isolates [531, 1295, 2144, 2202].
Microscopic Features
Septate hyaline hyphae, hyaline or slightly pigmented conidiophores, and conidia are visualized. The conidiogenous cells on the conidiophores are inflated, swollen, and ampulliform in shape. They bear a single conidium (14-20 µm in diameter) at their apex. Conidia are black, solitary, unicellular, slightly flattened horizontally, and have a thin equatorial germ slit [531, 1295, 2144, 2202].
Compare to
Humicola
Nigrospora is differentiated from Humicola by its very black conidia that originate from hyaline, inflated conidiophores.
Laboratory Precautions
No special precautions other than general laboratory precautions are required.
Susceptibility
No data are available
ref: http://www.doctorfungus.org/thefungi/Nigrospora.php Acessado em 12/06/12
Kocuria rosea
Kocuria rosea
Kocuria rosea and Micrococcus spp. (gram-positive bacteria) are widespread in nature and commonly found along with coagulase-negative Staphylococcus spp. on the skin of humans and mammals.
REF: http://www.usmicro-solutions.com/referencelibrary/bacteriallibrary.html Acessado em 11/06/12
Kocuria rosea and Micrococcus spp. (gram-positive bacteria) are widespread in nature and commonly found along with coagulase-negative Staphylococcus spp. on the skin of humans and mammals.
REF: http://www.usmicro-solutions.com/referencelibrary/bacteriallibrary.html Acessado em 11/06/12
Bacillus lentus
BioHazard Level:
1
Growth Temperature:
26oC
Appropriate growth media:
CASO agar
Genomic sources for restriction enzymes (at this website):
BlpI
Gram Stain:
Bacillus lentus is Gram stain positive
Respiration:
Bacillus lentus is aerobic
Taxonomic lineage:
Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus
Industrial uses or economic implications:
Bacillus lentus produces a commercially important alkaline protease.
Miscellaneous:
Bacillus lentus is a urea-decomposing soils bacteria.
Human health and disease:
Bacillus lentus is considered non-pathogenic.
REF: http://www.thelabrat.com/restriction/sources/Bacilluslentus.shtml Acessado em 11/06/12
Bacillus cereus
A 'Bacillus cereus' é uma bactéria beta hemolítica gram-positiva, de forma cilíndrica, endêmica, que vive no solo. Algumas cepas são prejudiciais aos seres humanos e causam intoxicação alimentar, enquanto outras cepas podem ser benéficas, como os probióticos para animais [1]. É a causa da Síndrome do "Arroz Frito", como as bactérias são classicamente contraídas a partir de pratos de arroz frito que têm estado à temperatura ambiente por horas (tal como em um 'buffet'). [2]. As bactérias B. cereus são organismos anaeróbios facultativos, e tal como outros membros do gênero Bacillus, podem produzir endósporos protetores. Seus fatores de virulência incluem a cereolisina e a fosfolipase C.
REF: http://pt.wikipedia.org/wiki/Bacillus_cereus Acessado em 11/06/12
REF: http://pt.wikipedia.org/wiki/Bacillus_cereus Acessado em 11/06/12
Alternaria spp
Taxonomic Classification
Kingdom: Fungi
Phylum: Ascomycota
Class: Euascomycetes
Order: Pleosporales
Family: Pleosporaceae
Genus: Alternaria
Description and Natural Habitats
Alternaria is a cosmopolitan dematiaceous (phaeoid) fungus commonly isolated from plants, soil, food, and indoor air environment. The production of melanin-like pigment is one of its major characteristics. Its teleomorphic genera are called Clathrospora and Leptosphaeria.
Species
The genus Alternaria currently contains around 50 species. Among these, Alternaria alternata is the most common one isolated from human infections. Some authorities suggest that Alternaria alternata is a representative species complex rather than a single species and consists of several heterogenous species. While Alternaria chartarum, Alternaria dianthicola, Alternaria geophilia, Alternaria infectoria, Alternaria stemphyloides, and Alternaria teunissima are among the other Alternaria spp. isolated from infections, some Alternaria strains reported as causative agents remain unspecified.
Synonyms
See the summary of synonyms and teleomorph-anamorph relations for the Alternaria sp
Pathogenicity and Clinical Significance
Alternaria spp. have emerged as opportunistic pathogens particularly in patients with immunosuppression, such as the bone marrow transplant patients [1581] [2297]. They are one of the causative agents of phaeohyphomycosis. Cases of onychomycosis, sinusitis, ulcerated cutaneous infections, and keratitis, as well as visceral infections and osteomyelitis due to Alternaria have been reported [66, 802, 1429, 2042]. In immunocompetent patients, Alternaria colonizes the paranasal sinuses, leading to chronic hypertrophic sinusitis. In immunocompromised patients the colonization may end up with development of invasive disease[2306]. It is among the causative agents of otitis media in agricultural field workers [2345].
Since Alternaria species are cosmopolitan and ubiquitous in nature, they are also common laboratory contaminants. Thus, their isolation in culture requires cautious evaluation [1847].
Macroscopic Features
Alternaria spp. grow rapidly and the colony size reaches a diameter of 3 to 9 cm following incubation at 25°C for 7 days on potato glucose agar. The colony is flat, downy to woolly and is covered by grayish, short, aerial hyphae in time. The surface is greyish white at the beginning which later darkens and becomes greenish black or olive brown with a light border. The reverse side is typically brown to black due to pigment production [462, 1295, 2144].
Microscopic Features
Alternaria spp. have septate, brown hyphae. Conidiophores are also septate and brown in color, occasionally producing a zigzag appearance. They bear simple or branched large conidia (7-10 x 23-34 µm) which have both transverse and longitudinal septations. These conidia may be observed singly or in acropetal chains and may produce germ tubes. They are ovoid to obclavate, darkly pigmented, muriform, smooth or roughened. The end of the conidium nearest the conidiophore is round while it tapers towards the apex. This gives the typical beak or club-like appearance of the conidia [462, 1295, 2144].
Histopathologic Features
Dark colored filamentous hyphae are observed in the sections of infected tissue stained with H&E. If the pigment formation is not obvious, Fontana-Masson silver stain, which is specific to melanin, may be applied [462].
REF: http://www.doctorfungus.org/thefungi/alternaria.php Acessado em 11/06/12
Acremonium spp
Acremonium spp. are filamentous, cosmopolitan fungi frequently isolated from plant debris and soil, they are known to result in invasive infections in the setting of severe immunosuppression. In this letter, we present a case of catheter-related fungaemia associated with Acremonium spp. in a patient with chronic renal failure. After removal of the subclavian catheter, the patient was treated successfully with voriconazole, with a loading dose of 400 mg followed by a maintenance dose of 200 mg bid. To the best of our knowledge, this is the first paper reporting Acremonium spp. associated fungaemia in a relatively immunocompetent host. We also discuss the diagnosis and treatment of Acremonium spp. associated infections in the context of current literature.
REF: A novel fungal pathogen under the spotlight--Acremonium spp. associated fungaemia in an immunocompetent host.
Purnak T, Beyazit Y, Sahin GO, Shorbagi A, Akova M.
http://www.ncbi.nlm.nih.gov/pubmed/19702621 Acessado em 11/06/12
REF: A novel fungal pathogen under the spotlight--Acremonium spp. associated fungaemia in an immunocompetent host.
Purnak T, Beyazit Y, Sahin GO, Shorbagi A, Akova M.
http://www.ncbi.nlm.nih.gov/pubmed/19702621 Acessado em 11/06/12
quinta-feira, 8 de março de 2012
Environmental monitoring: settle plates
One of the series of learning articles, an overview of settle plates for environmental monitoring.
Settle plates are Petri-dishes, typically of either 9cm or 14cm diameter, containing different fill volumes of agar (normally between 20 and 30 mL). Settle plates are designed to detect any viable micro-organisms that may directly settle on or in the product (that is micro-organisms that are carried in the air-stream, although a person who leans over a plate can also potentially deposit micro-organisms). At determined monitoring locations (ideally positioned and exposed either side of the testing environment) the lids of the dishes are removed and the plates are exposed to the air for a defined period of time. In theory, micro-organisms and units containing micro-organisms settle out of the air under gravity, and are deposited onto horizontally positioned agar plates. This theoretically works better in turbulent or laminar airflows. The efficiency can be described as the ‘settling rate’.
The settling rate depends partly on the characteristics of the particles and on the air-flows. Larger units will tend to settle faster (due to gravitational effects) and settling is facilitated by still air-flows (which should not occur within a correctly designed uni-directional air-flow zone). Smaller particles have a lower tendency to settle due to sir resistance and air currents. The principle behind settle plates is that most micro-organisms in air are in association with particles. Generally the ‘complete particle’ (micro-organism in association with the ‘carrier’) is 12mm diameter or larger[i].
Outside of uni-directional air, such as the main cleanroom itself, then the greater the degree of turbulence there is. The amount of air turbulence is proportional to the amount of time that particles remain suspended in the air. Thereby, the greater the amount of air turbulence then the longer the particles will remain suspended in the air (this is not always a bad thing, as particles can be blown away from a critical zone, depending upon the design of the room). This can, however, influence the reliability of the settle plate and here the additional use of active air-samplers can provide additional assurance for the microbiologist assessing the cleanroom cleanliness.
The phenomenon of gravitational settling is, however, a debatable issue. The prevailing view, as discussed above, is that as most micro-organisms are associated with physical particles they will be large enough to settle out of the air due to gravity i. The dissenting view is that micro-organism carrying particles or any micro-organisms not associated with units as being light enough to remain in the air-stream for several minutes and possibly be carried out of the air-stream and not settle[ii]. Much of this debate thereby centres on the size of the particles in the air and the airflow.
The exposure time of the settle plate can be varied, although there is probably little value in exposing plates for less than one hour. For consistency of sampling, for aseptic filling, the EU GMP Guide recommends a four hour exposure time. This time should not be exceeded without strong justification, and even then there will probably be a challenge from the regulatory authority. For exposure times under four hours, such as when a shorter activity is being monitored, the result obtained should be extrapolated using the simple equation:
Count x 240 = cfu / 4 hours
Time exposed (minutes)
The risk from any exposure is desiccation. The depth and condition of the agar are the key variables, as is the cleanroom environment. The agar in the plate will dry out faster if the airflow is excessively high or if the air humidity is low. Therefore the exposure time of settle plates under the conditions of use (a particular cleanroom or uni-directional airflow cabinet) must be validated.
[i] Whyte, W. (1986): ‘Sterility assurance and models for assessing airborne bacterial contamination’, Journal of Parenteral Science and Technology, 40, pp188-197
[ii] Sykes, G. (1970): ‘The control of airborne contamination in sterile areas’, Aerobiology: Proceedings of the 3rd International Symposium, in Silver, I. H. (ed.), Academic Press, London
REF: http://pharmig.blogspot.com/2010/07/environmental-monitoring-settle-plates.html ACESSADO EM 08/03/12
One of the series of learning articles, an overview of settle plates for environmental monitoring.
Settle plates are Petri-dishes, typically of either 9cm or 14cm diameter, containing different fill volumes of agar (normally between 20 and 30 mL). Settle plates are designed to detect any viable micro-organisms that may directly settle on or in the product (that is micro-organisms that are carried in the air-stream, although a person who leans over a plate can also potentially deposit micro-organisms). At determined monitoring locations (ideally positioned and exposed either side of the testing environment) the lids of the dishes are removed and the plates are exposed to the air for a defined period of time. In theory, micro-organisms and units containing micro-organisms settle out of the air under gravity, and are deposited onto horizontally positioned agar plates. This theoretically works better in turbulent or laminar airflows. The efficiency can be described as the ‘settling rate’.
The settling rate depends partly on the characteristics of the particles and on the air-flows. Larger units will tend to settle faster (due to gravitational effects) and settling is facilitated by still air-flows (which should not occur within a correctly designed uni-directional air-flow zone). Smaller particles have a lower tendency to settle due to sir resistance and air currents. The principle behind settle plates is that most micro-organisms in air are in association with particles. Generally the ‘complete particle’ (micro-organism in association with the ‘carrier’) is 12mm diameter or larger[i].
Outside of uni-directional air, such as the main cleanroom itself, then the greater the degree of turbulence there is. The amount of air turbulence is proportional to the amount of time that particles remain suspended in the air. Thereby, the greater the amount of air turbulence then the longer the particles will remain suspended in the air (this is not always a bad thing, as particles can be blown away from a critical zone, depending upon the design of the room). This can, however, influence the reliability of the settle plate and here the additional use of active air-samplers can provide additional assurance for the microbiologist assessing the cleanroom cleanliness.
The phenomenon of gravitational settling is, however, a debatable issue. The prevailing view, as discussed above, is that as most micro-organisms are associated with physical particles they will be large enough to settle out of the air due to gravity i. The dissenting view is that micro-organism carrying particles or any micro-organisms not associated with units as being light enough to remain in the air-stream for several minutes and possibly be carried out of the air-stream and not settle[ii]. Much of this debate thereby centres on the size of the particles in the air and the airflow.
The exposure time of the settle plate can be varied, although there is probably little value in exposing plates for less than one hour. For consistency of sampling, for aseptic filling, the EU GMP Guide recommends a four hour exposure time. This time should not be exceeded without strong justification, and even then there will probably be a challenge from the regulatory authority. For exposure times under four hours, such as when a shorter activity is being monitored, the result obtained should be extrapolated using the simple equation:
Count x 240 = cfu / 4 hours
Time exposed (minutes)
The risk from any exposure is desiccation. The depth and condition of the agar are the key variables, as is the cleanroom environment. The agar in the plate will dry out faster if the airflow is excessively high or if the air humidity is low. Therefore the exposure time of settle plates under the conditions of use (a particular cleanroom or uni-directional airflow cabinet) must be validated.
[i] Whyte, W. (1986): ‘Sterility assurance and models for assessing airborne bacterial contamination’, Journal of Parenteral Science and Technology, 40, pp188-197
[ii] Sykes, G. (1970): ‘The control of airborne contamination in sterile areas’, Aerobiology: Proceedings of the 3rd International Symposium, in Silver, I. H. (ed.), Academic Press, London
REF: http://pharmig.blogspot.com/2010/07/environmental-monitoring-settle-plates.html ACESSADO EM 08/03/12
Environmental monitoring: settle plates
One of the series of learning articles, an overview of settle plates for environmental monitoring.
Settle plates are Petri-dishes, typically of either 9cm or 14cm diameter, containing different fill volumes of agar (normally between 20 and 30 mL). Settle plates are designed to detect any viable micro-organisms that may directly settle on or in the product (that is micro-organisms that are carried in the air-stream, although a person who leans over a plate can also potentially deposit micro-organisms). At determined monitoring locations (ideally positioned and exposed either side of the testing environment) the lids of the dishes are removed and the plates are exposed to the air for a defined period of time. In theory, micro-organisms and units containing micro-organisms settle out of the air under gravity, and are deposited onto horizontally positioned agar plates. This theoretically works better in turbulent or laminar airflows. The efficiency can be described as the ‘settling rate’.
The settling rate depends partly on the characteristics of the particles and on the air-flows. Larger units will tend to settle faster (due to gravitational effects) and settling is facilitated by still air-flows (which should not occur within a correctly designed uni-directional air-flow zone). Smaller particles have a lower tendency to settle due to sir resistance and air currents. The principle behind settle plates is that most micro-organisms in air are in association with particles. Generally the ‘complete particle’ (micro-organism in association with the ‘carrier’) is 12mm diameter or larger[i].
Outside of uni-directional air, such as the main cleanroom itself, then the greater the degree of turbulence there is. The amount of air turbulence is proportional to the amount of time that particles remain suspended in the air. Thereby, the greater the amount of air turbulence then the longer the particles will remain suspended in the air (this is not always a bad thing, as particles can be blown away from a critical zone, depending upon the design of the room). This can, however, influence the reliability of the settle plate and here the additional use of active air-samplers can provide additional assurance for the microbiologist assessing the cleanroom cleanliness.
The phenomenon of gravitational settling is, however, a debatable issue. The prevailing view, as discussed above, is that as most micro-organisms are associated with physical particles they will be large enough to settle out of the air due to gravity i. The dissenting view is that micro-organism carrying particles or any micro-organisms not associated with units as being light enough to remain in the air-stream for several minutes and possibly be carried out of the air-stream and not settle[ii]. Much of this debate thereby centres on the size of the particles in the air and the airflow.
The exposure time of the settle plate can be varied, although there is probably little value in exposing plates for less than one hour. For consistency of sampling, for aseptic filling, the EU GMP Guide recommends a four hour exposure time. This time should not be exceeded without strong justification, and even then there will probably be a challenge from the regulatory authority. For exposure times under four hours, such as when a shorter activity is being monitored, the result obtained should be extrapolated using the simple equation:
Count x 240 = cfu / 4 hours
Time exposed (minutes)
The risk from any exposure is desiccation. The depth and condition of the agar are the key variables, as is the cleanroom environment. The agar in the plate will dry out faster if the airflow is excessively high or if the air humidity is low. Therefore the exposure time of settle plates under the conditions of use (a particular cleanroom or uni-directional airflow cabinet) must be validated.
[i] Whyte, W. (1986): ‘Sterility assurance and models for assessing airborne bacterial contamination’, Journal of Parenteral Science and Technology, 40, pp188-197
[ii] Sykes, G. (1970): ‘The control of airborne contamination in sterile areas’, Aerobiology: Proceedings of the 3rd International Symposium, in Silver, I. H. (ed.), Academic Press, London
REF: http://pharmig.blogspot.com/2010/07/environmental-monitoring-settle-plates.html ACESSADO EM 08/03/12
One of the series of learning articles, an overview of settle plates for environmental monitoring.
Settle plates are Petri-dishes, typically of either 9cm or 14cm diameter, containing different fill volumes of agar (normally between 20 and 30 mL). Settle plates are designed to detect any viable micro-organisms that may directly settle on or in the product (that is micro-organisms that are carried in the air-stream, although a person who leans over a plate can also potentially deposit micro-organisms). At determined monitoring locations (ideally positioned and exposed either side of the testing environment) the lids of the dishes are removed and the plates are exposed to the air for a defined period of time. In theory, micro-organisms and units containing micro-organisms settle out of the air under gravity, and are deposited onto horizontally positioned agar plates. This theoretically works better in turbulent or laminar airflows. The efficiency can be described as the ‘settling rate’.
The settling rate depends partly on the characteristics of the particles and on the air-flows. Larger units will tend to settle faster (due to gravitational effects) and settling is facilitated by still air-flows (which should not occur within a correctly designed uni-directional air-flow zone). Smaller particles have a lower tendency to settle due to sir resistance and air currents. The principle behind settle plates is that most micro-organisms in air are in association with particles. Generally the ‘complete particle’ (micro-organism in association with the ‘carrier’) is 12mm diameter or larger[i].
Outside of uni-directional air, such as the main cleanroom itself, then the greater the degree of turbulence there is. The amount of air turbulence is proportional to the amount of time that particles remain suspended in the air. Thereby, the greater the amount of air turbulence then the longer the particles will remain suspended in the air (this is not always a bad thing, as particles can be blown away from a critical zone, depending upon the design of the room). This can, however, influence the reliability of the settle plate and here the additional use of active air-samplers can provide additional assurance for the microbiologist assessing the cleanroom cleanliness.
The phenomenon of gravitational settling is, however, a debatable issue. The prevailing view, as discussed above, is that as most micro-organisms are associated with physical particles they will be large enough to settle out of the air due to gravity i. The dissenting view is that micro-organism carrying particles or any micro-organisms not associated with units as being light enough to remain in the air-stream for several minutes and possibly be carried out of the air-stream and not settle[ii]. Much of this debate thereby centres on the size of the particles in the air and the airflow.
The exposure time of the settle plate can be varied, although there is probably little value in exposing plates for less than one hour. For consistency of sampling, for aseptic filling, the EU GMP Guide recommends a four hour exposure time. This time should not be exceeded without strong justification, and even then there will probably be a challenge from the regulatory authority. For exposure times under four hours, such as when a shorter activity is being monitored, the result obtained should be extrapolated using the simple equation:
Count x 240 = cfu / 4 hours
Time exposed (minutes)
The risk from any exposure is desiccation. The depth and condition of the agar are the key variables, as is the cleanroom environment. The agar in the plate will dry out faster if the airflow is excessively high or if the air humidity is low. Therefore the exposure time of settle plates under the conditions of use (a particular cleanroom or uni-directional airflow cabinet) must be validated.
[i] Whyte, W. (1986): ‘Sterility assurance and models for assessing airborne bacterial contamination’, Journal of Parenteral Science and Technology, 40, pp188-197
[ii] Sykes, G. (1970): ‘The control of airborne contamination in sterile areas’, Aerobiology: Proceedings of the 3rd International Symposium, in Silver, I. H. (ed.), Academic Press, London
REF: http://pharmig.blogspot.com/2010/07/environmental-monitoring-settle-plates.html ACESSADO EM 08/03/12
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