sbnet 2010 proper autoclaving of solid wastes - mabritec autoclaving_solid_wastes.pdf · proper...

SBNet 2010 Proper autoclaving of solid wastes Bernard Jenni

Autoclaves: Hazards are often underestimated

Hazards

High temperatures

High pressure

Steam

Hot liquids

Biohazard

2 | SBNet 2010 | B.Jenni | 19.10.2010 | solid wastes |

USERS (the main hazards!)

Incorrect transport of wastes

Incorrect handling

Incorrect processing of materials

Incorrect operation

Inadequate maintenance

Explosion hazard

Explosion in an autoclave caused by cellulose nitrate tubes Nature 199, 102 (July 1963); doi:10.1038/199102a0, I. H. SILVER

Explosion in an autoclave and subsequent fire. Most likely cause: Incorrect disposal of chemical waste in the biowaste bin (2006, USMW).

Do not autoclave bleach (Javel)

Do not autoclave volatile chemicals

Do not autoclave radioactive materials.


Transport >> << CONFLICT >> << Autoclaving

Containers for BL2-biowastes must be closed during the transport - with a visible biohazard sign

They must be open during the autoclaving to allow the penetration of steam

Biohazard sign should not be visible for final discard

4 | | SBNet 2010 | B.Jenni | 19.10.2010 | solid wastes |

The basic principle of autoclaving

Saturated STEAM UNDER PRESSURE is the sterilizing agent

Hot steam = very high heat content = high killing power

Dry, saturated steam condenses on colder surfaces

Condensation releases high amount of heat heat transfer killing effect

Condensation decrease of volume lower pressure draws more steam

Potential issue: condensed water wets the surfaces (objets)


Why is an autoclave such an effective sterilizer?

The world of autoclaves http://esf.uvm.edu/uvmsafety/labsafety/biosafety/autoclaves/autoclaves.html


An autoclave is a large pressure cooker; it operates by using steam under

pressure as the sterilizing agent. High pressures enable steam to reach high

temperatures, thus increasing its heat content and killing power. Most of the

heating power of steam comes from its latent heat of vaporization. This is the

amount of heat required to convert boiling water to steam. This amount of heat

is large compared to that required to make water hot. For example, it takes 80

calories to make 1 liter of water boil, but 540 calories to convert that boiling

water to steam. Therefore, steam at 100º C has almost seven times more heat

than boiling water.

Steam is able to penetrate objects with cooler temperatures because once the

steam contacts a cooler surface it immediately condenses to water, producing a

concomitant 1,870 fold decrease in steam volume. This creates negative

pressure at the point of condensation and draws more steam to the area.

Condensations continues so long as the temperature of the condensing surface

is less than that of steam. These properties ensure rapid heating of surfaces,

good penetration of dense materials, and coagulation of proteins.

Liquid water,...,water vapour,…steam,…& air

Common belief indicates the contrary, but...


MW =

molecular weight

Gas MW

H2O 18

N2 28

O2 32

CO2 44

... AIR IS HEAVIER THAN STEAM! Air density (masse volumique) in kg/m3 under 1013 mbar

RH= relative humidity

Temp. 100% RH 1% RH

0 °C 1.29 1.29

21 °C 1.19 1.20

100 °C 0.59 0.95

... water vapour & steam are invisible!

(only condensed water droplets are visible, e.g., clouds)

Autoclaving of solid wastes

Air is the enemy since the heat power is the steam

Air does not mix readily with steam and sinks to the bottom of the autoclave chamber

Entrapped air pockets prevent steam from touching surfaces, impairing killing effect.

Air must be removed


Air removal by autoclaves without vacuum

Gravity displacement: limited efficiency to remove entrapped air pockets


Source:

Steam sterilization. Rolf Dittli (Systec Schweiz GmbH)

www.sidi.uzh.ch/activities/bio/Dittli.pdf

Air removal by vacuum assisted autoclaves

Air removal by pulsed, fractionated pre-vacuum

Usually multiple pre-vacuum pulse(s)

Vacuum steps below atmospheric pressure


- For solid loads!

- Perfect air removal by hollow

objects, tubings, pipette tips,

etc...

- Liquids will boil, splash and

spoil the autoclave chamber!

Air removal by pressure pulsation steps

Air removal by several pressure releases (not vacuum)

Pulses steps above atmospheric pressure


- Less efficient than fractionated,

pre-vacuum steps

- A better choice for „not-so-solid“

wastes, agar plates, etc...

Sterilization of agar plates

Pressure released steps, 124ºC, 90 min


Temperature inside load Temp chamber

Pressure

QA-run performed by Philippe Wirth

Sterilization of agar plates

Pressure released steps, 124ºC, 90 min


Agar melts and melting is an endothermic reaction

Temperature decreases and much time is needed

to reach the set temperature again.

QA-run performed by Philippe Wirth

The three phases of a cycle

Conditioning phase

- Air is removed and the load is heated to a set temperature.

Sterilizing phase

- Time, which is maintained at the set temperature.

Exhaust / cooling / post-vacuum drying

- Steam is replaced with air (ev. positive pressure to avoid boiling)

- Solid wastes are usually not actively cooled

- Return to atmospheric pressure

- Post-vacuum to minimize odors.


Program parameters, printout and graphs

A sterilizer without such data is a blackbox!

A sterilizer without somebody (BSO?) able to interpret these data is a blackbox as well!


Parameters for „semi-solid“ biowastes

Parameters for the different types of use.


Solids

(glassware, pipette

tips)

Semi-solid biowastes

(plasticware containing

liquids, agar plates,

etc...)

Liquids

(media ,

cell cultures)

Conditioning

phase

Pre-vacuum pulses Steam release pulses

Pre-vacuum pulses (okay for 90% of cases)

Gravitation

Single vacuum

pulse

Sterilizing

phase

Cooling No active cooling No active cooling Active cooling

Positive pressure

Drying Post-vacuum No vacuum No vacuum

Process indicators

Autoclave tapes: only „not treated / treated“

Chemical indicators

Biological indicators (spores of Geobacillus stearothermophilus)


Vials for liquids 3M Attest & paper strips: only for solids

irreversible reaction with heat, steam & time: CrCl3 (pink) + 6H2O + heat CrCl3·6H2O (green)

Proper autoclaving of wastes Questions:

What is the GOAL of the process?

1) GLP/GMP sterilisation?

2) „Make-safe“ inactivation for final discard?

Is validation with G. stearothermophilus necessary for the inactivation of (BL1-BL2) biowastes (which do not contain thermoresistant microorganisms)?

Would it not be sufficient to use a microorganism representative of the load, e.g., a mesophilic bacterium

like Bacillus subtilis or B. atrophaeus?


Message to take home

Autoclaving is a very complex process, which requires a lot of diverse knowledge.

Proper waste autoclaving needs a knowledgeable BSO, able to be an efficient platform between all involved parties (management, autoclaving service, lab users, autoclave suppliers, further external parties).

The BSO should know, understand and able to interpret the physical parameters and printouts of the autoclave cycles.


Dampfdesinfektion versus Dampfsterilisation

Din 58949-3 Deutsche Norm


Desinfektion - DampfDesinfektionsapparate

Teil 3: Prüfung auf Wirksamkeit

1. Anwendungsbereich

Diese Norm gilt für die Prüfung auf Wirksamkeit von Dampf-Desinfektionsapparaten

nach DIN 58949-2 einschliefllich des Desinfektionsprozesses.

Sie gilt auch für die Dampfdesinfektion von Abfällen, an die aus infektionspräventiver

Sicht besondere Anforderungen zu stellen sind, d. h. für Abfälle, die aufgrund von §17

Infektionsschutzgesetz behandelt oder gemäss Europäischem Abfallkatalog dem

Schlüssel 18 01 03 entsprechen.

ABFALLVERZEICHNIS : 18 01 03 = Infektiöse Abfälle

6.2 Biologische Indikatoren

Die Wahl des biologischen Indikators ist abhängig vom Desinfektionsverfahren. Für

das 75 °C-Verfahren mit einer Haltezeit von 20 min ist der biologische Indikator

DIN 58949-Bi-AB (Enterococcus faecium nach DIN 58949-4) zu verwenden. Für das

105 °C-Verfahren mit einer Haltezeit von 5 min ist der biologische Indikator DIN

58949-Bi-ABC (Bacillus subtilis nach DIN 58949-4) zu verwenden.

BACKUP SLIDE

Dampfdesinfektion mit B. subtilis als Bioindikator

D-Werte von B. subtilis


Quelle: 2. Vorlesung Steriltechnik - Galenische Pharmazie

BACKUP SLIDE

Boiling water for 10 minutes can reduce B. anthracis spore counts by at least 6log10. Inactivation of Bacillus anthracis spores.

Ellen A. Spotts Whitney et al. Emerging Infectious Diseases, 9, No. 6,623-627, 2003

Dampfdesinfektion mit B. subtilis als Bioindikator

Bioburden


Quelle: 2. Vorlesung Steriltechnik - Galenische Pharmazie

BACKUP SLIDE

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