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Myths

pharmaceutical
microbiology
Dr. Tim Sandle

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.

Aseptic technique

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.

Myth media
growth promotion
is not necessary

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

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/