Thursday 30 April 2015

Meaningful Performance Metrics for Compliance

A free chapter, from the book "Achieving Quality and Compliance Excellence in Pharmaceuticals: A Master Class GMP Guide", titled "Meaningful Performance Metrics for Compliance", written by Roger Janczak, can be accessed for free here.

The focus is:

Performance metrics are an essential element of the management review process. A well designed set of metrics enables managers to evaluate the performance of their business processes, make decisions based on the evaluation and take action. Quality metrics may include elements such as customer satisfaction, supplier performance, manufacturing defects, complaints, cycle times and many other internal or external processes. This chapter provides a framework for establishing the right quality indicators/metrics for evaluating the performance of the quality system. The primary objective of this chapter is to help the reader in designing appropriate tools that can assess the health of the quality system and to find the problems, ideally before they mature into a quality defect and fix them.

Posted by Tim Sandle

Wednesday 29 April 2015

Pharmig News #59

The latest edition of the Pharmaceutical Microbiology Interest Group (Pharmig) newsletter has been published.

In the current edition (#59) there is an article on the sanitization of pharmaceutical facilities by Tim Sandle, a profile of Julie Roberts, and an extensive round-up of industry and regulatory trends.

Copies have been sent to member organizations. To request a copy or to inquire about membership of Pharmig please contact the Pharmig office.

Tim Sandle's article is:

Sandle, T. (2015) Sanitization of Pharmaceutical Facilities, Pharmig News, number 59, pp2-7

Posted by Tim Sandle

Reverse osmosis for Water-for-Injections

The European Pharmacopeia is set to allow reverse osmosis to be used to manufacture Water-for-Injection (WFI) for the first time. Hitherto, only distillation has been permitted.

To explain what is involved, the European Commission has released a concept paper titled “Reverse osmosis in Ph. Eur. monograph Water for injections (0169).”

The introduction to the paper reads:

“The production of WFI is described in the European Pharmacopoeia (Ph. Eur.) monograph Water for injections (0169). In this monograph the method of production of WFI is limited to distillation only. This is currently distinct from the production methods described in the United States Pharmacopeia monograph, which allows for production of WFI by distillation or a purification process proven to be equal to or superior to distillation, and in the Japanese Pharmacopoeia, which allows for distillation or reverse osmosis (RO) followed by ultrafiltration (UF).

In the pharmaceutical industry, distillation has been the dominant method for producing WFI due to its ability to meet the required specifications and in part due to the regulatory environment. However, other industries with a requirement for high quality water, rather than distillation, employ RO and UF to produce water that is equivalent to or of a better quality than WFI described in the Ph. Eur.”

The paper can be accessed here.

You need to register for a copy. Alternatively please email Tim Sandle.

Posted by Tim Sandle

Tuesday 28 April 2015

Disease and health Infographics

Bioquell have issued as series of well-illustrated infographics relating to disease and health matters. The charts are available for download and make for a good educational resource.

The charts can be found here.

Posted by Tim Sandle

Monday 27 April 2015

Are bacteria as individual as people?

Bacteria are as individual as people, according to new research by Professor Peter Young and his team in the Department of Biology at the University of York. Bacteria are essential to health, agriculture and the environment, and new research tools are starting to shed more light on them.

The York team dug up a square meter of roadside verge on the University’s campus in search of a bacterium called Rhizobium leguminosarum. The name means "root dweller of the legumes," and these bacteria are natural fertilizer factories that extract nitrogen from the air and make it available to peas, beans, clover and their wild relatives.

In the laboratory, the team extracted the bacteria from the plant roots and established 72 separate strains. They determined the DNA sequence of the genome of each strain. Their research, published in Open Biology, shows that each of those 72 strains is unique -- each has different genes and is capable of growing on different food sources.

People are unique because each of us inherits half our genes from our mother and half from our father, but bacteria reproduce by binary fission, making two identical daughters. What bacteria are good at, though, is passing packages of genes from one cell to another. It is this process of horizontal gene transfer that made every rhizobium unique.

"We can think of the bacterial genome as having two parts," says Professor Young. "The core genome does the basic housekeeping and is much the same in all members of the species, while the accessory genome has packages of genes that are not essential to the operation of the cell, but can be very useful in coping with aspects of the real world.

"Bacteria are like smartphones. Each phone comes out of the factory with standard hardware and operating system (core genome), but gains a unique combination of capabilities through apps (accessory genes) downloaded through the internet (by horizontal gene transfer)."
We increasingly recognize the vital roles played by bacterial communities, such as those in our gut or on the roots of plants. Many researchers have used variation in a standard core gene to draw up lists of the species in a community, but the new research shows that a list of names is not sufficient.

"There may be 300 people called Baker in your city, but you can't assume that there are 300 people baking bread," explains Professor Young.

It is possible, with more sequencing effort, to look at all the genes in a bacterial community -- an approach called "metagenomics" -- but to understand how they are functioning, we also need to know which genes occur together in the same bacterium. This new study helps us to understand the way in which bacterial genomes are assembled.

Story Source: University of York.

Journal Reference:
Nitin Kumar, Ganesh Lad, Elisa Giuntini, Maria E. Kaye, Piyachat Udomwong, N. Jannah Shamsani, J. Peter W. Young, Xavier Bailly. Bacterial genospecies that are not ecologically coherent: population genomics of Rhizobium leguminosarum. Open Biology, January 2015 DOI: 10.1098/rsob.140133

Sunday 26 April 2015

Solid Dosage and Excipients eBook

Pharmaceutical Technology has released an eBook called “Solid Dosage and Excipients.” The examines how advances in technology, equipment, excipients, analytical techniques, software, and manufacturing practices are facilitating the development, scale up, manufacturing, efficacy, and delivery of solid- and semi-solid dosage forms.

Issue topics include:
  • Advances in Tableting
  • Analytical Techniques for Efficient OSD Formulation
  • Improving Pharmaceutical Palatability
  • Improving Efficiency in Hot Melt Extrusion and Spray Drying
  • Ionic Liquids and Formulations
  • IPEC Quality Agreements
  • New Technologies for Solid Dose Manufacturing

 For details see: Pharmaceutical Technology

Posted by Tim Sandle

Saturday 25 April 2015

Garlic Compound Eliminates Pseudomonas Biofilms

Pseudomonas aeruginosa is the predominant organism of chronic lung infections in cystic fibrosis (CF) patients. The organism is particularly difficult to treat because of its ability to colonize the lungs of its hosts and form a biofilm. Biofilms are far more resistant to antibiotic therapy than individual bacterial cells. Consequently, patients with CF are especially vulnerable to infection by P. aeruginosa and, despite intensive treatment, the organism is responsible for a high rate of morbidity and mortality.

There are several opportunistic pathogens, such as P. aeruginosa, capable of utilizing their arsenal of virulence factors in an organized fashion. These pathogens have evolved a chemical language referred to as quorum sensing (QS). QS enables bacterial cells to communicate more effectively, to keep track of their numbers and to minimize host responses until sufficient cell numbers have amassed to overwhelm the host immune system. Therefore, despite rigorous antibiotic therapy, CF patients have a life expectancy of about 40 years, with the main cause of death being complications associated with chronic P. aeruginosa infection.

However, there may now be a glimmer of hope in the fight against biofilms that is of particular significance to CF patients. Researchers at the University of Copenhagen and their collaborators have developed a mechanism to identify sources of potential, non-toxic QS-inhibitor compounds in certain foods. The presence of these naturally occurring compounds suggests that diet may offer a more holistic prophylaxis against infection by P. aeruginosa.

Garlic provides benefits to the cardiovascular and immune systems, but it is also an effective anti-fungal, anti-cancer, anti-viral, anti-protozoan and anti-microbial compound. Now, the team of researchers has identified a constituent of garlic that works to block QS-properties required for biofilm formation. The garlic-derived QS-inhibitor, ajoene, is the major constituent of sulfur-containing compounds released when garlic is crushed. During the study, ajoene was found to inhibit the expression of QS-controlled bacterial genes and, furthermore, worked to reduce the production of bacterial rhamnolipid, a key component in shielding biofilms from white blood cells. Moreover, by combining ajoene with tobramycin, the team was able to demonstrate a 90 percent reduction in biofilm forming cells and a rapid clearing of pulmonary P. aeruginosa infection in mice.

With documented success, the team now hopes to find a pharmaceutical partner to develop antimicrobial drugs based on ajoene that will provide a more effective treatment solution for CF patients. The notion of moving beyond the conventional concept of killing bacterial cells to a more dynamic approach of blocking bacterial communication may prove immediately beneficial in the treatment of P. aeruginosainfections, while paving the way for more advanced developments in antibiotic therapy.
Posted by Tim Sandle

Friday 24 April 2015

The Changing Role of the Pharmaceutical Microbiologist

A shift of seismic proportion is happening...but there is no need to gaze down at your feet. The role of the Pharmaceutical Microbiologist is slowly shifting from the perplexed tester to the perplexed risk assessor.

I've watched this change, from my vantage point, over the past twenty years. From when I began my career in microbiology as a bottle washer (well, my first task in a Microbiology laboratory was removing labels from media bottles which were to be recycled) to my current role heading up the microbiological function at a major pharmaceutical manufacturer.

Whether this change is driven by regulators or by Quality Assurance (QA) or by microbiologists themselves, struggling to complete a massive program of work offset against struggles to purchase equipment and the continual juggling of human resources, is arguable. What is clear is that there has been a shift of emphasis from testing towards risk assessment; from the pharmaceutical microbiologist at the bench (chained or otherwise) to the pharmaceutical microbiologist out in the factory.

The current ethos is to spend less time testing and accumulating a mass of data which is never properly analyzed or studied for trends towards more time formulating corrective and preventative actions and performing microbiological risk assessments.

The microbiologist is now called upon to have a far greater knowledge of physical parameters. For example, can the significance of results from a clean room, whether viable micro-organisms or non-viable particles, be truly understand without an understanding of other physical parameters? Physical tests, such as pressure differentials, clean-up times, airflows frame the context of the microbiological result. Likewise the microbiologist is required to have a greater understanding of engineering and engineering systems. For example, in assessing the results from a purified water system some knowledge of flow rates, valve design, re-circulation, heating and piping is required.

Once a sample has been read and speciated (and been taken through a reasonably lengthy confirmation that it is not a laboratory error) further evaluation is required as part of the out-of-limits (OOL) procedure. OOL is a more preferable term than out-of-specification (OOS): it is an anathema to the microbiologist to be told that one erroneous surface RODAC plate result is an OOS! Old philosophies of test, re-test (and carry on re-testing) until a satisfactory result is obtained are redundant approaches or much reduced in emphasis. The new language is that of risk assessment.

The types of risk assessment that the microbiologist is required to become involved with are either an assessment of the significance of an above action level result where corrective and preventative actions (CAPA) are employed. Or, more commonly, an assessment of the controls and measures in place to ensure that the above action result does not occur in the first place. In other words being proactive rather than reactive.

Tools for performing such assessments include risk analysis tools borrowed from other industries or professions including HACCP (hazard analysis critical control points) from the food industry; FMEA (failure modes and effects analysis) and FTA (fault tree analysis) taken from engineering industries, such as, car production. These approaches share a number of things in common:

• Constructing diagrams of work flows
• Pin-pointing areas of greatest risk
• Examining potential sources of contamination
• Deciding on the most appropriate sample methods
• Helping to establish alert and action levels
• Taking into account changes to the work process / seasonal activities

In order to understand these and to assess them it is important that the microbiologist builds up detailed knowledge of the production system and processes and gets to walk around the factory and manufacturing environment.

An example of using these approaches can be applied to environmental monitoring in establishing a testing regime:

• Monitoring in areas which have a more 'dirty' activity taking place in an adjacent room
• Varying the frequencies for surface monitoring compared to viable air monitoring
• Examination of the movement of people (corridors and changing rooms are often routes of the spread of contamination and a monitoring program may focus more heavily on these areas)
• Assessing routes of transfer / in-coming goods
• Focusing on key areas like component preparation
• Having higher frequencies of monitoring for areas at ambient temperature with high amounts of water compared to cold rooms
• Intensifying monitoring towards final formulation / purification / secondary packaging / product filling
• Establishing a monitoring program designed to test the effectiveness of cleaning regimes
• More frequent monitoring for open, compared to closed, processes
• Monitoring areas of potential contamination, for example door handles

One approach may be to establish a 'criticality factor' where different rooms, with different activities, can be rated. Therefore one room where product purification takes place, would be given a higher criticality factor and be monitored weekly, whereas a wash-up area, would be given a lower criticality factor, and be monitored monthly.

Therefore, in my view, the role of the pharmaceutical microbiologist has changed. Many of the techniques for testing remain the same but it is the way that the data is used and the necessary pre-thinking before the testing begins which is different. The only thing lagging behind is the status of the microbiologist in the organization. This person, one most able to offer a global view and to assess the impact of process and contamination risk, is too often found hidden in the laboratory.

Posted by Tim Sandle

Wednesday 22 April 2015

The Human Microbiome: Beyond the Gut

From The Scientist - FREE Webinar — Tuesday May 5, 2015. 2:30 p.m. - 4:00 p.m. Eastern Time

The human body hosts myriad distinct bacterial colonies that play important functions in both health and disease. These dynamic microbial colonies fluctuate over time and even form niche colonies in close proximity to one another. This webinar will highlight new research on the human microbiome and how this knowledge is being used to understand health and disease. The panelists will discuss advances in research on lung and skin microbiome. Attendees will have an opportunity to interact with the experts, ask questions, and seek advice on topics that are unique to their research.

For details, see The Scientist

Posted by Tim Sandle

Importance of Hand Santisation

Hands, whether gloved or ungloved, are one of the main ways of spreading infection or for transferring microbial contamination. The use of hand disinfectants is part of the process of good contamination control for personnel working in hospital environments, or those involved in aseptic processing and within cleanrooms. Although there are many different types of hand sanitizers available there are differences with their effectiveness and several do not meet the European standard for hand sanitization.

Personnel working in hospitals and cleanrooms carry many types of microorganisms on their hands and such microorganisms can be readily transferred from person to person or from person to equipment or critical surfaces. Such microorganisms are either present on the skin not multiplying (transient flora, which can include a range of environmental microorganisms like Staphylococcus and Pseudomonas) or are multiplying microorganisms released from the skin (residential flora including the genera of Staphylococcus, Micrococcus and Propionibacterium). Of the two groups, residential flora are more difficult to remove. For critical operations, some protection is afforded by wearing gloves. However gloves are not suitable for all activities and gloves, if not regularly sanitized or if they are of an unsuitable design, will pick up and transfer contamination.

Therefore, the sanitization of hands (either gloved or ungloved) is an important part of contamination control either in hospitals, to avoid staff-to-patient cross contamination or prior to undertaking clinical or surgical procedures; and for aseptic preparations like the dispensing of medicines. Moreover, not only is the use of a hand sanitizer needed prior to undertaking such applications, it is also important that the sanitizer is effective at eliminating a high population of bacteria. Studies have shown that if a low number of microorganisms persist after the application of a sanitizer then the subpopulation can develop which is resistant to future applications.

There are many commercially available hand sanitisers with the most commonly used types being alcohol-based liquids or gels. As with other types of disinfectants, hand sanitizers are effective against different microorganisms depending upon their mode of activity. With the most common alcohol based hand sanitizers, the mode of action leads to bacterial cell death through cytoplasm leakage, denaturation of protein and eventual cell lysis (alcohols are one of the so-called 'membrane disrupters'). The advantages of employing alcohols as hand sanitizers include a relatively low cost, little odour and a quick evaporation (limited residual activity results in shorter contact times). Furthermore alcohols have a proven cleansing action.

In selecting a hand sanitiser the pharmaceutical organisation or hospital will need to consider if the application is to be made to human skin or to gloved hands, or to both, and if it is required to be sporicidal. Hand sanitisers fall into two groups: alcohol based, which are more common, and non-alcohol based. Such considerations impact both upon cost and the health and safety of the staff using the hand sanitiser since many commonly available alcohol based sanitisers can cause excessive drying of the skin; and some non-alcohol based sanitisers can be irritating to the skin. Alcohol hand sanitizers are designed to avoid irritation through possessing hypoallergenic properties (colour and fragrance free) and ingredients which afford skin protection and care through re-fatting agents.

Alcohols have a long history of use as disinfectants due to inherent antiseptic properties against bacteria and some viruses. To be effective some water is required to be mixed with alcohol to exert effect against microorganisms, with the most effective range falling between 60 and 95% (most commercial hand sanitizers are around 70%). The most commonly used alcohol based hand sanitisers are Isopropyl alcohol or some form of denatured ethanol (such as Industrial Methylated Spirits). The more common non-alcohol based sanitisers contain either chlorhexidine or hexachlorophene. Additives can also be included in hand sanitizers in order to increase the antimicrobial properties.

Before entering a hospital ward or clean area hands should be washed using soap and water for around twenty seconds. Handwashing removes around 99% of transient microorgansisms (although it does not kill them) (4). From then on, whether gloves are worn or not, regular hygienic hand disinfection should take place to eliminate any subsequent transient flora and to reduce the risk of the contamination arising from resident skin flora.

The technique of hand sanitisation is of great importance as the effectiveness is not only with the alcohol but also relates to the 'rub-in' technique. For example:

-Dispense a small amount of hand gel onto the palm of one hand by
-pressing down on the pump dispenser
-Put hands together and proceed to rub the hand gel into both hands. Pay particular attention to the following areas:
-Back of hands
-Between webs of fingers
-Allow hands to dry, this should take no more than 60 seconds

Regular applications of the hand sanitizer are required and also prior to carrying out critical activities. This is because alcohols are relatively volatile and do not provide a continual antimicrobial action. Although microorgansisms are removed from material like latex more readily than from skin, a regular frequency of hand sanitization should still be applied to gloves.

There are very few safety concerns with hand sanitizers and the occupational exposure is relatively low, although this can build up in enclosed spaces. Care should be taken when using sanitizers near naked flames (which can occur where gas burners are used in laboratories).

In conclusion, hand sanitisation is an important procedure for staff to follow in healthcare and pharmaceutical settings. Hand sanitization is one of the main methods for preventing the spread of infection in hospitals and contamination within pharmaceutical operations. This required level of control requires the use of an effective hand sanitizer.

Posted by Tim Sandle

Tuesday 21 April 2015

April is Oral Cancer Awareness Month

Close to 43,250 Americans will be diagnosed with oral or pharyngeal cancer this year. April is Oral Cancer Awareness Month, what can patients without dental insurance do to check and prevent oral cancer? A national dental plan rolling out across America is changing the way we view dental insurance and helping those who are in need of insurance. Quality Dental Plan is an affordable family dentistry that opens its doors to those who are unemployed or do not have insurance. It is a national company that is run by local dentist to help promote good dental health and understand the importance of dental care for both children and adults.

Posted by Tim Sandle

Introduction to Disinfectants

A disinfectant is a chemical agent that is used to reduce the number of viable microorganisms on pharmaceutical surfaces to an acceptable level. Disinfectants have a variety of properties that include spectrum of activity, mode of action, and effectiveness. Some are bacteriostatic, where the ability of the bacterial population to reproduce is halted. In this case, the disinfectant can cause selective and reversible changes to microbial cells by interacting with nucleic acids and inhibiting enzymes, or permeating into the cell wall. Once the disinfectant is removed from contact with bacterial cells, the surviving bacterial population can potentially grow. Other disinfectants are bactericidal in that they destroy bacterial cells and cause irreversible damage through different mechanisms that include structural damage to the cell, cell lysis, and autolysis, resulting in leakage or coagulation of cytoplasm. The destruction of bacterial and fungal spores is a property which a given disinfectant may or may not possess. This type of chemical agent is called a sporicide. A chemical agent does not have to be sporicidal in order to be classified as a 'disinfectant' or as a 'biocide'. The bacteriostatic, bactericidal and sporicidal properties of a disinfectant is influenced by many variables.

Disinfectants can be categorized into groups by chemical nature, spectrum of activity, or mode of action. Some disinfectants, on entering the microbial cell either by disruption of the membrane or through diffusion, proceed to act on intracellular components. Actions against the microbial cell include: acting on the cell wall, the cytoplasmic membrane (where the matrix of phospholipids and enzymes provide various targets) and the cytoplasm. This section provides a summary some of the more common disinfectants used the pharmaceutical environment. The two principle categories consist of non-oxidizing and oxidizing disinfectants.

Non-Oxidizing Disinfectants: The majority of disinfectants in this group have a specific mode of action against microorganisms and generally have a lower spectrum of activity compared to oxidizing disinfectants. These disinfectants include alcohols. Alcohols have an antibacterial action against vegetative cells. The effectiveness of alcohols against vegetative bacteria increases with their molecular weight (i.e., ethanol is more effective than methanol and in turn isopropyl alcohols are more effective than ethanol). Alcohols, where efficacy is increased with the presence of water, act on the bacterial cell wall by making it permeable. This can result in cytoplasm leakage, denaturation of protein and eventual cell lysis (alcohols are one of the so called 'membrane disrupters'). The advantages of using alcohols include a relatively low cost, little odor and quick evaporation. However, alcohols have very poor action against bacterial and fungal spores and can only inhibit spore germination at best.

Oxidizing Disinfectants: This group of disinfectants generally has non-specific modes of action against microorganisms. They have a wider spectrum of activity than non-oxidizing disinfectants with most types able to damage bacterial endospores. The disinfectants in this group pose greater risks to human health. This group includes oxygen-releasing compounds like peracetic acid and hydrogen peroxide. They are often used in the gaseous phase as surface sterilants for equipment. These peroxygens function by disrupting the cell wall causing cytoplasm leakage and can denature bacterial cell enzymes through oxidation. Oxidizing agents are clear and colorless, thereby eliminating staining, but they do present significant health and safety concerns particularly in terms of causing respiratory difficulties to unprotected users.

Posted by Tim Sandle

Monday 20 April 2015

Pharmig speaking at Making Pharmaceuticals

Pharmig are presenting on three subjects at Making Pharmaceuticals on April 28, 2015.

Making Pharmaceuticals brings together pharmaceutical developers, R&D, manufacturers, contract manufacturers, quality engineers & microbiologists, process engineers, licence holders and brand owners with suppliers of equipment, services, expertise and know-how to address the complex demands in the developmental life cycle leading to successful commercial manufacturing of pharmaceuticals.

The event is free to attend and it is taking place on April 28 and 29 at the National Motorcycle Museum in Birmingham, U.K.

The Pharmig seminars are:

09:15   -          Best Practices for the Environmental Monitoring Programme, presented by Tim Sandle
09:35   -           Regulatory Requirements for Disinfectants, presented by Rachel Blount
09:55   -           Microbiological Aspects of Risk Assessment, presented by Tim Sandle

Certificates of Attendance will be available on site for anyone who needs or can use them for CPD purposes.

For further details and to register, see Making Pharmaceuticals.

Posted by Tim Sandle

Key factors affecting cleanroom wipes

Cleanroom wipes are among the most important consumables in the cleanroom environment and are integral to the cleaning process in relation to removing contaminants.

To ensure efficient cleaning, cleanroom wipes must be able to absorb a wide range of surface contaminants in a relatively quick time and without themselves distributing further pollution in the form of particles, fibres, ions or microbiological contaminants

With wipes, there are several key issues:
  • The wipe must have a low bioburden or be sterile
  • The efficacy of the disinfectant when impregnated into the wipe should be known, since this could be less efficacious than the disinfectant in solution
  • The particle load and particle generation should be assessed, especially for wipes to be used in cleanrooms
  • The efficacy of the wipe on different types of surfaces should be understood
  • The wiping technique must be standardised 
In relation to this, Tim Sandle has written an article for the journal European Medical Hygiene. The reference is:

Sandle, T. (2015) Cleanroom Wipes, European Medical Hygiene, Issue 9, pp24-29

If you are interested in reading a copy, please contact Tim Sandle

Posted by Tim Sandle

Sunday 19 April 2015

Identifying Bacteria - Introducing the Gram Stain

Microorganisms found in pharmaceutical and healthcare environments require identification in order to determine the species. This is important so that the origin of contamination can be assessed and the origin of contamination determined. This is commonly performed by using a standing technique called the Gram stain, which is based is a type of "phenotypic identification method" and it undertaken so that the microbiologist can understand the general profile for microorganisms.

The first step of most identification schemes is to describe the colony and cellular morphology of the microorganism. Colony morphology is normally described by directly observing growth on agar, where the colony will appear as a particular shape (such as raised, crenated, spherical and so on) and the colony will have a particular pigment. Some microbiologists will attempt to identify the microorganism based on such visual identification. This is not normally encouraged as considerable experience is required to do this and the variety of microflora cannot be characterised with any degree of accuracy. Furthermore, the characteristics of a microorganism are often dependent upon the type of culture medium used. Nevertheless, a description of the morphology can assist with further stages of identification.

Cellular staining provides important information relating to the composition of the microbial cell wall, as well as the shape of the organism. Of these, the most frequently used method is the Gram stain.
The Gram stain method employed includes the four-step technique: Crystal violet (primary stain); iodine (mordant); alcohol (decolorizer); and safranin (counter stain). Done correctly, Gram-positive organisms retain the crystal violet stain and appear blue; Gram negative organisms lose the crystal violet stain and contain only the counter-stain safranin and thus appear red. Common pitfalls in this method are that heat fixation may cause Gram-positive cells to stain Gram-negative and older cultures may give Gram-variable reaction; using too much decolorizer could result in a false Gram-negative result and not using enough decolorizer may yield a false Gram-positive result.

The Gram reaction is based on the differences in the cell wall composition for the two cellular 'groups'. The bacteria that retained the stain (the Gram-positive bacteria) have a higher peptidoglycan and lower lipid content than those that do not retain the stain (the Gram-negative bacteria). The effect of the solvent is to dissolve the lipid layer in the cell wall of the Gram-negative bacteria, thereby causing the crystal violet to leach out; whereas for Gram-positive bacteria the solvent dehydrates the thicker cell walls, blocking any diffusion of the violet-iodine complex, which closes the pores of the cell and retains the stain. There are now several automated Gram stain devices available on the market that can reduce the labour requirement required when performing several multiple Gram stains and, possibly, improve accuracy.

In addition to the difference based on cell wall, microscopic examination of the stains allows the cellular shape to be determined. Bacteria commonly fall into the categories of coccus (spherical), rod, vibrio (curved), spirilla (spiral) and plemomorphic (variable).

Posted by Tim Sandle

Saturday 18 April 2015

Malaria cells produce odors that attract mosquitoes

Malaria causing parasites produce chemical compounds that give off odors. These odors attract mosquitoes to come and bite an infected animal, thereby ensuring the cycle of infection continues.

Scientists have discovered that one malaria causing parasite Plasmodium falciparum produces chemical compounds called terpenes. Terpenes are a large and diverse class of organic compounds. In the chemical industry, they are the major components of resin, and of turpentine produced from resin.
These chemicals are key to producing the odors that attract mosquitoes. These chemicals have been detected in the sweat and breath of people who have malaria.
The researchers detected the chemical traces by infecting human blood cells with the parasites and holding them, with growth media, in air-tight bags. The gas produced by the respirating cells was then sampled and send for chemical analysis.
Further investigation showed that the parasite uses a chemical pathway (termed MEP) to produce the terpenes. Two terpenes were dominant: one called limonene (which has a slight lemon-y smell) and pinanediol (which has a slight pine-like odor). These smells appeal to mosquitoes and the insects are equipped to pick these out from distance.
It is hoped that the discovery will lead to the development of a breathalyzer test to determine if someone has malaria. Such tests would be far easier, and more pleasant for the patient to endure, than a blood test.
The information should also help scientists to understand why one person becomes infected with malaria and another does not, or why some people are more prone to being bitten by mosquitoes that others.
The research has been published in the journal mBio. The paper is titled “Malaria Parasites Produce Volatile Mosquito Attractants.”
In related news, mosquitoes carrying "tropical diseases" could become widespread across the U.K. over the next 20 or 30 years. This is assuming that European temperatures continue to rise.

Posted by Tim Sandle

Friday 17 April 2015

Canada Launches GMP Inspection Database

Canada's Health Minister Rona Ambrose announced the launch of a new publicly accessible database for manufacturing inspections on 13 April 2015.

The new Drug and Health Product Inspection Database will allow Canadians to search for timely information on good manufacturing practice (GMP) inspections conducted by Health Canada.

The press release states that the new database will contain information on the inspections the regulator conducted since 2012. Health Canada says it will still update the inspection tracker it launched in March to keep tabs on "emerging issues."

The Drug and Health Product Inspection Database is designed for use by the general public and features "plain language [and] timely information on inspections." Users can search the database by establishment name, reference number, location, inspection dates and compliance rating.

Once a search has been initiated, users can select the establishment they wish to view for details about the establishment, including the number of times the site has been inspected in the past three years. From there they can select the establishment's rating to see a summary of the inspection, including the observations cited in the inspection report.

Posted by Tim Sandle

Thursday 16 April 2015

Unusual bacteria discovered in deep ocean trench

Researchers from Japan discovered microscopic bacteria thrive in the canyon called Challenger Deep, which is the lowest point on Earth's surface and the deepest part of the Mariana Trench, the team reports in the journal Proceedings of the National Academy of Sciences. In particular, they found an unusual community of bacteria there called heterotrophs, or microbes that cannot produce their own food and must eat what they find in the water.

The heterotrophs in the Challenger Deep likely derive food from sinking particles, such as dissolved fecal pellets or dust, or possibly from geologic processes such as earthquake-triggered landslides, which could send organic-rich sediments tumbling into the canyon's depths.

The research found that ocean's microbial diversity varied with depth. Genetic-fingerprinting techniques identified different microbes based on certain genes, and also indicated the relative abundance of different species.

The ocean's invisible life was found at all depths, but microbes were most abundant near the surface and on the ocean floor, where they can find the most food. The ocean was stratified into layers, with a warm, salty layer on top and a colder, less salty layer starting about 1,300 feet (400 m) below the surface. The deepest water was about 1 degree Celsius (34 degrees Fahrenheit).

Plantlike phytoplankton crowded the surface waters. (Light only penetrates into the upper 328 feet, or 100 m, of the ocean.) Chemolithotrophs, or microbes that survive by converting compounds such as sulfur and ammonia into food, were abundant in the nutrient-poor abyssal zone but declined below a depth of 19,685 feet (6,000 m), to be replaced by heterotrophs, the study found. The abyssal zone ranges from 6,560 feet to 16,400 feet (2,000 m to 5,000 m).

For further details see Microbe World.

Posted by Tim Sandle

Wednesday 15 April 2015

Dissemination of Scientific Awareness through Digital media

These are exciting times to be communicating science as developments in technology, increasing de-regulation and the legacy of previous high-profile science-based issues combine to produce new opportunities for dialogue, engagement and deliberation.

Microbioz India has an interesting article about the use of digital media for the promotion of science news.

Here is an extract:

"At present our society depends on scientific awareness about disease and other discovery and using of this knowledge through different ways in his common life.The important role of science in our daily schedule is only mobilized when new discoveries raises ethical questions. For these reasons, the public needs to be properly informed, so that it can make up its mind on the issues.In present era the digital scientific media focuses in building of huge level of audience ships which no doubt plays a great role in dissemination of science across the globe."

The complete article can be found here.

Posted by Tim Sandle

Use of fungi to make bio-ethanol

Fungi that digest wood in novel ways could fuel new avenues of research on cellulosic ethanol, and suggest a need to move beyond traditional classification systems.

Fungi that digest wood are typically categorized as white rots, which degrade both lignin and cellulose, or brown rots, which only have enzymes that act on cellulose. But two newly sequenced species are capable of digesting lignin, even though they lack the enzymes typically found in white rots, according to a study published recently in PNAS.

The species, Botryobasidium botryosum and Jaapia argillacea, appeared to be white-rot fungi based on the microscopic patterns they created in decomposing wood. However, at the molecular level, researchers found that the key enzymes considered markers of white-rot fungi were missing.

The results suggest a continuum rather than a dichotomy between the white-rot and brown-rot modes of wood decay, and highlight the need for a more nuanced categorization of rot types, according to the authors. Identifying the decay mechanisms in these new species could also have practical applications in the production of cellulosic biofuels.

The research is titled “Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi.”

Posted by Tim Sandle

Tuesday 14 April 2015

HEPA filters in safety cabinets

Control of airborne particulates in indoor environments is critical to develop quality products, protect employees from contact with hazardous materials, or prevent health problems from prolonged exposure to allergens. How airborne particulates are controlled varies from industry to industry and from an occupational setting to a home environment. To better understand why HEPA filters are used in the biological safety cabinet industry, it is necessary to explore particle sizes, types of filters available for home and occupational use, efficiency and penetration, filter standards and performance testing.

In relation to this topic, Suzy Whitt has written a paper about biological safety cabinets. The paper can be accessed here.
Posted by Tim Sandle

Monday 13 April 2015

Teixobactin: A New Class of Antibiotic

A new antibiotic has been discovered: Teixobactin. This in itself represents a significant breakthrough since it is the first new type of antibiotic to be discovered since the late 1980s. The discovery carries more significance especially in the era of antibiotic crisis, in which a rising number of microbial strains are evolving resistance to common antibiotic (and in the case of a narrower but more potent range, “multi-drug resistant” which infers resistance to more than one type of antibiotic.) Though we welcome the new antibiotic, their discovery reports are addressed in much of the popular media (and, unfortunately, in some of the scientific press) are always tainted by hyperbole. (And, unfortunately, in some of the scientific press) is tainted with hyperbole. 

This is the opening of a new paper by Tim Sandle, considering the issues around the headline grabbing new antibiotic discovery.

The reference is:

Sandle, T. (2015) Teixobactin: A New Class of Antibiotic, SOJ Microbiology & Infectious Diseases, 3 (1): 1-2

To view a copy, please see: Antibiotic

Posted by Tim Sandle

Sunday 12 April 2015

Plants detect bacterial endotoxin in similar process to mammals

Plants possess an innate immune system that protects them from invading pathogens. Molecular structures that only occur in pathogens enable their recognition and trigger the immune response. Lipopolysaccharide (endotoxin) is one such substance, occurring in the outer membrane of certain bacteria. A team of scientists has now described the first endotoxin immunosensor in plants.

Plants possess an innate immune system equipped with various immunosensors that detect substances occurring only in microorganisms and trigger a defensive reaction. In mammals, for instance, toll-like receptor 4 (TLR4) detects lipopolysaccharide, which is also referred to as endotoxin and is the main component of the outer membrane of many bacterial pathogens.

Since plants also mount an immune response to lipopolysaccharide, the assumption was that they too possess an immunosensor for this substance. The nature of this sensor was previously unknown. Researchers have succeeded in decoding the detection mechanism for bacterial lipopolysaccharide in the model plant Arabidopsis thaliana (thale cress).

The search for the plant's lipopolysaccharide sensor was complicated by the fact that the endotoxin does not consist of a single defined molecule, but of a complex mixture of similar lipopolysaccharide molecules. For that reason, it has also not yet been possible to synthesize it for testing.

For further details see:

Stefanie Ranf, Nicolas Gisch, Milena Schäffer, Tina Illig, Lore Westphal, Yuriy A Knirel, Patricia M Sánchez-Carballo, Ulrich Zähringer, Ralph Hückelhoven, Justin Lee, Dierk Scheel. A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana. Nature Immunology, 2015; DOI: 10.1038/ni.3124

Posted by Tim Sandle

Saturday 11 April 2015

Identification of Clostridium species

The genus Clostridium belongs to the family Clostridiaceae and it currently contains 203 species and 5 subspecies, with only a few species being pathogenic to humans. Of these species, 21 have been reclassified to other genera, 5 have been reclassified within the genus and 1 has been de-accessioned.

Clostridium are phylogenetically heterogeneous and are Gram positive but can decolourise easily and appear Gram negative or Gram variable, spore formers and non-spore formers, rods and cocci and anaerobic and non-anaerobic bacteria.

Medically significant Clostridium strains tend to be Gram positive rods (some are Gram variable), 0.3 – 2.0 x 1.5 – 20.0μm which are often arranged in pairs or short chains, with rounded or sometimes pointed or square ends. They are commonly pleomorphic and vary considerably in their oxygen tolerance. Some species such as Clostridium novyi type A and Clostridium haemolyticum may require extended incubation on pre-reduced or freshly prepared plates and total handling in an anaerobic chamber. Conversely, Clostridium tertium, Clostridium histolyticum and Clostridium carnis are aerotolerant and will form colonies on blood agar plates incubated in an atmosphere of air with 5-10% added CO2.

Virtually all of the members of the genus, except Clostridium perfringens, are motile with peritrichous flagellae and form oval or spherical endospores that may distend the cell. They may be saccharolytic or proteolytic and are usually catalase negative. Many species produce potent exotoxins.
In 1994 the heterogeneity of this species was confirmed by 16S rRNA gene sequencing. This has been reaffirmed by the work of Yutin et al that 16S rRNA and ribosomal protein sequences are better indicators of evolutionary proximity than phenotypic traits. This genus like several others has undergone a number of revisions with the increasing availability of genomic data. An analysis of proteins from a number of members of this genus suggested another revision3. The main findings from the proposal suggested that:

The Selenomonas-Megasphaera-Sporomusa group are still members of the genus Clostridium
Clostridium difficile and its close relatives are placed within the family Peptostreptococcaceae. Under this proposal, the species Clostridium difficile would become Peptoclostridium difficile
Members of the family Ruminococcaceae belong to the genus Clostridium
It was also proposed to create six new genera to accommodate the 78 validly described species that fell outside the family Clostridiaceae. These genera are: Erysipelatoclostridium, Gottschalkia, Lachnoclostridium, Peptoclostridium, Ruminiclostridium and Tyzzerella
The type species is Clostridium butyricum.

For more about the identification of Clostridium species, see a special technical note from Public Health England.

Posted by Tim Sandle

Special offers