Sterilization and validation

Richard Marchand MD
Medical Microbiologist and Infectious
Diseases
Assistant Professor
University of Montreal
One hospital (The CSSSSVLDLJ)
Somewhere in Quebec

Plan
History
Basic

definitions
A lot of little quizzzz
Properties of heat, steam and bugs
Process validation
Biological Indicators
Chemical Indicators
Water, water, water

Disinfection or Sterilisation ?
What

is the needed temperature to inactivate

bacteria ????

Answer : It depend on the bacteria because

some are highly resistant to heat.
(Ex.: Geothermal bacteria like hot spring bugs withstand 250 oC )
Most human pathogens were thought to be killed by temperature
lower than 150 oC.

Little Quizz
Question

: Where came from these weird

standards like

Reduction of 106
Temperatures of 121, 132 and 134 oC ?????

Answer: From the post world war II food

industry (mostly canning )
FDA : FOOD and Drug Agency

Steam sterilization origins

HISTORY

1862
1880
1906
1925
1932

Invention of the Autoclave

The first indicator : a potato
Creation of the FDA
Waxy pellets that melt at 121oC
(ATI) CI with lead sulphite
passes from black to white

1940

(ATI) CI Chromium TriChloride

passes from purple to green

Pasteurisation and tyndallisation

Around 1860 Louis Pasteur demonstrated that the heating

between 50 and 60 oC without air for 30 minutes prevent
deterioration of wine during transport. He also demonstrated that
a previous heating of the malt before yeast inoculation prevented
beer contamination.
Later a process called tyndallisation was developed by Tyndall
and consisted in a series of burst of increased temperature up to
70 oC at regular intervals (originally once a day for 3 days). This
is to activate the resistant forms to germinate in order to kill them
with the next heat burst. Milk pasteurisation was to follow by
using 30 minutes heat burst at 63 oC followed later by a 15
minutes heat burst at 73 oC.

HISTORY

39-45 World War II (many cases of food poisoning)

1950-60

Development of validation concepts

for the canning industry (Clost. bot)

1960-70

Development of the Dvalue concept

using heat resistant spores

The canning industry monitoring

Spores

(Bacillus and Clostridia) are the most

heat resistant organisms
Spores are to be killed with a good and
reliable probability
So
Lets use non infectious spores to ascertain
that the beans and the sardines are safely
canned

How does heat kill micro organisms ?

Heat

coagulates proteins : egg white

Heat has a mild oxidative effect
What is oxidation ?
Will discuss that later

Water Boils at
100 C : fresh water in a pan with a lid
97 C : fresh water in a pan without a lid
104 C : sea water (depending on salt concentration)

Boiling never guarantees that the temperature was high enough for
bacterial proteins to coagulate

The very low water content of spores and other substances ( Heat
shock proteins also called stress proteins) protects them from denaturation

Sporulated pathogens (like Clostridium sp.) resists up to 8H30 at

100C. (Lowering risks of infections other than tetanus or gas gangrene )

Boiling Temperature is linked to

pressure

100

C at 1 ATM
121 oC at 2 ATM (1 barr)
132 oC at 3 ATM (2 barr)
80 oC at 0.5 ATM (Mont Blanc)
70 oC at 0.35 ATM (Mont Everest)
40 oC at 0.02 ATM (Mechanical vacuum)
o

Quizz Pasteur ?
Why 2 or more exposures to heat ?
Answer : For germination to happen
Why no air ?
Answer : Because the air blocks the energy
transfer toward organic matter.

The origin of Biological indicators BI

Although

most do not, some bacterial

species die in a predictable manner, specially
some sporulated thermophillic bacilli
So lets screen them and use the most resistant
and predictable safe bug that can be
canned to design safe processes.

SURVIVAL PROBABILITY

How to define resistance to heat ?

The

D value concept was invented !

D value: principle and logic

More it is hot, faster the micro organisms die

Faster the bugs die, faster is the process

The speed of the process is expressed with the D value

value = exposition time required (in minutes)

to kill 1 log (90%) of the micro organisms

A Dvalue of 6 means that it takes 6 minutes to reduce par a

factor of 10 (90%) or 1 log the number of micro organisms.

N.B. Faster does not mean more efficient, because a bug killed more slowly is not less dead.

Effect of probability for a BI exposed

to heat
For a BI of : 2.0 X 106 et Dvalue* of 1 minute
Survival

After 1.0 minute = 200,000

After 2.0 minutes = 20,000
After 3.0 minutes = 2000
After 4.0 minutes = 200
After 5.0 minutes = 20
After 6.0 minutes = 2
After 7.0 minutes = 0.2

Total time to reduce to zero = 6.5 minutes

*At that time the most resistant Bacillus know was the stearothermophilus
with a Dvalue of 1 to 1.5

Effect of probability for many cans

Cycle of 6.5 minutes

1 can/1 BI (2.0 X 10 )
= 0-0.2 survivor
10 cans/10 BI (2.0 X 10 ) = 2 survivors (or to 7.5 min)
100 cans/100 BI (2.0 X 10 ) = 20 surv. (or to 8.5 min)
103 cans/103 BI (2.0 X 10 ) = 200 surv. (or to 9.5 min)
104 cans/104 BI (2.0 X 10 ) = 2000 surv. (or 10.5 min)
105 canss/105 BI (2.0 X 10 ) = 20000 surv.(or 11.5 min)
6

10

11

More we have cans, more we have contaminated cans !

Then what about safety ?

Cans

batches are generally less than a million

at a time

So lets double the time, to bring back the survival

probability once again around 0 0.2
(The overkill approach was born)

Why most micro organisms do not die

in a linear fashion ?
The

mechanisms of death are not mono

molecular (different proteins are degraded or
coagulated at different speeds)
The proportion of life essential proteineous
targets varies with the age of the micro
organisms, their life cycle, their food supply
etc..
Susceptibility to heat varies thereof none
linearly therefore with limited predictability

Why is it written everywhere that all

bugs die on linear scale fashion ?
The

fifty percent principle applies

(eg. Half of what is written in textbooks is false,

the problem is we dont know which one)
Confusius

Evidence

In

base medicine is applied

(eg.: A concept is an evidence when everybody

say the same thing, true or not.)

fact most European textbooks recognize

this fact, while only few American books does

Pasteurisation yesterday
In

1964 it was demonstrated that hotter but

shorter heat bursts have less deleterious
effect on organic material without loosing its
effects on microbial flora.
HOWEVER : This process is not a sterilization
process because it kills only the heat
sensitive flora.

HISTORY cont.

1960-70

Development of the Dvalue concept

using heat resistant spores for
sterilisation + Fvalue + Zvalue

1965

Proposal by Sweden of the SAL for a

definition of sterility

1979

Proposal by Canada of a legal

definition of sterility

Z value: principle and logic

If the temperature is lowered, bugs are killed more slowly and the D
value increases (because it takes more time to kill)

Conversely if the temperature is higher, faster the bugs die, and

lower is the D value

Z value = the number of degree of temperature

required to obtain a variation of 1 log of the D value

For a given micro organism, A Zvalue is a measure of its

resistance to heat because higher the Zvalue, more heat is needed
to augment the Dvalue by a factor of 1.

F value: principle and logic

If the D value is measured at different temperature and pressure it can be seen that
a D value varies with the pressure. More the Dvalue decreases with a specific
increase of pressure, more powerful is considered the process.

F value = a measure of the capacity to inactivate

bacteria in function of the temperature.

Mathematically the F value is expressed by the rate of mortality per

minute in function of temperature for a given pressure.

This concept applies de facto to steam sterilization only and is a

measure of the power of a sterilizer
are faster than a kettle.)

(Big boilers and big pipes

Little quizzz
Question : Is all processes D values a time
dependant measure ?
Answer : No, for radiation and ozone
sterilization the Dvalue is dose dependant

The reference cycle

1965 The Swedish National Health Board proposed :

Sterility Assurance Level (SAL) 10-6
121 oC (gravity) and 1.05 bar
106 spores with a Dvalue of 1.0 to 1.5 min

Overkill Cycle of 12 to 18 minutes + conditioning and

rising time (Temperature and Pressure )

At that time because of gravity cycle technology, average

cycle took : 30 minutes

SURVIVAL PROBABILITY

STERILITY : LEGAL DEFINITION proposed by Canada

A medical device can be qualified of sterile if: the

probability of survival of a micro organism is less then
:

FOR IMPLANTABLES : 1 on 1,000,000 (SAL of

10-6)

FOR TOPICALS :
10-3)

+ 2 other conditions : endotoxins and biomechanical properties

SAL = Sterility Assurance Level

1 on 1000

(SAL of

STERILITY ASSURANCE LEVEL

Effect of probability for a BI

For a BI of : 2.0 X 106 et Dvalue of 1 minute
Survival

After 1.0 minute = 200,000

After 2.0 minutes = 20,000
After 3.0 minutes = 2000
After 4.0 minutes = 200
After 5.0 minutes = 20
After 6.0 minutes = 2
After 7.0 minutes = 0.2

Total time to reduce to zero = 6.5 minutes

Effect of probability for many packs

Cycle of 6.5 minutes

1 pack/1 BI (2.0 X 10 )
= 0-0.2 survivor
10 packs/10 BI (2.0 X 10 ) = 2 survivors (or to 7.5 min)
100 packs/100 BI (2.0 X 10 ) = 20 surv. (or to 8.5 min)
103 packs/103 BI (2.0 X 10 ) = 200 surv. (or to 9.5 min)
104 packs/104 BI (2.0 X 10 ) = 2000 surv. (or 10.5 min)
105 packs/105 BI (2.0 X 10 ) = 20000 surv.(or 11.5 min)
6

10

11

More we have packs, more wont pass !

What should be done to insure safety

with big loads ?
Lets double the time (overkill approach)
BUT : many devices cannot withstand a doubling
in time !

Cannot be applicable by the industry for many things

(Industrial sterilizers can handle thousands of BIs)

What if the real bioburden is greater than a million

What if the bugs are dead but their toxins still there ?
The bioburden evaluation approach was born when
regulatory bodies agreed to adapt normalized and
individualised cycles to devices characteristics and lots

The bioburden method in short

Is

not application to usual hospital settings

Require microbiological evaluations of the
microbial burden on a representative
sampling of devices
Require an adaptation of cycles for devices
and lot characteristics
Stringent monitoring of all critical parameters

How does the SAL should apply to

hospital overkill cycles ?

First : do not take into account the conditioning and

rise up phases (they add up on the killing therefore
increasing the safety margin)
Second, because an overkill approach is used, make
sure that the SAL objective is attained before
doubling the time

This means that the 10-6 objective should be acquired at half

cycle
The whole cycle should be capable to attained a 10-12
The cycle should be long enough to give some margin for
delayed heating ups by heat barriers

Effect of liquids or heat barriers

Quizz
Question : Can you name a heat barrier material ?
Answer : polymers (plastics) like non porous
plastic containers, wood, rubber etc..

Other cycles later developed

Vacuum

reduces cycle to approximately 20

minutes.
Taking into account the Fvalue :

132 oC
2.0 bar
Time drops to 4 minutes

Preferred by many for orthopaedic

metallic devices

steel and

In the real . . .
Pre
conditioning

Sterilization
3.5 to 15 min

Dry time 15 to
20 minutes max.

Why 132 oC for metals ?

Less

damaging for the passivation layer

Less oxidation (pin point oxidation) on edges
(sharpness is kept longer)
Less water deposition and water stains
Less fatigue because of shorter exposure to
heat
Less micro fractures
Faster

Pasteurisation cont. : Today

= energy bursts of all kinds
Physical

methods : heat (with or without

steam), UVs, ultrasounds, pressure bursts etc..
Chemical Methods : peroxyde, ozone, cold
plasma, glow discharge plasma etc..
132oC for 4 minutes is basically the same
concept
In all cases, for them to work, water is required to
a certain level. No water molecules, to little or
to much will hamper these process.

Little quizzzzz :
Name the critical parameters of steam
sterilization
Temperature (121 oC)
Time (duration)
Humidity
Pressure (includes vacuum)
Phases of cycle (manufacturer and Europe)

134oC SAL of 10-6 (total cycle of 3.5 minutes)

BIOLOGICAL KILL
(HALF-CYCLE)

0 minute
1 minute

2 minutes

3 minutes

S.A.L. 10-6

This is presuming all mechanical aspects of your

process are working the way they should and you are
getting adequate saturated steam.

The challenge of short cycles

1 minute in a 3.5 minutes cycle is a variation of 30 percent

1 minute in a 20 minutes cycle is a variation of 5 percent

Shorter cycles

need a much more precise monitoring and highly sensitive

indicators
are greatly influenced by heat barriers
are at greater risks of errors if the quality of steam is not optimal
are much more susceptible to condensation if a proper preconditioning is not done

Little Quizzzzzz

f
o
t
lo

e
d
e
r
o
t
s

!
y
g
ne r

What is the difference between steam at 100 oC

and humidity at 100oC ?
What is the difference between 0oC ice solid
water and et 0oC liquid water ?
Can water exist as a gas at temperature lower
than 0oC ?
Can water not be steam at temperature higher
y
than 100oC ?
t
i
d
i

m
u
h
,
s
e
Y

Humidity versus Vapour

For a given temperature
Humidity

and vapour are both constituted of

water molecules in a gas state but,
with very different levels of stored energy.

All gases in presence of water have some of it

in a gaseous state called humidity.

Is humidity a must ?

Humidity is an intermediate between the steam

(the energy source) and the organic material
(the target)
Humidity acts as the transient energy buffer
transferring state that permits proteins
denaturation called hydrolysis

Role for humidity

The sterilant (the steam) transport the energy

(1kg = 540 kCal)

The energy is not transferred efficiently to the

organic matter or the device to sterilise by the air or
by a gaz. ( remember dry heat is a poor process)
Lack of humidity = lack of transport buffer = poor
energy transfer
Surplus of humidity = difficulty to transfer the energy
to the target because you put the energy in the buffer
that you have to fill first (the water absorbs the
energy of the sterilant)

Dry heat (Purkins 1960)

170

C (340 oF)
160 oC (320 oF)
150 oC (300 oF)
140 oC (285 oF)
121 oC (250 oF)
o

:
:
:
:
:

60 minutes
120 minutes
150 minutes
180 minutes
12 to 14 hours

It takes 538 calories to convert one mL of

water at 100oC to steam at 100oC
T
E
M

STEAM
Low energy
vapour

High energy
vapour

water

100

200

300

400

500

600

700

Instruments cause a temperature

drop.
One or two degrees of
temperature drop here

100

200

300

Releases hundreds of
calories of heat here.

400

500

600

700

The energy contained and

released is called :
HEAT OF CONDENSATION

100

200

300

400

500

600

700

To condense or not to con dance

100

200

300

400

500

600

700

If to much energy is released the steam goes

back to liquid water ( full condensation) = the
devices will be wet.

To dance rather than condense

The

best : If enough energy is kept in the

system, the energy release is up to a level
where the water is still in a gaseous state
(preventing water deposition)

100

200

300

400

A very fine tuning line to control

500

600

700

What conditions favours full

condensation ?
Cold

instruments when the steam comes in

(improper pre-conditioning)
Low energy steam (insufficiently heated
boilers, bad insulated pipes, calcium
deposits)
Poor quality steam with lots of debris and
salts (favours total release of the energy)
To much humidity (Wet steam)

Steam quality is important

Saturated steam

Dry steam

98% steam, 2% water vapor

Superheated

Wet steam

Supersaturated

STERILISATION VALIDATION
Sterilisation validation is arbitrarily laid on the
construction of an cycle based on the
behaviour of biologic indicators hopped to be
predictable

BASIC USAGE OF BIs

For

-) cycle developement
-) cycle
validation
-) routine monitoring

Cycle

development = numeration analysis and

negative fraction analysis
In accordance with the cycle philosophy
Exceeding

force method (overkill)

Bioburden evaluation method (bioburden)

Variability of BI
The

D value varies in function of

the support
The D value varies in function of
the wrapping (and assembly)
Viability varies with the culture media
Viability with the recovering technique
Viability goes down with time

WHAT IS AN INDICATOR ?
On

paper
Self -contained
Sealed ampulla (spores + broth)
Spores suspension
Tube witness (pt of fusion)

It is a device conceived to
verify if the process operated as expected

Validation of biological indicators

The

reality :

We

do not use the most resistant organisms

The predictive behaviour is generally linear only for one process
Manufacturers seldom use more practical strains (read : less
linear strains for more economical, inoffensive, self resistance
and stability)

There

is no such thing as a universal biological

indicator
The choice of any particular strain is therefore a
manner of arbitrary choice

Rapid BIs and EIs

Rapid

Bis do the same thing as conventional Bis

but give answers in 1 to 3 hours
Rapid Bis use chemical or fluorometric markers
to signify the presence of living organisms
Enzymatic Indicators use enzymes mimicking
life essential proteins as inactivation targets.
Answers are obtained in 20 seconds (speed for
$$$$$)

Role of Biological Indicators

BIs,

albeit their weaknesses are still the best

tools to develop and validate the construction
of a cycle or a process from beginning to
end.
BI manufacturing is therefore submitted to
stringed norms and regulation by
governmental agencies.

3M Rapid Indicator

3M Rapid Indicator

Commercial BI
Spores

from all spore vendors are produced

by 3 major suppliers
Manufacturers of BI must specify :
Type

of bacterial population
Quantity of spores
Storage conditions and expiration date
Usage condition (culture media and incubation time)
Performance characteristics

Sportrol

(1.4 X 105 Dvalue 1.5)

Survivors

0 min 140,000
1.5 min
3.0 min
4.5 min
6.0 min
7.5 min
1.4
9.0 min 0.14

Complete

14,000
1,400
140
14

kill is achieved in 8.5 minutes or 70 % of

the required time for 12 log
(with a Dvalue of 1.0, or
47% with a Dvalue of 1.5)

Attest

Survivors

(2.5 X 105 Dvalue 1.9)

0 min
1.9 min
3.8 min
5.7 min
7.6 min
9.5 min
11.4 min

250,000
25,000
2,500
250
25
2.5
0.25

Complete kill is achieved in 10.7 minutes or 90 % of

the 12 log reference cycle

Proof plus

Survivors

(1.3 X 104 Dvalue 1.9)

0 min
1.9 min
3.8 min
5.7 min
7.6 min
9.5 min

13,000
1,300
130
13
1.3
0.13

Complete kill is achieved in 8.7 minutes or 75 % or

the 12 log reference cycle

Chemspor

(2.5 X 102 Dvalue 4.9)

Survivors

0 min
4.9 min
9.8 min
14.7 min

250
25
2.5
0.25

Complete kill is achieved in12.1 minutes or 102 %

of the 12 log reference cycle

BI in overkill method

With few exceptions, most commercial BIs do not

verify the proposed 12 log reference cycle
Most commercial BIs pass between 55 and 95 % of
the reference cycle
They wont tell you if your sterilizer operate suboptimally
A fail tells you Houston we have a problem
Are not useful to tell where it is and how to judge its
is important.

Chemical Indicators (CIs)

Except for wax, most CIs are based on a pH change

resulting from organic acid evaporation by heat and
revealed by a colorimetric indicator
The oldest CI
Pb + S + H
PbS
The
reaction do not occur without heat
Paper CIs (chemical ink) are much more stable and
predictable than CIs requiring assembly

Indicators :

ISO Class 11140-1

Class

1 External indicator charged to signal

if the pack has been exposed or not.
Example : autoclave tape.

Class

2 Indicator for a specific parameter

Qualitative Example : Bowie Dick (vaccum 121 oC)
Class 3 Indicator for a unique parameter :
Quantitative Example : Melting wax pellet at 121 oC

ISO 11140-1

Class 4

Indicator sensitive to 2 parameters (ex.:

time and temperature) within 25 % of expected targets.

For class 4 and higher, indicators must change abruptly

The change of color must happen within 25 % of the
expected time and within 2 oC
Example : An indicator that accept (OK or Pass) at 132oC pour 4 min.
MUST reject (Fail) a 130oC for 3 minutes.

ISO 11140-1

Class 5 An indicator/integrator sensitive to 2 or more

parameters, and reacting within 15 % of expected
targets.

In this category, the change of color must be abrupt, happens within 15

% of expected targets and MUST NOT happen if the targeted
temperature is not achieved within 1oC.
Example : An indicator

that accept (OK or Pass) at 132 oC for 4 min MUST reject (Fail) a 131 oC for 3 minutes and
22 seconds.

Classe 6 An indicator/integrator sensitive to 2 or more

parameters, and reacting within 6 % of expected targets.

In this category, the change of color must be abrupt, happens within 6%

of expected targets and MUST NOT happen if the targeted temperature
is not achieved within 1oC.
Example : An indicator that accept (OK or Pass)
at 132oC for 4 min MUST reject (Fail) a 131 oC for 3 minutes and 45 seconds.

Enemy of steam sterilization

: air

Why, why, why

For the same reasons we wash the plates in
standing position (specialy with gravity
cycles) (unwrapped material)
Avoid stacking up
Avoid asparagus assembly
Question : Are you sure that things are sterile under the
rubber bands

My daughters trick ?

Air displacement
By

gravity
By dilution (flash)
By pressure pulse
By vacuum
By pressure pulse and vacuum
(Steam Flush Pressure Pulse)

By high pulse pressure

(Above Atmospheric Steam Flush Pressure Pulse)

Water, eau, H2O

Water is a source of life and headaches

Water is source or stains and deposits
Hard water : contains a lot mineral salts and
generally has a basic pH

calcium deposits , pipe crusting

A basic pH means less hydrogen ions availability

Soft water: few mineral salts but is generally

much more corrosive being most of the time
much more acid.

Canada
Red

stain : iron
Brown stain : Manganese, Magnesium
Green or blue stain : copper
Viscous and green Stuff : metallothropic
bacteria
Hardness
according to
regions

Micro organisms found in potable water

Divers viruses (Norwalk like, enterovirus)

Pseudomonas (aeruginosa in particular)
Acinetobacter spp
Burkolderia cepacia
Aeromonas
Legionella
non TB Mycobacteria
Toxoplasma gondii
Cryptosoridia

Water Surveillance
Water

quality requirements in hospital should

be high because :

Immunosupressed patients
Types of treatments
Needs for cleaning, disinfection and sterilization

Unacceptable usages for tap water

Endoscope
Washing
Drug

of wounds and ulcers ; (Why?)

nebulizers ; (Why ?)

Dialysis
CSR

rinsing after disinfection ; (Why ?)

; (Why ?)

nebulizers and ultrasonic baths : (Why?)

Question period

Is your brain also steaming with

heat

Thank you