Introduction - 7 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? 5. Environmental monitoring parameters can they be pre-set? 6. Bunsen burners needed to create aseptic space or not? 7. Identification results always believable? 1. 2. 3. 4.
Myths What is a myth? Myth ~ a traditional or legendary story with or without a determinable basis of fact or a natural explanation.
Myths around the
Colony Forming Unit
Myth CFUs 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
Myth CFUs 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.
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.
Myth Clean air
devices are secure and contamination free
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.
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
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
Myth microbes in cleanrooms are free floating
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 m in size. Argument for positioning settle plates inside UDAFs.
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
Myth There are
universal conditions for environmental monitoring
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.
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
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.
Myth Bunsen Burners are needed to create aseptic space
Myth Bacteria dont lie
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.
Myth Microbiological identification is infallible
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.
Myth if Ive 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
Summary 1. Colony Forming Units what 2. 3. 4. 5.
6. 7.
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 preset? Bunsen burners needed to create aseptic space or not? Identification results always believable?
Thank you Pharmaceutical microbiology: http://www.pharmamicroresources.com/
