Bioburden Estimation of Reusable and Single Use Medical Devices

Bioburden estimation of reusable
and single use medical devices –

methodology and evaluation
Egil Lingaas
Department of Infection Prevention
Rikshospitalet
Oslo
Norway
Sykehushygiene 05/2004
Universitetsklinikk Egil Lingaas
Sterilization is a special process
The effectiveness of the process
can not be fully verified by
subsequent inspection and testing
of the product
Bioburden
Population of viable microorganisms
on or in product and/or a package
Origins of bioburden
● raw materials
● manufacturing of components
● assembly process
● manufacturing environment
● assembly/manufacturing aids
● cleaning process
● packaging of finished products
Use of bioburden determination
● Validation and revalidation of sterilization
processes
● Routine monitoring of:
● raw materials
● components
● packaging
● manufacturing processes
● Assessment of the efficiency of cleaning
processes
● Environmental monitoring
Sterilization in industry
Bioburden determination common

as part of sterile production
Sterilization in hospitals
Bioburden determination not common
➨ In validation of sterile production
➨ For routine monitoring
Medical devices
– Multiple use
– Single use
Microorganisms on surgical instruments
Proportion of instruments (%)
120
100
80
Bef ore w ashing
60 Af ter w ashing I
40 Af ter w ashing II
20
0
< 10 > 10 > 100 > 1000
CFU per instrum ent
Modified from Nyström B. J Hosp Infect 1981;2:363

Bioburden on surgical instruments
before sterilzation
Proportion of insttruments (%)
80
70
60
50
40
30
20
10
0
0-10 11-100 > 100
Microorganisms per instruments (CFU)
Modified from Rutala WA, et al. AJIC 1998;26:143

Bioburden on surgical instruments
after use and after washing
Number of instruments 35
30
After use
25
After washing
20
15
10
5
0
< 10 10-100 100-1000 1000-10000
CFU
Modified from Chu NS, et al. AJIC 1999;27:315

Change of bioburden on surgical
instruments during washing
16
Number of instruments
14
12
10 Decrease: 32% Increase: 45%
8
6
4
2
0
-3 -2 -1 0 1 2 3
Log10 change
Modified from Chu NS, et al. AJIC 1999;27:315

Bioburden determination and
sterilization in hospitals
Non-medical devices
– Multiple use
– Single use
ISO 11737
Sterilization of health care products
- Microbiological methods
Part 1: Determination of a population of
microorganisms on product
Part 2: Tests of sterility performed in the

validation of a sterilization process
Part 3: Guidance on evaluation and the

determination of bioburden data
Selection of product
● Representative of the batch

● Whole product, if feasible
● If not: As large a portion as
possible
Selection of an appropriate method
● Removal of microorganisms
● Culturing
● Enumeration
● Characterization
TECHNIQUES FOR REMOVAL
OF MICROORGANISMS
FROM PRODUCT
Adhesion of microorganisms
to surfaces
● Surface characteristics
● Microorganisms involved
● Origin of contamination
● Presence of other substances
Removal techniques
● Stomaching
● Ultrasonication
● Shaking (mechanical or manual)
● Vortex mixing
● Flushing
● Blending (disintegration)
● Swabbing
Stomacher
Removal techniques
● Stomaching
● Ultrasonication
● Vortex mixing
Removal techniques
● Stomaching
● Ultrasonication
● Vortex mixing
● Flushing
● Blending (disintegration)
● Swabbing
Eluents
Diluents
Transport media
No proliferation or inactivation
of microorganisms
Eluents and diluents
● Sodium chloride 0.25 – 0.9 %

● Ringer
● Buffered peptone water
● Phosphate buffered saline
● Thiosulphate Ringer
● ---------------
● ---------------
Additives to eluents and diluents
● Surfactant
●Polysorbate (Tween) 80
● Neutralizers for microbicidal and
microbistatic substances
Release of substances from product
Substances released into suspending fluid

should neither promote nor inhibit
the growth of microorganisms
 bacteriostasis test
Methods where removal of
microorganisms is not employed
● Contact plating
● Agar overlaying
Contact plate
Contact plate
-- after incubation
Medical device ?
TRANSFER TO CULTURE
MEDIUM
Membrane filtration
● Filtration of an eluent
followed by incubation of the filter
on appropriate growth medium
● Plates are incubated
Membrane filter
Growth of moulds on filter
Growth of bacteria on filter
Membrane filtration
Particularly useful for:

➨ Suspensions with low concentrations
of microorganisms
➨ Suspensions with microbicidal or

microbistatic substances
Pour plating
● Defined aliquots of suspension mixed

with molten agar medium at 45 oC
● Agar allowed to solidify in the plate
Spread plating
● An aliquot of defined volume is spread

on the surface of a nutritient medium
Spiral plating
● A defined volume spread in a spiral track

on a rotating agar plate
Media and incubation conditions
Several combinations of media and

incubation conditions are usually required
Bacteria
Yeasts and moulds
Anaerobic bacteria
30 - 35 oC
20 - 25 oC
ENUMERATION
Enumeration procedures
● Detecting small colonies

● Counting and reporting unusual colonies
e.g. spreaders
● Enumerating and reporting crowded plates
● Reporting counts from multiple dilutions
CHARACTERIZATION
Characterization
● staining properties
● cell morphology
● colony morphology
● use of selective culturing
● biochemical or other means of identification
● genetic analysis
Spore forming bacteria
Validation of method for
removing microorganisms
● Repetitive recovery
● Inoculating a known number

of microorganisms onto product
Repetitive recovery
Treatment no. Colonies

1 86
2 6
3 1
4 1
Total 94
Removal by first treatment 80,8%
Correction factor 1,24
Routine monitoring of bioburden
● Sample size
● Sampling frequency
● Acceptable limits and actions taken
if a limit is exceeded
● Trend analysis
Bioburden in air/water channel of flexible
endoscopes after disinfection
700
600
Bioburden (CFU)
500
400
Mean:
Median:
300 39 CFU
0 CFU
200
100
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27
Endoscope no
Ashurst L, Kjos B, Lingaas E. 2004

Evaluation and corrective action
Does bioburden determination
tell the truth ??
Sampling deficiency?
Adhesion
Biofilm
Incomplete dispersion
Does bioburden determination
tell the truth ??
Lack of growth ?
Germination of spores
”Dormant” bacteria
Small colony variants
------
Raw materials
Cleaning/lubrication/manufacturing liquid
Transport/holding containers
Work surfaces
Personell attire/hygiene practices
Handling/assembly
Packaging materials and procedures
Storage conditions
Characterization of organisms recovered
But what is the relevance?
Can the problem be solved
simply by adding
another minute
to the sterilization cycle?
Bioburden, inactivation factor
and sterility assurance level (SAL)
1.000.000
Inactivation required:
Number of organisms
10.000
: 1012 : 10 9 : 106
100
0.01
0.0001
0.000001
SAL 10 -6
Exposure to inactivation process
Universitetsklinikk
Dead microorganisms can
also create problems
Endotoxin
Proteins
Teichoic acid
---
What is the maximum
allowable number of dead bacteria
on a sterile device?
?
● 0
● 10
● 100
● 1000
● 10000
● 10.0000
● 1.000.000
● 10.000.000
● 100.000.000

Bioburden Estimation of Reusable and Single Use Medical Devices

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Bioburden Estimation of Reusable and Single Use Medical Devices

Uploaded by

Copyright:

Available Formats

Bioburden estimation of reusable

and single use medical devices –

Bioburden determination common

Modified from Nyström B. J Hosp Infect 1981;2:363

Modified from Rutala WA, et al. AJIC 1998;26:143

Modified from Chu NS, et al. AJIC 1999;27:315

Modified from Chu NS, et al. AJIC 1999;27:315

Part 2: Tests of sterility performed in the

Part 3: Guidance on evaluation and the

● Representative of the batch

● Sodium chloride 0.25 – 0.9 %

Substances released into suspending fluid

Particularly useful for:

➨ Suspensions with microbicidal or

● Defined aliquots of suspension mixed

● Agar allowed to solidify in the plate

● Plates are incubated

● An aliquot of defined volume is spread

● Plates are incubated

● A defined volume spread in a spiral track

● Plates are incubated

Several combinations of media and

● Detecting small colonies

● Inoculating a known number

Treatment no. Colonies

Ashurst L, Kjos B, Lingaas E. 2004

You might also like