1. Be sure the operator read the Instruction Manual of the cabinet and understand the Panel Instructions

2. Switch on the BSC / UAC 5/15 minutes before to start the activity

3. Check that all functions of the BSC /UAC are regular

4. Dress the clean overall with button on the back

5. Dress sterile gloves, covering the wrists

6. Wipe all inside surfaces of the BSC / UAC with suitable disinfectant (e.g.: sterile 70% alcohol)

7. Wipe all material with sterile 70% alcohol before transfer it inside the bench and before to start the activity

8. Avoid creating air turbulence by fast movement of the arms

9. Avoid speaking, sneezing, and coughing

10. Never move or work over open bottles, flasks, dishes, plates

11. Never block air flow through the ventilation holes

12. No unnecessary equipment and material in the clean working bench area

13. Three separate areas inside the working bench: right “clean”, centre “working”, left “dirty”

14. Immediately remove spilled liquids in the clean bench

15. Sterilize by autoclaving the working surfaces, when necessary

16. Clean and disinfect all the surface of the inside BSC at the end of activity

17. Leave the BSC / UAC on for 15 minutes at the end of activity

18. A regular technical check-up of the BSC should be performed annually.

http://www.asm.org/index.php?option=com_content&view=article&id=7498

Can the microbial good and evil be told apart? Yes, UCLA life scientists and an international team of researchers report in the online journal PLOS ONE.

“We have shown that a certain group of Burkholderia, which have just been discovered in the last 12 years as plant-growth promoting bacteria, are not pathogenic,” said the study’s senior author, Ann Hirsch, a professor of molecular, cell, and developmental biology in the UCLA College of Letters and Science. “This opens up the possibility of using these particular species for promoting plant growth through the process of nitrogen fixation, particularly in areas of climate change. This will have a major impact, especially on people in the developing world in producing protein-rich crops.”

Font: Bioscience Technology

One of the greatest contributions to the field of microbiology came from the kitchen. In 1881, scientist Walter Hesse was searching for a solid medium that could be used to cultivate bacteria. Unlike gelatin, the growth medium of choice at that time, the material had to be stable at high temperatures, allow a variety of microorganisms to be separated easily, and resist digestion or liquefaction by certain microbial species. Fanny Angelina, Hesse’s wife and laboratory assistant, had the answer: Agar Agar, a gelling agent that she used in her jellies and puddings. This simple kitchen ingredient revolutionized the science of microbiology, allowing the separation and culturing of microbes to become a routine procedure. Now, almost 125 years later, microbiology agar is the most important and widely used microbial growth medium available today. Fanny would be proud… but should we be proud as well?

Although the growth of microbial cells on agar surfaces provides the laboratory with critical information about the amount and the type of organisms that may be present in a sample under evaluation, the time to result is usually longer than what is desired. Days and even weeks may elapse before microbial colonies are visually detected, and in most cases, confluent growth prevents individual organisms from being isolated, necessitating sub-culture onto additional agar media, delaying the time to result even further. Additionally, many laboratories are discovering that microorganisms, when stressed due to nutrient deprivation, or following exposure to sub-lethal concentrations of antimicrobial agents, such as preservatives, disinfectants, heat or decontaminating gases, may not replicate when cultured on artificial media, because the environment is not truly optimal for the resuscitation and subsequent proliferation of organisms that may be present. For these and many other technical, quality and business reasons, the modern microbiological laboratory has been motivated toward developing innovative approaches for the detection, quantification and identification of microorganisms, and this includes the implementation of rapid microbiological methods.

The recently published Volume 4 of the Encyclopedia of Rapid Microbiological Methods is playing an important role in providing guidance for companies wishing to implement rapid methods as alternatives to conventional microbiological assays. Since the first three volumes were published in 2005, many new technologies that have been developed, validation and statistical strategies have improved, and most importantly, regulatory guidance and new policies have paved the way for an easier path to acceptance and implementation.  The following is a brief overview of what is discussed:

- The application of modern microbial methods to the Quality Control testing of probiotics, including master and working cell banks, release and stability testing, viable cell counts, identification and strain typing, and absence of bacterial pathogens.

- Considerations when aligning a RMM with an end-user’s particular needs, such as the drivers for rapid methods, time savings, same day results, sample compatibility, automation, using a qualitative method as a screening tool, validation, identification, integration with LIMS and other data management platforms.

- Overview of rapid and automated microbial identification systems, including MALDI-TOF and SELDI-TOF mass spectrometry, FT-IR, elastic and inelastic light scattering, ribotyping, PCR, gene sequencing, microfluidics and microarrays.

- A case study for using MALDI-TOF mass spectrometry for the identification of microorganisms. Sample preparation, OQ, PQ, accuracy, precision, robustness and computer validation are discussed.

- A second case study on microbial identification focusing on genotypic methods, amplification of DNA, automation and validation (accuracy, precision, robustness and specificity).

- A novel case study of a new growth-based rapid microbiological method that detects the presence of specific organisms
 and provides an estimation of viable cell count. Data from the validation studies, inclusivity and exclusivity testing, and a comparison to USP <61> are provided.

- An end-user case study that describes an evaluation of a relatively new growth-based rapid method that utilizes a membrane filtration workflow coupled with a viability staining technique. A review of the technology and evaluation results are offered followed by a discussion of the system’s use for monitoring mammalian cell cultures.

- A comprehensive end-user evaluation using an optical spectroscopy technology for the real-time and continuous monitoring of airborne microorganisms in cleanroom and isolator environments.

- Validation and workflow of an ATP bioluminescence RMM for the release testing of both sterile (sterility testing) and non-sterile products (bioburden assessments).

- An end-user’s validation approach for a rapid, growth-based detection system as an alternate sterility test for cellular immunotherapy products.

- A case study by an end-user of a rapid, solid-phase cytometry technology. The validation strategy, use of statistical models, and considerations for stressed cells and sample matrix effects are offered.

- Understanding how to use statistics when validating an alternative sterility test, including probabilities and multiplicity, limit of detection and what is statistically “different” versus what is statistically “equivalent.” This is a must read for anyone wanting to validate a rapid sterility test and how to design the studies and use statistics to justify the results.

- The use of a novel qPCR-based system for the rapid detection of specific microorganisms.

- An end-user case study using a nucleic acid amplification platform for the detection of Mycoplasma. A review of regulatory requirements for nucleic acid amplification systems is also presented.

- An analysis of the science and workflow of a new nucleic acid amplification and microarray-based rapid method for the detection of Mycoplasma.

- An end-user’s overview of rapid viral detection methods. Experiences with Vesivirus, MVM and other public viral incidents are explored, as well as other topics associated with the future directions in using molecular methods for viral detection.

- A review of biopharmaceutical manufacturing, regulations, testing requirements and contamination events, and how the application of rapid methods fits in with the future of bioprocessing.

In addition to the chapters listed above, the new volume starts with an excellent introduction by FDA’s Dr. Bryan Riley, New Drug Microbiology Staff at the Center for Drug Evaluation and Research.  Dr. Riley explains that modern approaches to process control (including Process Analytical Technology) require the availability of results in real-time (or at least close to real-time) to enable the operator to use the test results to make process decisions and adjustments. Furthermore, current rapid microbiological test methods are now able to start providing some of the advantages (from a process control and economic return standpoint) long enjoyed by our colleagues in the clinical and food microbiology labs. He concludes that pharmaceutical microbiologists would be well served by considering which of their samples would provide a benefit with a more rapid result and then assessing the current alternate microbiological methods to see if any of them are a good fit for their needs.

Volume 4 of the Encyclopedia of Rapid Microbiological Methods provides new insights, validation and implementation guidance, and most importantly, encouragement for the pharmaceutical industry to embrace the next generation of microbiology testing platforms and strategies.

- Volume 4 may be obtained through the PDA at https://store.pda.org/ProductCatalog/Product.aspx?ID=1899. 
- Volumes 1-3 may be accessed at https://store.pda.org/ProductCatalog/Product.aspx?ID=513.

By Michael J. Miller, Ph.D.
President, Microbiology Consultants, LLC and Owner of rapidmicromethods.com

http://visit.microbiologics.com/l/8752/2013-07-24/dbjn3

The purpose of the Growth Promotion Test is to determine the suitability of culture media that is used in pharmaceutical tests. The test is performed by inoculating the media with a small number of microorganisms (less than 100 colony forming units) to ensure the nutritive properties of the media are adequate to support even a small number of microorganisms. Pseudomonas aeruginosa is a fastidious microorganism that is notoriously difficult to grow. For this reason, it is an ideal strain to use for the Growth Promotion Test because it will meaningfully challenge the media.

Fonte: Microbiologics

At the center of this study is Geobacter sulfurreducens, a biofilm-forming, metal-reducing bacterium. Like other metal-reducing bacteria, Geobacter give away their electrons as part of a series of electron exchanges that drives energy, or ATP, production.

Understanding bacteria-metal electron exchange is important because it provides insight into how metals behave in their environment and how electrons might be captured to produce electricity. For their studies, the research team employed a novel electrochemical-nuclear magnetic resonance (EC-NMR) microimaging system, which allows biofilms to grow inside an NMR magnet.

EC-NMR, designed and built by EMSL staff and users, makes it possible for the first time to conduct simultaneous electrochemical and NMR studies on biofilms, revealing details about  metabolic activity, structure, porosity, nutrient movement, and nutrient concentrations in multiple dimensions.

Coupling EC-NMR with X-ray absorption spectroscopy to measure the transfer of electrons to a uranium probe for validation, the team showed that the Geobacter biofilms contained a top layer of metabolically active bacteria sitting on an inactive layer. Surprisingly, the inactive layer serves as an extension of the electrode, providing a bioscaffold that allows electrons to flow from the biofilm top to the electrode surface on which the biofilm grew. This new finding will influence the design of bioelectrochemical systems, such as for producing hydrogen or for enhancing methods to treat waste water while simultaneously generating electricity. It will also improve predictive models that describe electron transfer and provide a more rounded understanding of the behavior and role of biofilms in the environment.

Font: Environmental Molecular Sciences Laboratory

“This represents the first result of current production solely on hydrogen,” says Amit Kumar, a researcher on the study who, along with his co-authors are part of the Lovley Lab Group at the university.

Under the leadership of Derek Lovley the lab group has been studying Geobacter bacteria since Lovley first isolated Geobacter metallireducens in sand sediment from the Potomac River in 1987. Geobacter species are of interest because of their bioremediation, bioenergy potential, novel electron transfer capabilities, the ability to transfer electrons outside the cell and transport these electrons over long distances via conductive filaments known as microbial nanowires. 

Kumar and his colleagues studied a relative of G. metallireducens called Geobacter sulfurreducens, which has the ability to produce electricity by reducing organic carbon compounds with a graphite electrode like iron oxide or gold to serve as the sole electron acceptor.  They genetically engineered a strain of the bacteria that did not need organic carbon to grow in a microbial fuel cell.

“The adapted strain readily produced electrical current in microbial fuel cells with hydrogen gas as the sole electron donor and no organic carbon source,” says Kumar, who notes that when the hydrogen supply to the microbial fuel cell was intermittently stopped electrical current dropped significantly and cells attached to the electrodes did not generate any significant current.

This research was supported by funding by the U.S. Department of Energy and the Office of Naval Research.

# # #

This research was presented as part of the 2013 General Meeting of the American Society for Microbiology held May 18-21, 2013 in Denver, Colorado.  A full press kit for the meeting, including tipsheets and additional press releases, can be found online at http://bit.ly/asm2013pk

“In this work, we used synthetic biology approaches to probe and rewire photoautotrophic (exclusively relying on carbon dioxide and light energy for growth) cyanobacterial metabolism for the ability to grow without light energy,” says Jordan McEwen, the lead researcher on the study.  He is part of Shota Atsumi’s lab at the university, a research group focused on developing synthetic organisms capable of converting carbon dioxide directly to biofuels.

The cyanobacterium strain Synechococcus elongatus strain PCC 7942 has been well characterized as a model photoautotroph.  Previous work by Atsumi’s lab has engineered this organism to recycle carbon dioxide into a variety of biofuels and valuable chemicals in the presence of light.  Any cost-effective, cyanobacterial biofuel production scheme would use natural lighting conditions, limiting how much biofuel could be produced in a 24-hour period.

“To overcome this constraint, we installed foreign genes into S. elongatus to allow this cyanobacterium to grow and generate biofuels in diurnal (light or dark) conditions,” says McEwen.  “With recent, increased focus on cyanobacteria-based industrial applications, this advancement is desirable for more efficient, economical and controllable bioproduction systems.”

This work was funded by a grant from the National Science Foundation (1132442).

# # #

This research was presented as part of the 2013 General Meeting of the American Society for Microbiology held May 18-21, 2013 in Denver, Colorado.  A full press kit for the meeting, including tipsheets and additional press releases, can be found online at http://bit.ly/asm2013pk.

The American Society for Microbiology is the largest single life science society, composed of over 39,000 scientists and health professionals. ASM’s mission is to advance the microbiological sciences as a vehicle for understanding life processes and to apply and communicate this knowledge for the improvement of health and environmental and economic well-being worldwide

“It was a great surprise to find the nitrogen-rich crystals inside the algae,” says corresponding author Anders Meibom of the École Polytechnique Fédérale de Lausanne, Switzerland. “Itall makes perfect sense now. The algae suck up the ammonium and nitrate like a sponge when the concentration of these molecules increases, then store this nitrogen as uric acid crystals for later use.”  

Like all reef-forming corals, the species they studied, Pocillopora damicornis, is actually a symbiosis of two different organisms: the coral provides protection to a species of photosynthetic algae called dinoflagellates, which, in turn, provide sugars and nitrogen to the coral host. The symbiosis allows the coral to thrive in clear, tropical waters that are naturally nutrient-poor. In many places, however, coral reefs are suffering from an excess of nutrients – pollution from sewage and fertilizers that impacts the symbiotic relationship and the health of coral in unknown ways.

To better understand these exchanges of materials and to determine how an excess of nutrients might affect the balance, the researchers exposed pieces of coral to varying concentrations of isotopically-labeled nitrogen-rich compounds. Using the facilities at the Aquarium Tropicale Porte Dorée in Paris, France, the scientists applied a relatively new analytic technique called nano-scale secondary ion mass-spectrometry (NanoSIMS) to follow the path of the nitrogen. NanoSIMS enabled them to visualize and quantify the uptake, movement, and accumulation of this labeled nitrogen within the coral.

When supplied with nitrogen in the form of ammonium, nitrate or aspartic acid the dinoflagellates responded by rapidly storing the nitrogen as crystals of uric acid within its cells. But the dinoflagellates don’t hang onto the nitrogen for long. Starting at about six hours after exposure, the microbes begin translocating nitrogen-rich compounds to the coral host, where the nitrogen is used in specific cellular compartments all over the surface layers of the coral.

This storage and release process helps explain how these corals get through the ups and downs of nitrogen concentrations, says Meibom. “This gives the coral-algae symbiosis a very efficient way to deal with strong fluctuations in nitrogen availability,” writes Meibom. “When the nitrogen availability suddenly becomes high, the algae can take-up large amounts of nitrogen on a timescale of a few hours, store it into crystals inside the algae cells and then release this stored nitrogen for metabolic processes and growth when the nitrogen levels become normal again.”

To follow up on this work, Meibom says he and his colleagues are now studying how carbon-based nutrients are taken up and distributed in the same coral-algae symbiosis.

The article can be found online at http://bit.ly/mbio0513b.

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