دورة مختصرة عن المعمل الميكروبيولوجى ودوره فى شركات ومصانع الادوية
المحتوى :
- Introduction to Microbiology
- Microbiology lab. Overview
- Microbiology Lab. Role
- Pharmaceutical Microbiology
- Microbiological tests for pharmaceuticals
الميكروبيولوجى ببساطة
Introduction to Basic Pharmaceutical MicrobiologyChittaranjan Das
Contains basic of pharmaceutical microbiology and major microflora in the cleanroom. Microorganisms like bacteria and fungi. Common microorganisms in the cleanroom and diseases they produce. Biofilm in the pharmaceutical cleanroom.
دورة مختصرة عن المعمل الميكروبيولوجى ودوره فى شركات ومصانع الادوية
المحتوى :
- Introduction to Microbiology
- Microbiology lab. Overview
- Microbiology Lab. Role
- Pharmaceutical Microbiology
- Microbiological tests for pharmaceuticals
الميكروبيولوجى ببساطة
Introduction to Basic Pharmaceutical MicrobiologyChittaranjan Das
Contains basic of pharmaceutical microbiology and major microflora in the cleanroom. Microorganisms like bacteria and fungi. Common microorganisms in the cleanroom and diseases they produce. Biofilm in the pharmaceutical cleanroom.
Contributions of Various scientist for the development of Microbiology field.
1. Antony Van Leeuwenhoek
2. Edwerd Jenner
3. Louis Pasteur
4. Joseph Lister
5. Robert Koch
6. Paul Ehrlich
7. Alexander Fleming
The word MICROBIOLOGY describes exactly what the discipline is: the study of small living things. MICRO = small, BIO = living, and LOGY = to study. Microbiology (or specifically, bacteriology) is still a very young science and not yet completely understood.
Contributions of Various scientist for the development of Microbiology field.
1. Antony Van Leeuwenhoek
2. Edwerd Jenner
3. Louis Pasteur
4. Joseph Lister
5. Robert Koch
6. Paul Ehrlich
7. Alexander Fleming
The word MICROBIOLOGY describes exactly what the discipline is: the study of small living things. MICRO = small, BIO = living, and LOGY = to study. Microbiology (or specifically, bacteriology) is still a very young science and not yet completely understood.
An introduction to the international cleanroom standard ISO 14644 and the 2015 revisions to Parts 1 and 2. The focus is on particulate and contamination control.
Different medications must be absorbed to be effective. For absorption, the drug must be administered in proper manner. To choose a route of administration we need to relate the dosage form, the advantages and disadvantages etc.
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?
Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology.
Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both.
It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined medium.
It is often essential to isolate a pure culture of microorganisms
A pure culture theoretically contains a single bacterial species. There are a number of procedures available for the isolation of pure cultures from mixed populations. A pure culture may be isolated by the use of special media with specific chemical or physical agents that allow the enrichment or selection of one
organism over another.
safety data sheet, an introduction to cell culture, safety equipment, safe laboratory practices, ascetic techniques, sterile work area, good personal hygiene, sterile reagents and media, sterile handling, planning of cell culture labs.
The term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment. It becomes necessary to maintain the viability and purity of the microorganism by keeping the pure culture free from contamination.
PLANT TISSUE CULTURE
K. Vanangamudi
History of plant tissue culture
Terms and terminology of plant tissue culture
Techniques of plant tissue culture
Stages of micro propagation
Diagrammatic representation of stages of micropropagation
Advantages of micro propagation
Demerits of micropropagation
Commercially propagated plants through micro propagation in India
Explants and medium used
1. INTRODUCTION TO CELL CULTURE
2. SOURCES & TYPES OF CONTAMINATION
3. MONITORING OF CONTAMINATION IN CELL CULTURE
4. CROSS CONTAMINATION
5. ANTIBIOTIC USE
Similar to Myths of pharmaceutical microbiology (20)
Microbiologists carry out a lot of environmental montoring, but is this sufficiently focused? Are too many samples taken? Are samples taken in the wrong locations or at the wrong frequency? Some ideas are presented.
Overview of the key requirements ofelectronic data management systems in relation to pharmaceuticals and healthcare facilities. This includes the importance of computerised systems controls and defenitions of data. The presentation includes the importance of validation and quality assurance aspects.
Risk management tools and techniques for environmental monitoring:
Application of HACCP for selecting environmental monitoring locations; Use of risk filtering to determine frequencies of monitoring ; Applying FMEA to assess risks from process equipment – a sterility testing isolator.
Overview of the apporach to non-compliances and related matters. Appropriate training for analysts on how to perform the tests and steps to take when obtaining OOS results should be implemented . The use of root cause analysis tools when finding an OOS should also be available for review.
Application of FMEA to a Sterility Testing Isolator: A Case StudyTim Sandle, Ph.D.
Presentation on Failure Modes and Effects Analysis, in the pharmaceutical context. Covering:
Introduction to risk assessment
What are risks?
Advantages and disadvantages of FMEA
Applying FMEA to review a sterility testing isolator – case study
Pharmaceutical Microbiology: Current and Future Challenges Tim Sandle, Ph.D.
The changing environment for pharmaceutical microbiology
Limitations of methods
Need for new (rapid) methods
Separating people form processes
Single-use technologies
Environmental monitoring programme
Best practices
Rapid methods
Contamination control strategy
Objectionable organisms
Burkholderia cepacia complex
Why use reference materials?
The importance of reference materials
Different categories of reference materials.
Different classes of reference materials.
Standards for reference materials.
How reference materials are prepared and assessed.
How reference materials are used.
GxP is a general abbreviation for the "good practice" quality guidelines and regulations. These slides provide an overview of current regulations, with a focus on pharmaceuticals and healthcare.
What is likely to go into the revised Annex 1, including:
Terminal sterilisation vs aseptic processing
WFI produced by reverse osmosis
Guidance for media simulation trials
This remains speculative
Key question:
Could the plague ever re-emerge on a similar level in the twenty-first century?
Due to the potential seriousness of the disease this is a subject worthy of epidemiological consideration and research.
The two most commonly used within microbiology are
HACCP (which originated in the food industry) and FMEA
(developed for engineering). This article explores these two
approaches, first with a description of HACCP, followed by a
description and case study of FMEA in sterility testing.
Considering: Environmental monitoring guidance, Background to USP <1116>, Main changes and debates Method limitations, Incident rates, Frequencies of monitoring, Locations of monitoring, Other changes, Regulatory issues and Rapid methods
Presentation on the examination of microbiological data for assessment and trending.
Includes: normalizing data, graphs, and assessment of alert and action levels.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
2. Introduction - 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?
3. Myths
What is a myth?
Myth ~ a traditional or legendary story with or without a
determinable basis of fact or a natural explanation.
4.
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.
8.
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.
12.
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
15.
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
18.
19. 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.
20. 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
21. 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.
22.
23. Myth – Bacteria don’t 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.
24.
25. 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.
26. 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
27. Summary
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?
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.
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 – 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.
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’
OK – what we have looked at in 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