Pharma micro myths (sandle)
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Introduction – the ‘great’ myths Colony Forming Units – what are they? Microbiology laboratory cabinets – always work? Media growth promotion – can it be skipped? Microbial distribution in cleanrooms – free floating? Environmental monitoring parameters – can they be pre-set? Bunsen burners needed to create aseptic space– or not? Identification results– always believable?
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Pharma micro myths (sandle)
- 2. Introduction – the ‘great’ myths 1. Colony Forming Units – what are they? 2. Microbiology laboratory cabinets – always work? 3. Media growth promotion – can it be skipped? 4. Microbial distribution in cleanrooms – free floating? 5. Environmental monitoring parameters – can they be pre-set? 6. Bunsen burners needed to create aseptic space– or not? 7. Identification results– always believable?
- 3. Myths What is a myth? Myth ~ a traditional or legendary story with or without a determinable basis of fact or a natural explanation.
- 5. Myth – CFU’s tells me how many bacteria there are? #1 Not always: Traditional culture based microbiological methods are variable, Plate counts are an approximation of what is present, Many microorganisms will not grow on standard media or their physiological state does not promote recovery, Dilution errors lead to poor recovery e.g.: Over dilution, Under dilution = confluent growth Aim of the ‘countable range’ cf Sutton “Accuracy of Plate Counts”, Journal of Validation Technology, 17 (3): 42-46 Counting errors can occur
- 6. Myth – CFU’s tells me how many bacteria there are? #2 Often a CFU is not a single bacterium A colony could arise from one cell or several. Issue can occur through: Poor sample mixing e.g. bacteria clumping together, Poor plate mixing, Settle plate picking up skin detritus.
- 7. Myth – sampling from anywhere within a colony is equal With pure colonies, cells experience different local conditions: Near the middle of the colony, cells starve for nutrients, and accumulate wastes, Cells in the middle of the colony are in stationary phase, Leading edge cells are in log phase, Mutations can occur - genetic diversity.
- 9. Myth – microbiological workstations always are laminar Are they unidirectional? Only do when they are empty. Materials and equipment disrupt air flow and cause the air to swirl. This can spread bacteria across surfaces or to other objects in the hood. To avoid contamination, clutter must be minimized.
- 11. Myth - Isolators never leak Isolators Aseptic manufacturing Compounding Sterility testing Leakage Loss of air Leaks: Isolators leak a given amount of their volume per hour. Gloves are a vulnerable point.
- 13. Myth – let the manufacturer perform media growth promotion testing #1 Vendor: Challenges lots plate media with a type culture from a culture collection Uses a low level challenge (< 100 CFU) Tests against previously released media Compare growth rates
- 14. Myth – let the manufacturer perform media growth promotion testing #2 In-house testing: Good practice to consider environmental isolates. There can be a case for reduced testing, but: Need to verify the supplier Need to account for different temperatures of use Need to consider if all appropriate control strains are included Transport issues Heat shock
- 16. Myth – microorganisms are free floating #1 Microorganisms in cleanrooms are rarely ‘free floating’ Most are found on skin flakes shed by operators. Or attached to dust Typical number (Whyte) = 4 organisms. Argument for assessing particles >0.5 µm in size. Argument for positioning settle plates inside UDAFs.
- 17. Myth – microorganisms are free floating #2 Microorganisms in air Do not grow, air is not a natural biotope. Die off: Relative humidity Lack of oxygen UV light Those attached to water droplets can survive, potentially grow and travel long distances. Travel through passive movement
- 19. Cleanroom gloves Myth - Only natural latex gloves can give me an allergy. Chemical allergy is more commonly encountered than natural latex allergy, but is often confused with the latter. With natural latex and nitrile gloves, chemical allergy is frequently derived from the accelerators that are used in the vulcanisation process. Myth - All gloves have the same barrier properties. Prior to use, gloves may exhibit equivalent barrier properties as defined by AQL. But, in-use, glove material, thickness, degradation etc. will influence the potential to develop holes. To assess the barrier performance of your gloves, you can perform your own test by wearing a pair for a defined time then filling them up with water to see whether they leak.
- 21. Universal conditions for environmental monitoring #1 Do “universal conditions” for environmental monitoring exist? Issues: Not all microorganisms are culturable; Those that are culturable will not grow on all types of media; Those that are physiologically weak (‘stressed’) will take longer to grow than others; Our ‘microbiome’ is more complex than previously thought, Environmental monitoring methods are limited in meteorology and variable in application. Therefore, we cannot expect to capture or to grow everything but we need a standard set of conditions.
- 22. Universal conditions for environmental monitoring #2 Some decisions required: Whether to select? A general medium incubated across suitable temperature range, or Two media – typically ‘bacterial’ and ‘fungal’, Consideration of periodic selective agar / incubation conditions use. Once agar has been selected, establish appropriate incubation times. References: Sandle, T., Skinner, K. and Yeandle, E. (2013). Optimal conditions for the recovery of bioburden from pharmaceutical processes: a case study, European Journal of Parenteral and Pharmaceutical Sciences, 18 (3): 84-91 Sandle, T. (2014) Examination of the Order of Incubation for the Recovery of Bacteria and Fungi from Pharmaceutical Cleanrooms, International Journal of Pharmaceutical Compounding, 18 (3): 242 – 247
- 23. Universal conditions for environmental monitoring #3 How much does this matter? Accept the limitations, Aim for optimal recovery, Be consistent: Locations of monitoring, Frequencies of monitoring, Times of monitoring, Cleanroom conditions for monitoring.
- 25. Myth – Bacteria don’t lie Are all biological indicator failures indicative of a failed sterility cycle? Often, but rogue biological indicators exist. Cause false positives, due to: Clumping of spores, Overlaying of spores, Incorrect D-values, Inoculated discs having roughened edges. One solution is to use multiple BIs in one location.
- 27. EM is a good predictor of product quality Myth – Through environmental monitoring, I can tell if my product is safe This is not so. Environmental control is always more important. EM data only reflects a snapshot in time, representing a transient condition that may or may not persist. A single data point cannot necessarily be extrapolated. Trending is far more important. The ‘art’ or ‘science’ of interpretation. With aseptic processing, there is not often a clear connection between EM data and media fill results- and this would be the same for product fills.
- 30. Low endotoxin recovery Low recovery of lipopolysaccharide (LPS), as used for the LAL test control endotoxin, occurs in certain conditions, such as chelating buffers and detergents. But, this issue does not affect all products. The issue at hand is low lipopolysaccharide recovery rather than ‘low endotoxin’ recovery. Endotoxin is more sophisticated than LPS. The use of a uniform screening test can reveal conditions of concern and legitimize the use of alternative naturally occurring endotoxin preparations for endotoxin challenge studies.
- 31. Cleaning and disinfection Myth - Cleaning and disinfecting are the same thing Cleaning refers to removing dirt and dust from surfaces, which also removes (but does not kill) some of the bacteria present. Disinfecting kills bacteria and fungi on surfaces through contact them with the active ingredient. Myth - Disinfecting alone is more powerful than cleaning. Cleaning and disinfecting go hand-in-hand. Before you disinfect, you should always apply a detergent — remove surface dust, dirt and grime.
- 32. Disinfectant application Myth – Disinfectants work instantly Disinfectants are essential for cleanroom control, but when applied to a surface the products do not kill microbes instantly. Merely wiping a surface will not destroy the organisms there. Need to note the contact time. May need to reapply to keep a surface ‘wet’.
- 33. More about disinfectant application Myth - Saturating a surface through spraying is just as good as wiping Technique matters - liquid chemical disinfectants work through direct contact with cell membranes. The mechanical action of wiping and mopping ensures this happens. Wiping helps to address variables like surface materials and complexity, size and accessibility of areas.
- 34. Disinfectant rotation Myth – Rotation of disinfectant prevents resistant strains There is no data to show that microorganisms can develop resistance to disinfectants (as with antibiotics). The purpose of rotation is to broaden the spectrum of activity. Where one disinfectant is sporicidal, this enhances the overall contamination control programe.
- 35. Alcohols kill spores Myth – Applying an alcohol-based disinfectant, like 70% IPA, will kill bacterial spores Alcohols will kill some of the vegetative bacteria that can form spores, but alcohols cannot penetrate the endospore coat. A true sporicidal agent is required to prevent address the survival of spore-formers into the cleanroom
- 36. More about mutations and resistance Myth - Alcohol hand sanitisers cause bacterial mutation and help create resistant strains The alcohol-based antibacterial rubs are effective enough that they do not create resistant strains, although antibacterial soaps may present a hazard. But some antimicrobial soaps might be a problem. While the alcohol rub stays on the hands and is not meant to be rinsed off, the antibacterial triclosan is rinsed off before it can do all its work and then enters the water supply. In addition, products like triclosan can cause problems once they are in the water supply, and resistant strains of bacteria have been created in labs using triclosan, although it remains to be seen if it will happen in the natural environment. Generally, antibacterial soap does not do enough to justify its use. Given that regular soap and water removes the organisms, there is often no need for an antibacterial agent, and it probably will not work anyway. Hand sanitisers are best reserved where hand washing facilities are not readily accessible.
- 38. Myth – Burners and aerosols Is it best not to "flame the mouth of the flask" when transferring fluids, or when pouring autoclaved media into petri plates? Can increase the risk through generation of aerosols /air current contamination transfer Best technique: Rapid transfer, Holding the flask or tube horizontal to avoid dust settling.; Use single-use sterile disposable items.
- 39. Bunsen burners and aerosols Depending on the organism handled, this can create an element of risk when using a Bunsen burner, in relation to aerosols. Particles of less than 5 um are most effective in establishing airborne infection in laboratory animals. Particles in the 2.0- to 3.5- um range appear to offer equal opportunities for both upper respiratory and alveolar deposition. Moreover, the idea that the Bunsen burner creates an upward draft of air to prevent contaminants in the air from settling on the work surface below does not always result in more robust practices, as sometimes airflow disruption leads to particles settling out. Also, where Bunsen burners are used, it is optimal to flame a loop in the airspace underneath the burner flame.
- 41. Myth – if controls work, the ID is sound Gram-stain Easy to get a mixed colony, Old colonies lean towards Gram-positives, Over decolorisation can occur, Bacillus species can appear Gram-negative. Automated systems Phenotypic systems are affected by phenotypic changes, All systems are only as good as their databases, Cross-contamination can occur.
- 42. Myth – if I’ve found organism x it must be x Question the result of the identification Is it expected from the sample source? Have I really got Bacillus anthracis? Or Prochlorococcus spp.? Or Thermus brockianus? Most identification systems work on the basis of matching and probability Mixed cultures produce odd results
- 43. Cultures from culture collections Myth - Freeze-dried cultures are safe The number of particles aerosolized upon opening lyophilized cultures depends upon the consistency of the end product. Most of the particles aerosolized upon opening lyophilized cultures are larger than 5 um. Dropping a lyophilized culture creates an extremely concentrated aerosol composed predominantly of particles larger than 5 um. Two parameters must be established to assess the risk of handling laboratory cultures: the biological decay rate of aerosols under laboratory conditions, and the human infectious dose by the respiratory route. Several common organisms survive at least 1 hour in droplet nuclei at standard laboratory relative humidity.
- 44. Pure cultures Myth - We understand microbes best in pure culture and as single colonies Much of our knowledge about microbial biochemistry, genetics, physiology, etc. comes from culture-based work. However, there are several limitations to this approach. Culturing organisms in a Petri dish meaning growing them in non-natural conditions… cut off from the normal community of microbes surrounding them in nature. We now know that only around 1% of the microorganisms in a typical environment can even be cultured at all. Sequence-based techniques such as metagenomics and rRNA sequencing can help address these concerns to varying degrees, and are an important complement to culture-based techniques.
- 45. Summary The biggest 7 myths? 1. Colony Forming Units – what are they? 2. Microbiology laboratory cabinets – always work? 3. Media growth promotion – can it be skipped? 4. Microbial distribution in cleanrooms – free floating? 5. Environmental monitoring parameters – can they be pre- set? 6. Bunsen burners needed to create aseptic space– or not? 7. Identification results– always believable?
- 46. Dr. Tim Sandle Pharmaceutical microbiology: http://www.pharmamicroresources.com/
Hinweis der Redaktion
- Introduction
- Difficult to select myths. Time available, topics selected are: Colony Forming Units – what are they? Microbiology laboratory cabinets – always work? Media growth promotion – can it be skipped? Microbial distribution in cleanrooms – are they free floating? Environmental monitoring parameters – can they be pre-set? Bunsen burners needed to create aseptic space– or not? Identification results– always believable?
- A myth is something that can be widely accepted, but which doesn’t stand up to scrutiny or collected facts. As pharmaceutical microbiology has advanced, some things that have been commonly held as ‘correct’ aren’t necessarily so…or at least not as certain as previously thought. With these myths, some things will be a surprise to some of you; to others you’ll know everything…but hopefully they’re interesting areas to re-visit.
- First – colony forming units – the basis of pharmaceutical microbiology for decades, although now challenged by some rapid methods.
- Should we assume the colony forming unit represents all of the microorganisms in a sample? Or those that could be recovered? No, because of several reasons - Our methods, especially traditional culture based ones, are variable Many microorganisms will not grow on standard media – either ‘viable but nonculturable’ (‘active but non-culturable’) or they are too stressed or the media or incubation parameters are not suitable. Depending on the test method…It is easy to over-dilute, leading to under estimation It is easy to have too many colonies on a plate, leading to confluent growth or over crowding – good paper by Scott Sutton – want an optimal countable range of 25 – 250 CFU per plate We can all make counting or calculation errors I’ll talk about media type, incubation time and temperature later
- Also, a CFU should not be thought of as a single bacterium or fungus – it is a colony forming unit. The ‘unit’ could be made up from one cell or many. There are several situations where this can arise. Example: Poor mixing of a sample before plating out, where cells stick together or become bound to the sample. Bacillus species, for example, are notorious for clumping; This can also be the result of poor mixing of agar plates; Also, with environmental monitoring, if a skin flake lands on a settle plate, this is often carrying more than one organism….I’ll come back to this later.
- Let’s throw in another ‘myth’. Each visible colony on a plate is composed of around one million cells but the organisms within a colony are not all in the same state. Pure colonies come from a single ancestor, but progeny cells are different based on the location. Cells near the middle of the colony can be starved of nutrients and affected by toxic wastes. These cells may not grow when subcultured. Also, central cells are in the stationary phase and will take longer to grow when subcultured. BUT cells at the edge are in the log phase, and should grow faster. Mutations can occur, especially with cells adjacent to each other, leading to genetic diversity – can be an issue with some identification methods...also can lead to transfer of genetic material and antimicrobial resistance. Improvement in obtaining pure, healthy cultures from quadrant plate technique.
- Next myth – clean air devices in labs– are they always contamination free?
- First off – do clean air cabinets always have unidirectional airflow? Well, only when they’re empty. Materials and equipment disrupt unidrectional flow and cause the air to swirl, which can actually spread contamination across surfaces. To avoid contamination, clutter must be minimized
- Here are some illustrations of things that can disrupt unidirectional airflow devices: Objects, Bunsen flames, Broken filter faces, Other obstructions etc. It is important to assess the working area and to check the air velocity is within range where key aseptic operations are undertaken.
- Another clean air device in a lab could be an isolator, as might be used for sterility testing. Although isolators present a barrier, all isolators, contrary to some opinion, leak. What matters is by how much do they leak. This should be assessed before each decontamination cycle using a pressure decay test. This is expressed as pressure drop over time. The size of the leak, if excessive, needs investigation. Here the location of the leak is as much indicative of the contamination risk as the size. A common risk area is with gloves, especially around the cuffs or with pin prick holes. Many users assess gloves post-use by water intrusion.
- Next myth – do we really need to carry out media growth promotion?
- At one level, why do it? Arguments against are: The vendor does it, using type cultures for consistency: Here media growth properties are demonstrated from a low level challenges. And media growth performance can be assessed against a previously released lot, so we can compare growth rates and patterns.
- There could be a case to reducing testing, but here it is important to: Verify the supplier, Ensure the vendor tests the media at all of the temperatures of use that you will use it at, Check that all representative control strains are used and if you are concerned with a particular objectionable microorganism, that this is included, Ensure transportation issues have been assessed e.g. heat shock if the freight lorry breaks down on a hot day. Personally I think confirmatory testing by the receiving laboratory is important to address these factors. Also, if environmental isolates are necessary for inclusion, this can only really be done by the user. There’s no time for the environmental isolate debate.
- Next myth, which relates a little to the colony forming units, is about how microorganisms are distributed in cleanrooms.
- Here it is rare for microorganisms in cleanrooms to be ‘free floating’ Most microbes are found on particles, like skin flakes or dust. Work by Bill Whyte suggests 4 bacteria are typically found on one skin cell, and these are typically around 12 microns in size. This is perhaps an argument for looking for larger particles in cleanrooms. Also, the risk of larger particles falling put of the air through gravity or air striking an object supports the use of settle plates (in addition to air samplers) - if settle plates are in the correct locations. Smoke studies help to decide this.
- The biggest risk from air is mainly that it acts as a vector for contamination and this is a concern in a poorly designed cleanroom. Because microbes do not grow in air and many will eventually die in cleanrooms, unless they are endospores, then where air ends up and whether particles will fall out is important. Airflow design is key to contamination control. With air particles, many microbes will survive for longer on water droplets compared with dust, which means places like wash bays need to be controlled.
- Only natural latex gloves can give me an allergy. Chemical allergy is more commonly encountered than natural latex allergy, but is often confused with the latter. With natural latex and nitrile gloves, chemical allergy is frequently derived from the accelerators (e.g. thiazoles, dithiocarbamates, thiurams etc) that are used in the vulcanisation process. Plasticisers such as phthalates are often found in vinyl gloves and can also trigger a chemical allergy. All gloves have the same barrier properties. Gloves are often worn as a barrier for personal protection or for process protection and often for both. Prior to use, gloves may exhibit equivalent barrier properties as defined by AQL. The latter refers to the statistical probability of holes in the gloves. In-use, glove material, thickness, degradation etc. will influence the potential to develop holes. A simulated use study by Kerr revealed failure rates of respectively 35% and 9% in vinyl and latex. To assess the barrier performance of your gloves, you can perform your own test by wearing a pair for a defined time then filling them up with water to see whether they leak.
- Next myth is about environmental monitoring and that there are universal incubation conditions that we can all follow.
- Actually there are no universal conditions for environmental monitoring because: Not all microorganisms can be cultured. Those that can grow on one culture medium will not grow on all culture media Some organisms can grow, in theory, but will not grow under the physiological state found, or they might grow slowly. The limitations of culture media have been emphasised recently by knowldged the human microbiome of the skin. Much more is found on the skin surface than we recover (see Tony Cundell). The methods we use are limited in terms of accuracy and variability.
- In doing so we need to make some decisions: Will we use one general culture medium –like TSA – and incubate it across a suitable temperature range or use a dual incubation step? Or will we use two media, including one designed to detect fungi and use separate temperature ranges? Do we need to enhance this with selective media if we are concerned about an objectionable microorganism or have a concern about a particular type e.g. anaerobes where nitrogen comes into contact with product. Once we’ve unravelled these, we need to decide on incubation times. This is a huge debate and there is no time to explore it here. I have provided some references on the slide which might be useful.
- How much does this matter? Well, we have to accept the limitations – we can’t capture everything We should run some studies to know we are close to optimal recovery. But most importantly, we need to be consistent. We can do this with our: Locations of monitoring, Frequencies of monitoring, Times of monitoring, Cleanroom conditions for monitoring, And using data to look for trends.
- Next myth – we should always believe the results microorganisms tell us. I’m illustrating this with biological indicators.
- The key question is – “Are all biological indicator failures indicative of a failed sterility cycle?” Yes, in most cases – but rogue biological indicators exist. This can cause false positives, and can arise due to: Clumping of spores, Overlaying of spores, Incorrect D-values, Inoculated discs having roughened edges. One solution is to use multiple BIs in one location. The reason to use multiple BIs at each test location is it allows an assessment of microbial kill through assessing the ‘Most Probable Number.’
- Myth – Through environmental monitoring, I can tell if my product is safe This is not so. Environmental control is always more important as the revised Annex 1 emphasizes. EM data only reflects a snapshot in time, representing a transient condition that may or may not persist. A single data point cannot necessarily be extrapolated. Trending is far more important. The ‘art’ or ‘science’ of interpretation. You need a qualified microbiologist. With aseptic processing, there is not often a clear connection between EM data and media fill results- and this would be the same for product fills. For example, media fills can fail (show growth) where EM is all zero CFU; or medial fills can pass, and yet EM can be poor. A further reason why control matters is due to the presence of viable but non-culturable organisms.
- No one disputes that low recovery of lipopolysaccharide (LPS), as used for the LAL test control endotoxin, occurs in certain conditions, such as chelating buffers and detergents. However, this issue does not affect all products. Furthermore, the issue at hand is low lipopolysaccharide recovery rather than ‘low endotoxin’ recovery. Endotoxin is more sophisticated, being composed of a hydrophilic polysaccharide covalently linked to a highly conserved, hydrophobic lipid region. LPS and endotoxin do not behave in the same way (LPS activity varies due to the presence of various salts and detergents). Furthermore, the use of a uniform screening test can reveal conditions of concern and legitimize the use of alternative naturally occurring endotoxin preparations for endotoxin challenge studies. However, to do required regulatory approval.
- Cleaning and disinfecting are the same thing. According to the Pharmig disinfection guide, cleaning refers to removing dirt and dust from surfaces, which also removes (but does not kill) some of the bacteria present. Cleaning is important because it reduces the number of bacteria in the environment. By removing soil it enables the disinfectant to make contact. Disinfecting, on the other hand, kills germs on surfaces by dousing them with chemicals. The active ingredient needs to be in contact for a sufficient length of time. Hence another myth is that disinfecting alone is more powerful than cleaning. Cleaning and disinfecting actually go hand-in-hand. Before you disinfect, you should always clean with a detergent— remove surface dust, dirt , protein, and grime. Surface dirt can react with the chemicals in disinfecting products and render them unable to kill bacteria and fungi effectively. Do not let your microbial-killing efforts go to waste by failing to thoroughly clean before applying disinfectant!
- We apply disinfectants to surfaces, but it’s important to know these products do not kill microorganisms instantly. Merely swiping a surface will not destroy the microbes there. It is important to read the product label carefully to find the instruction titled “to disinfect.” This instruction will tell you how many minutes to leave the product on the surface to kill microbes. The contact time is the time required for the disinfectant to bind to the microorganism, cross the cell wall and act at the target site. Ideally you will have verified this through surface efficacy studies. You may need to saturate the surface and leave it wet for several minutes.
- Myth - Saturating a surface through spraying is just as good as wiping Technique matters - liquid chemical disinfectants work through direct contact with cell membranes. The mechanical action of wiping and mopping ensures this happens. Wiping helps to address variables like surface materials and complexity, size and accessibility of areas.
- There are some expressed concerns, although not support by any major study, that genetically acquired disinfectant resistance might occur in a similar way to antimicrobial resistance. Whilst the phenomenon of microbial resistance is an issue of major concern for antibiotics there are few data to support development of resistance to disinfectants. Most disinfectants do not have a complete spectrum of activity effective against all microorganisms (spectrum of activity is the ability of the disinfectant to kill different types of microorganisms and microorganisms which are in different physiological states). The disinfectants commonly used are often effective against vegetative cells but are not sporicidal. To maintain an effective contamination, control the elimination of bacterial endospores through a sporicidal disinfectant is a recommended
- Unfortunately some users think that 70% IPA will kill spores. Alcohols only work against vegetative cells; they cannot enter a bacterial spore. Spores are resistant to many biocides: This is because of the relative impermeability of the polypeptides which make up the spore coat. These can stop many disinfectant from penetrating. If disinfectants get through, the inner layers – cortex and protoplast – limit chemical diffusion. Superoxide dismutase – a protein involved in spore coat formation – helps to create the thick, striated outer layer. It also helps protect bacteria from certain chemicals
- The alcohol-based antibacterial rubs are effective enough that they do not create resistant strains, although antibacterial soaps may present a hazard. While the alcohol rub stays on the hands and is not meant to be rinsed off, the antibacterial triclosan is rinsed off before it can do all its work and then enters the water supply. In addition, products like triclosan can cause problems once they are in the water supply, and resistant strains of bacteria have been created in labs using triclosan, although it remains to be seen if it will happen in the natural environment. Generally, antibacterial soap does not do enough to justify its use. The objective of hand washing, by rinsing in soap and water for at least 20 seconds, is not to kill bacteria, but simply to get germs and viruses off our hands. Using a sink and washing hands thoroughly 15 to 20 seconds with regular soap and then rinsing that is the most effective method of 'de-germing', or removing bacteria and viruses from your hands. Hand washing with soap and water does not remove all the microbes from our hands, because some are an important part of our skin, and even if we did kill them, they would return. Given that regular soap and water removes the organisms, there is often no need for an antibacterial agent, and it probably will not work anyway. Hand sanitisers are best reserved where hand washing facilities are not readily accessible.
- Next myth – I’ll look at how useful are Bunsen burners for aseptic testing. I’m not referring to loop or slide flaming here; more to the so-called ring of protection.
- The question here is whether flaming flasks for fluid transfer or having Bunsen burners on when carrying out bioburden testing, is necessary? Although it remains common for many labs to use Bunsens, a paper issued back in 1972 argued that it is best not to "flame the mouth of the flask" when transferring fluids, or when pouring autoclaved media into Petri dishes. This is because: This increases the risk of generation of aerosols and create air currents for contamination transfer So a better technique might be: Rapid transfer, Holding the flask or tube horizontal to avoid dust settling.; Use single-use sterile disposable items; Some cases testing in UDAFs.
- Many microbiologists find that the use of pre-sterilized disposable tools to transfer media and bacterial cultures result in lower contamination rates compared with reusable inoculating loops. The latter is a process known to create aerosols that may increase potential air contaminants. Depending on the organism handled, this can create an element of risk. Several studies have shown that particles of less than 5 um are most effective in establishing airborne infection in laboratory animals; and particles of 1 to 5 um can be deposited in the alveoli, with preferential deposition occurring with 1- to 2-um particles. Particles larger than 3.5 are most probably deposited in the upper respiratory tract. Particles in the 2.0- to 3.5- um range appear to offer equal opportunities for both upper respiratory and alveolar deposition. Where Bunsen burners are used, it is optimal to flame a loop in the airspace underneath the burner flame. Moreover, the idea that the Bunsen burner creates an upward draft of air to prevent contaminants in the air from settling on the work surface below does not always result in more robust practices, as sometimes airflow disruption leads to particles settling out.
- Final myth –should we always trust the results from microbial identification systems?
- With identifications there are a number of things that can go wrong. Take the Gram-stain. Errors include: Easy to get a mixed colony, Old colonies lean towards Gram-positives, Over decolorisation can occur through poor technique, Bacillus species can appear Gram-negative. Things can go awry with automated systems: Phenotypic systems are, by their name, affected by phenotypic changes, All systems are only as good as their databases, Cross-contamination can occur.
- With the actual result, the results obtained might be correct under the conditions of the test but is it the result reflective of the organism found? Always question the result of the identification Ask yourself if it is expected from the sample source? E.g. a Gram-negative rod from an aseptic filling area Have I really got Bacillus anthracis? Remember - phenotypic identification systems work on the basis of matching and probability – what you have is the best ‘guess’
- Freeze-dried cultures are safe until they are reconstituted The number of particles aerosolized upon opening lyophilized cultures depends upon the consistency of the end product. For example, fluffier end products tend to create more concentrated aerosols. Most of the particles aerosolized upon opening lyophilized cultures are larger than 5 um. Dropping a lyophilized culture creates an extremely concentrated aerosol composed predominantly of particles larger than 5 um. The inclusion of mother liquor in the suspending menstruum reduces the aerosol concentration approximately fourfold. Two parameters must be established to assess the risk of handling laboratory cultures: the biological decay rate of aerosols under laboratory conditions, and the human infectious dose by the respiratory route. Several common organisms survive at least 1 hour in droplet nuclei at standard laboratory relative humidity.
- A common image associated with microbiology is that of the plastic Petri dish. The ability to grow microorganisms like this is in fact the foundation of microbiology and much of our knowledge about microbial biochemistry, genetics, physiology, etc. comes from culture-based work. However, there are several limitations to this approach, some of which can now be addressed with alternative methods. Culturing organisms in a Petri dish meaning growing them in non-natural conditions, not only are the organisms in an artificial environment with a pre-defined collection of nutrients, they are usually grown in isolation… cut off from the normal community of microbes surrounding them in nature. In addition, we now know that only around 1% of the microorganisms in a typical environment can even be cultured at all. New sequence-based techniques such as metagenomics and rRNA sequencing can help address both of these concerns to varying degrees, and are an important complement to culture-based techniques. Helping with examining biofilms etc.
- OK – what we have looked at quite a few myths. I’ve selected my top seven from this presentation: Colony Forming Units – more than meets the eye Microbiology laboratory cabinets – must not be obscured Media growth promotion – probably best to do something Microbial distribution in cleanrooms – risk is with larger size particles Environmental monitoring parameters – need to accept limitations and be consistent. Bunsen burners– probably not necessary Identification results– need to do a reality check on each isolate
- Thank you!!!