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Specifications for laboratory autoclaves
J?ournal of Hospital Infection (1981) 2, 377-384
Specifications The
Public
Health
for laboratory
Laboratory
autoclaves
Service Subcommittee* Autoclaves-f
on Laboratory
Public Health Laboratory Service, Head Ofice, 61 Co&dale Avenue, London NW9 5EQ, U.K. Summary: As a survey of autoclaving practice in microbiology laboratories had previously shown autoclaving performance to be unsatisfactory, specifications have been drawn up that are intended to improve safety and efficiency in autoclave processing. Recommended specifications are defined for new horizontal autoclaves intended either for the sterilization of discard loads alone or for the dual purpose of sterilizing discard material and for the preparation and sterilization of microbiological media. The snecifications are considered suitable for laboratories which handle Category B and C pathogens but are not regarded as appropriate for high security laboratories handling Category A pathogens. Experimental data are presented to support the recommendations made.
Introduction A survey was undertaken by a Subcommittee of the Public Health Laboratory Service (PHLS) during 1975-76 into routine laboratory autoclaving practice to assess how far this met acceptable criteria for safety and reliability. Tests at 27 laboratories, mostly PHLS, on 46 downward displacement autoclaves showed that the sterilizing performance was unsatisfactory on 10 of 62 occasions. The results of this survey together with advisory notes on the safe and efficient use of laboratory autoclaves have been published (PHLS Subcommittee on Laboratory Autoclaves, 1978). In the light of these and other previously published observations by its members on problems connected with autoclaving for laboratory purposes (Gillespie and Gibbons, 1975; Everall and Morris, 1975, 1976; Everall, Morris and Yarnell, 1978), the Subcommittee drew up what it saw as the essential requirements for autoclaves suitable for use in medical laboratories, other than those handling Category A pathogens, and invited tenders for the manufacture of a multipurpose horizontal autoclave conforming to these requirements. Tenders from two manufacturers were accepted and two machines were installed, one at the PHLS laboratory, Shrewsbury, the other at the PHLS laboratory, Coventry, as prototypes for experimental evaluation. *J. E. M. Whitehead (Chairman), W. Bruce, J. V. Dadswell, P. H. Everall, S. A. Gibbons, C. A. Morris (Secretary), P. R. Mortimer, M. W. Scruton and J. B. Towell. tReprint requests to: Dr C. A. Morris, Public Health Laboratory, Mytton Oak Road, Shrewsbury SY3 8XH, U.K. or95-6701/8r/oqo377
+ 08 $or.oo~o
@ 1981 Academic
377
Press
Inc.
(London)
Limited
378
PHLS subcommittee
on laboratory
autoclaves
Material
Features of the prototype autoclave The prototype was a rectangular horizontal autoclave designed to sterilize culture media and discard materials. Fully automatic programming provided three pre-set cycles for the former and a single pre-set cycle for the latter; the selection and setting up of the time/temperature relationships being infinitely variable but adjustable only by a supervisor and not by the operator. An automatic door safety mechanism activated by the chamber pressure and also by the load temperature ensured that the door could not be opened until it was safe to do so. Assisted air removal from discard loads in a manner which would minimize possible dispersal of infected materials into the surroundings and assisted cooling were other requirements. The autoclaves were fitted with a chart temperature recorder with sensing probes, one indicating the chamber drain temperature, the other the load temperature. Air ballasting to reduce fluid loss from bottles during cooling was also included. Method
Both autoclaves were evaluated similarly according to protocols drawn up by the Subcommittee. Their evaluation and assessment by experiment and routine use enabled the Subcommittee to draw up recommended specifications for new autoclaves either for discard loads only (Specification A) or for media sterilization as well (Specification B), see Appendix. Results
Only a summary of the more important findings obtained during the course of experiments is given below. It is representative of the results obtained and is included to support the recommendations made. Control of the time/temperature Detailed SpeciJications)
relationship
in the load (see items 4.3 and 4.4 of
As a general principle the load temperature lags behind the chamber drain temperature in the early part of the cycle, conforms to that of the drain during the holding period and exceeds the chamber drain temperature during the cooling period. If the temperature within the chamber is controlled by a signal from the drain which is used to modulate the incoming steam supply, the correct temperature will be reached in the load, provided there are no problems in air removal (see below). To ensure that the load receives the correct amount of heat for the chosen time, a thermocouple in a unit of the load is employed to activate the process timer when the required temperature within the load has been reached. The load thermocouple, or a suitable simulator, is also used to ensure that the door release mechanism is not activated before the load has cooled to a safe temperature. A single pressurereducing valve set to give steam at the required temperature is a cheap and satisfactory system to give temperature control for a single cycle discard machine.
Specifications
for laboratory
autoclaves
379
Air removal from discard loads (see item 4.5 of Detailed Specifications) Simple downward displacement. In the survey of autoclaving practice which has been referred to earlier, eight of the 10 failures to achieve satisfactory sterilizing conditions were observed in discard loads: a strong indication that simple downward displacement of air through a chamber drain fitted with a trap set could not be relied upon to remove air from many laboratory loads. Later experience indicated that this was particularly the case when the load consisted of a number of small items (e.g. tissue culture tubes or bijou bottles) contained in a box or bucket, or of polystyrene ware (e.g. Petri dishes) which on melting can entrap air and infected materials in the cocoon-like structure thus formed. A measure of the efficiency, or the inefficiency, of air removal is the load lag time (the time elapsing between the drain reaching the intended sterilizing temperature and the load reaching the same temperature). It will be seen from Table I that, using simple downward displacement load lag times varied from 28 to 69 min with a mean of 42 min. A simple prevacuum of O-2 bar* absolute pressure does not overcome this problem (see Table I) and therefore reliance has to be placed on other methods of air removal. Dhect air displacement by steam (DADBS). This had been in routine use for some time previously at Shrewsbury and the prototype machine was modified to permit its use once it was obvious that neither simple downward displacement nor a single prevacuum were going to be effective. In this technique (Everall, Morris and Yarnell, 1978) an additional steam supply, obtained by inserting a T piece into the conventional steam inlet pipe, is taken into the chamber through a suitable port and terminated there as a manifold fitted with four Hansen valves. Air is displaced from discarded materials by injecting steam into the bottom of the containers. This steam is taken from the Hansen valve by means of silicone rubber tubes which are connected to the steam injection funnels with which the units of the load are provided. The results obtained using this technique on test loads consisting of 3 gross of 4 oz screw-capped (bijou) bottles contained in either stainless steel buckets or plastic boxes are shown in Table I. It will be seen that load lag times have fallen to zero. Free steaming andful’l$owfree steaming. The resistance to flow caused by numerous unnecessary fittings in the exhaust line of the autoclave at Coventry did not allow satisfactory free steaming. These restrictions were such that 1.4 bar g steam pressure was reached in the chamber even when the exhaust line was fully opened to allow free steaming. The chamber drain bypass and exhaust line were redesigned to eliminate many of these restrictions and 8 inch copper pipes were replaced by 34 inch tubing to improve the free steaming capability, i.e. full flow free steaming. Assisted cooling (see items 4.8 and 4.9 of Detailed Specifications A and B, resjectively) Assisted nutritive tures for as used
cooling shortens overall cycle times and minimizes the deterioration in the qualities of culture media which may occur when exposed to high temperaprolonged periods (Everall and Morris, 1978). Spray cooling with water, in pharmaceutical autocalves, is unsuitable as some loads will include
*105N/m2 (or pascals) = 1 bar = 14.7 lb/square
inch.
380
PHLS subcommittee
on laboratory
autoclaves
Table I. Load lag times and times to start of holding period
Method of air removal (A) Downward Shrewsbury
No. of cycles
Time to start of holding period (min) average (range)
Load lag time (min) average (range)
displacement
Coventry (B) Steam ejector Shrewsbury (0.2 bar abs.)* Coventry (0.15 bar abs.)* (C) ‘Free steaming’ Shrewsbury Coventry (1.4 bar g t)* (D) DADBS Shrewsbury
)E] (W
;
2
0-9 (C) W) (C)
: ;
g]
i
[ii!; (E) Full flow free steaming Coventry
36 (28-44) 30 (29-30) 61 (52-69)
46 (37-54) 44 (42-46) 63 (55-70)
29
46
I? (0.5-2.5) 10 (8-20)
7; (18-21) 27 (22-38)
12 (8-16) 12 (10-14)
56 (43-75) 16 (13-17)
1s
00
11 (7-16) 10
:
1 (0.1-2.0) 1 (05-2.0)
20 (16-26) 10 (9-10)
H, Hot chamber, autoclave used previously during the same working day; C, cold chamber, autoclave not used previously that working day. *105N/ma (or pascals) = 1 bar = 14.7 lb/square inch. tSee text, p. 379.
unsealed containers. Jacket cooling (Table II) was found to be efficient. Other methods, for example a water cooled coil in the chamber and refrigerated air, now exist which the Subcommittee did not have the opportunity to assess. Table II. The effect of jacket cooling on the cooling period Duration
Location Coventry Shrewsbury Coventry
Sterilization temperature (“C) 121 115
of cooling period (min)
No air ballast, no cooling
With air ballast, no cooling
No. of cycles Time
No. of cycles Time
1 1
247 267
1
246
-
With air ballast, with cooling No. of cycles Tie
1
241
1 1
66 61
2
214 218
2
z:
Air ballasting (see item 4.10 of Detailed Specifications) This is recommended as standard on dual purpose laboratory autoclaves and as an optional extra for single cycle machines designed solely for the sterilization of discard loads. The option should be exercised if there is any likelihood that the
Specifications
for laboratory
autoclaves
381
autoclave may be required to process large bottles of media. The effect of air ballasting on fluid losses is illustrated in Table III. Table III. Air ballast andfluid
losses (all cycles jacket cooled)
Average loss per bottle (g)
Operating Shrewsbury Shrewsbury Coventry
Water E;:
50 50 50
x x x
Shrewsbury
Agar/
50
x
300 ml in 600 ml bottles
110
water Awl
50
x
300 ml in 600 ml bottles
115
2.4
17.5
x
400 ml in 600 ml bottles
110
15.4
31-9
Load
No./vohune/bottle
water Shrewsbury
MacConkey agar
50
Containment
of autoclave
efluent
capacity
(see item 4.5 of Detailed
Air ballast
No air
temperature (“c> 400 ml in 600 ml bottles 110 400 ml in 600 ml bottles 115 400 ml in 600 ml bottles 115
Laboratory
o-7 ;:; (2 cycles) 5.5
ballast 112 14.2 20.3 9.3
Spec$ications)
Absolute containment of effluent is not considered necessary for laboratories which do not handle Category A pathogens. The effluent from an autoclave at the start of a cycle is not sterile. In some ‘worst case’ experiments, Bacillus stearothermophilus was found in the effluent for as long as 20 min, and even non-sporing bacteria and bacteriophage for as long as 5 min. Experimentally it was shown that organisms may be displaced from the surface of media by falling drops of condensate and/or air/ steam turbulence. It was further shown that the contamination of effluent is greatly reduced if the autoclave chamber is hot at the start of the cycle and infected material is contained so far as is practicable, i.e. buckets should not be overfilled, Petri dishes should be neatly stacked, not tossed haphazardly into the container. However, the load must not be enclosed to the extent that steam penetration is seriously impaired. The effect of some of these factors on the discharge of bacterial spores from horizontal downward displacement autoclaves is shown in Figures 1-3. Conclusion
This paper presents some experimental data by way of explanation for specifications which have been prepared to assist those who need to purchase laboratory autoclaves. Where there is restricted laboratory space available or financial limitation, a multipurpose autoclave capable of sterilizing both discard material and media can be particularly valuable. Fully automatic control goes a long way to ensure predictable and reproducible function of the autoclave and reduces operator errors. Control through sensing temperature from within load simulators ensures firstly that the load is held for an adequate time to achieve sterilization and, secondly, that it has cooled sufficiently to allow the door locking mechanism to be released and the autoclave opened safely. Assisted cooling leads to more efficient usage by reducing the
382
PWLS subcommittee
on laboratory
autoclaves
Figure 1. Effect of containment on displacement of spores from single culture plate* (no baffle at steam inlet; autoclave used previously the same day). (a) Open 250 x 250 mm plate (mean of six experiments); (b) 250 x 250 mm plate in open box (300 x 260 x 210 mm deep) (mean of seven experiments). *Agar surface culture of Bacillus globigii (30-40 x lOlo spores/plate).
cycle time and also reduces heat denaturation of culture media; cooling also contributes to safety. Fully variable time/temperature relationships under the control of the supervisor but not the operator ensure versatility without loss of safety from illadvised modifications to a cycle during the course of a run. Assisted air removal from a discard load promotes safety by hastening heat penetration by steam into the load and reduces the time during which viable organisms may be present in the autoclave e&ent. Air ballasting is a useful option for sterilization of bottle loads of media, contributing to economy by reducing loss from overspill during cooling and lessening the problems of alterations in the ratio of media constituents during processing. Some features, for example prevacuum to the chamber, included in the prototype autoclaves proved of no advantage in practice and so were omitted from the final specifications. A summary of the specifications is appended to this paper. Fuller versions (‘Detailed Specifications’) written in a form and detail necessary for competitive tendering, are available on request. Their use by intending purchasers of new
Figure 2. Discharge of spores in effluent related to hot or cold start. Challenge load: 1.25 1 of liauid culture containing 10’ snores Bacillus glob&ii/ml in enamel tray (240 X 190 X 50 mm): (a) ‘Hot’ start = autoclave used previously iame day (mean of t&o experiments); (b) ‘cold’ start = autoclave not used previously that day (mean of two experiments).
Specifications
3ooor
for laboratory
(a)
autoclaves
383
r(b)
Minutes
Figure 3. Arrangement of Petri dishes and presence of spores in effluent (autoclave used previously same day). (a) Filled discard box (300 x 260 x 210 mm deep) containing 78 scattered Petri dishes (90 mm diam.)* (mean of 14 experiments); (b) filled discard box (300 x 260 x 210 mm deep) containing 84 neatly piled Petri dishes (90 mm diam.)* (mean of 21 experiments). *Dishes containing agar surface cultures of Bacillus globigii (30-40 x log spores/dish).
laboratory autoclaves should result in the supply of autoclaves with a performance which is appreciably safer and more efficient than that achieved by many instruments at present in use. The Subcommittee is grateful to Mr P. H. Everall and Mr M. W. Scruton for much of the experimental work undertaken on its behalf; to Mr W. Bruce of the Animal Virus Research Institute, Pirbright for additional experiments on survival of bacteriophage; to Mr S. A. Gibbons of the Trent Regional Health Authority for technical advice; and to Mr K. Oates, Mr D. Hurrell and Mr J. C. T. Williamson of the Scientific and Technical Branch of the Department of Health and Social Security for their cooperation. References Everall, P. H. & Morris, C. A. (1975). Some observations on cooling in laboratory autoclaves. Journal of Clinical Pathology 28, 664-669. Everall, P. H. & Morris, C. A. (1976). Failure to sterilise in plastic bags. Journal of Clinical Pathology 29, 1132. Everal!, P. H., Morris, C. A. & Yarnell, R. (1978). Sterilisation in the laboratory autoclave using direct air displacement by steam. Journal of Chkical Pathology 31, 144-147. Gillespie, E. H. & Gibbons, S. A. (1975). Autoclaves and their dangers and safety in laboratories. Journal of Hygiene, Cambridge 75,475-487. PHLS Subcommittee on Laboratory Autoclaves. (1978). Autoclaving practice in microbiology laboratories: report of a survey. Jourr:al of Clinical Pathology 31,418-422. Appendix Spec@ations The detailed specifications referred to in the text are not appended to this paper but are available on application to the Director, Microbiological -safety Reference Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG. The specifications include the following: Specification 2. Specification for a laboratory &toclave for discard loads. This is for a single cvcle horizontal autoclave designed to sterilize discarded materials and general laboratory wake/equipment, but not culture media, and is for a free standing uni? (but capable of panel mounting) and of at least 285 I(10 cu ft) nominal capacitv. (The size of chamber recommended is Chosen as one suitable for PILLS laboratory nee‘ds but should not be taken to indicate that autoclave chambers of smaller capacity do not have a place in other laboratories.) Details are provided on construction, materials, autoclave housing, thermal
384
PEJLSsubcommittee
on laboratory autoclaves
insulation, the door and its closing and securing mechanism; safety provisions (including illuminated ‘door locked’ signal) for sliding and other doors; general safety requirements, provision of T pieces for testing; thermocouple connections; instrumentation including indicating thermometer sensing from the chamber drain, process control indicators, pressure gauges and temperature recording device independent of the indicating thermometers equipped with two sensing probes, one sensing from the drain and the other from the load; and timers. Means of checking accuracy of thermometers; the method of temperature and process control; the means of air removal from discard loads in a manner which will avoid dispersal of infected material into the working area; performance tests; chamber drain trap bypass; automatic air admission at the last stage of cycle; assisted cooling; fully automatic programming; air ballasting; servicing; commissioning tests and checks to be carried out at works and after installation on site; technical documents and maintenance are also included. Specification B. Specification for a dual purpose laboratory autoclave suitable for the sterilization of media and of discard loads. Construction and general safety provisions are similar to those of Specification A but included is an over-temperature cut out when the load temperature exceeds 5°C of the set working temperature; and fully automatic programming with manual reversion. The method of temperature and process control is also specified in detail. Appendices are included on the following: (1) Standards for materials used in the manufacture of the autoclave chamber and door. (2) Provisional minimum performance tests. (3) Commissioning tests. (4) Summary of tender. The specifications include a number of footnotes giving additional practical advice to assist a purchaser in selecting the most appropriate autoclave offered on the market at the time of choice.
Specifications The
Public
Health
for laboratory
Laboratory
autoclaves
Service Subcommittee* Autoclaves-f
on Laboratory
Public Health Laboratory Service, Head Ofice, 61 Co&dale Avenue, London NW9 5EQ, U.K. Summary: As a survey of autoclaving practice in microbiology laboratories had previously shown autoclaving performance to be unsatisfactory, specifications have been drawn up that are intended to improve safety and efficiency in autoclave processing. Recommended specifications are defined for new horizontal autoclaves intended either for the sterilization of discard loads alone or for the dual purpose of sterilizing discard material and for the preparation and sterilization of microbiological media. The snecifications are considered suitable for laboratories which handle Category B and C pathogens but are not regarded as appropriate for high security laboratories handling Category A pathogens. Experimental data are presented to support the recommendations made.
Introduction A survey was undertaken by a Subcommittee of the Public Health Laboratory Service (PHLS) during 1975-76 into routine laboratory autoclaving practice to assess how far this met acceptable criteria for safety and reliability. Tests at 27 laboratories, mostly PHLS, on 46 downward displacement autoclaves showed that the sterilizing performance was unsatisfactory on 10 of 62 occasions. The results of this survey together with advisory notes on the safe and efficient use of laboratory autoclaves have been published (PHLS Subcommittee on Laboratory Autoclaves, 1978). In the light of these and other previously published observations by its members on problems connected with autoclaving for laboratory purposes (Gillespie and Gibbons, 1975; Everall and Morris, 1975, 1976; Everall, Morris and Yarnell, 1978), the Subcommittee drew up what it saw as the essential requirements for autoclaves suitable for use in medical laboratories, other than those handling Category A pathogens, and invited tenders for the manufacture of a multipurpose horizontal autoclave conforming to these requirements. Tenders from two manufacturers were accepted and two machines were installed, one at the PHLS laboratory, Shrewsbury, the other at the PHLS laboratory, Coventry, as prototypes for experimental evaluation. *J. E. M. Whitehead (Chairman), W. Bruce, J. V. Dadswell, P. H. Everall, S. A. Gibbons, C. A. Morris (Secretary), P. R. Mortimer, M. W. Scruton and J. B. Towell. tReprint requests to: Dr C. A. Morris, Public Health Laboratory, Mytton Oak Road, Shrewsbury SY3 8XH, U.K. or95-6701/8r/oqo377
+ 08 $or.oo~o
@ 1981 Academic
377
Press
Inc.
(London)
Limited
378
PHLS subcommittee
on laboratory
autoclaves
Material
Features of the prototype autoclave The prototype was a rectangular horizontal autoclave designed to sterilize culture media and discard materials. Fully automatic programming provided three pre-set cycles for the former and a single pre-set cycle for the latter; the selection and setting up of the time/temperature relationships being infinitely variable but adjustable only by a supervisor and not by the operator. An automatic door safety mechanism activated by the chamber pressure and also by the load temperature ensured that the door could not be opened until it was safe to do so. Assisted air removal from discard loads in a manner which would minimize possible dispersal of infected materials into the surroundings and assisted cooling were other requirements. The autoclaves were fitted with a chart temperature recorder with sensing probes, one indicating the chamber drain temperature, the other the load temperature. Air ballasting to reduce fluid loss from bottles during cooling was also included. Method
Both autoclaves were evaluated similarly according to protocols drawn up by the Subcommittee. Their evaluation and assessment by experiment and routine use enabled the Subcommittee to draw up recommended specifications for new autoclaves either for discard loads only (Specification A) or for media sterilization as well (Specification B), see Appendix. Results
Only a summary of the more important findings obtained during the course of experiments is given below. It is representative of the results obtained and is included to support the recommendations made. Control of the time/temperature Detailed SpeciJications)
relationship
in the load (see items 4.3 and 4.4 of
As a general principle the load temperature lags behind the chamber drain temperature in the early part of the cycle, conforms to that of the drain during the holding period and exceeds the chamber drain temperature during the cooling period. If the temperature within the chamber is controlled by a signal from the drain which is used to modulate the incoming steam supply, the correct temperature will be reached in the load, provided there are no problems in air removal (see below). To ensure that the load receives the correct amount of heat for the chosen time, a thermocouple in a unit of the load is employed to activate the process timer when the required temperature within the load has been reached. The load thermocouple, or a suitable simulator, is also used to ensure that the door release mechanism is not activated before the load has cooled to a safe temperature. A single pressurereducing valve set to give steam at the required temperature is a cheap and satisfactory system to give temperature control for a single cycle discard machine.
Specifications
for laboratory
autoclaves
379
Air removal from discard loads (see item 4.5 of Detailed Specifications) Simple downward displacement. In the survey of autoclaving practice which has been referred to earlier, eight of the 10 failures to achieve satisfactory sterilizing conditions were observed in discard loads: a strong indication that simple downward displacement of air through a chamber drain fitted with a trap set could not be relied upon to remove air from many laboratory loads. Later experience indicated that this was particularly the case when the load consisted of a number of small items (e.g. tissue culture tubes or bijou bottles) contained in a box or bucket, or of polystyrene ware (e.g. Petri dishes) which on melting can entrap air and infected materials in the cocoon-like structure thus formed. A measure of the efficiency, or the inefficiency, of air removal is the load lag time (the time elapsing between the drain reaching the intended sterilizing temperature and the load reaching the same temperature). It will be seen from Table I that, using simple downward displacement load lag times varied from 28 to 69 min with a mean of 42 min. A simple prevacuum of O-2 bar* absolute pressure does not overcome this problem (see Table I) and therefore reliance has to be placed on other methods of air removal. Dhect air displacement by steam (DADBS). This had been in routine use for some time previously at Shrewsbury and the prototype machine was modified to permit its use once it was obvious that neither simple downward displacement nor a single prevacuum were going to be effective. In this technique (Everall, Morris and Yarnell, 1978) an additional steam supply, obtained by inserting a T piece into the conventional steam inlet pipe, is taken into the chamber through a suitable port and terminated there as a manifold fitted with four Hansen valves. Air is displaced from discarded materials by injecting steam into the bottom of the containers. This steam is taken from the Hansen valve by means of silicone rubber tubes which are connected to the steam injection funnels with which the units of the load are provided. The results obtained using this technique on test loads consisting of 3 gross of 4 oz screw-capped (bijou) bottles contained in either stainless steel buckets or plastic boxes are shown in Table I. It will be seen that load lag times have fallen to zero. Free steaming andful’l$owfree steaming. The resistance to flow caused by numerous unnecessary fittings in the exhaust line of the autoclave at Coventry did not allow satisfactory free steaming. These restrictions were such that 1.4 bar g steam pressure was reached in the chamber even when the exhaust line was fully opened to allow free steaming. The chamber drain bypass and exhaust line were redesigned to eliminate many of these restrictions and 8 inch copper pipes were replaced by 34 inch tubing to improve the free steaming capability, i.e. full flow free steaming. Assisted cooling (see items 4.8 and 4.9 of Detailed Specifications A and B, resjectively) Assisted nutritive tures for as used
cooling shortens overall cycle times and minimizes the deterioration in the qualities of culture media which may occur when exposed to high temperaprolonged periods (Everall and Morris, 1978). Spray cooling with water, in pharmaceutical autocalves, is unsuitable as some loads will include
*105N/m2 (or pascals) = 1 bar = 14.7 lb/square
inch.
380
PHLS subcommittee
on laboratory
autoclaves
Table I. Load lag times and times to start of holding period
Method of air removal (A) Downward Shrewsbury
No. of cycles
Time to start of holding period (min) average (range)
Load lag time (min) average (range)
displacement
Coventry (B) Steam ejector Shrewsbury (0.2 bar abs.)* Coventry (0.15 bar abs.)* (C) ‘Free steaming’ Shrewsbury Coventry (1.4 bar g t)* (D) DADBS Shrewsbury
)E] (W
;
2
0-9 (C) W) (C)
: ;
g]
i
[ii!; (E) Full flow free steaming Coventry
36 (28-44) 30 (29-30) 61 (52-69)
46 (37-54) 44 (42-46) 63 (55-70)
29
46
I? (0.5-2.5) 10 (8-20)
7; (18-21) 27 (22-38)
12 (8-16) 12 (10-14)
56 (43-75) 16 (13-17)
1s
00
11 (7-16) 10
:
1 (0.1-2.0) 1 (05-2.0)
20 (16-26) 10 (9-10)
H, Hot chamber, autoclave used previously during the same working day; C, cold chamber, autoclave not used previously that working day. *105N/ma (or pascals) = 1 bar = 14.7 lb/square inch. tSee text, p. 379.
unsealed containers. Jacket cooling (Table II) was found to be efficient. Other methods, for example a water cooled coil in the chamber and refrigerated air, now exist which the Subcommittee did not have the opportunity to assess. Table II. The effect of jacket cooling on the cooling period Duration
Location Coventry Shrewsbury Coventry
Sterilization temperature (“C) 121 115
of cooling period (min)
No air ballast, no cooling
With air ballast, no cooling
No. of cycles Time
No. of cycles Time
1 1
247 267
1
246
-
With air ballast, with cooling No. of cycles Tie
1
241
1 1
66 61
2
214 218
2
z:
Air ballasting (see item 4.10 of Detailed Specifications) This is recommended as standard on dual purpose laboratory autoclaves and as an optional extra for single cycle machines designed solely for the sterilization of discard loads. The option should be exercised if there is any likelihood that the
Specifications
for laboratory
autoclaves
381
autoclave may be required to process large bottles of media. The effect of air ballasting on fluid losses is illustrated in Table III. Table III. Air ballast andfluid
losses (all cycles jacket cooled)
Average loss per bottle (g)
Operating Shrewsbury Shrewsbury Coventry
Water E;:
50 50 50
x x x
Shrewsbury
Agar/
50
x
300 ml in 600 ml bottles
110
water Awl
50
x
300 ml in 600 ml bottles
115
2.4
17.5
x
400 ml in 600 ml bottles
110
15.4
31-9
Load
No./vohune/bottle
water Shrewsbury
MacConkey agar
50
Containment
of autoclave
efluent
capacity
(see item 4.5 of Detailed
Air ballast
No air
temperature (“c> 400 ml in 600 ml bottles 110 400 ml in 600 ml bottles 115 400 ml in 600 ml bottles 115
Laboratory
o-7 ;:; (2 cycles) 5.5
ballast 112 14.2 20.3 9.3
Spec$ications)
Absolute containment of effluent is not considered necessary for laboratories which do not handle Category A pathogens. The effluent from an autoclave at the start of a cycle is not sterile. In some ‘worst case’ experiments, Bacillus stearothermophilus was found in the effluent for as long as 20 min, and even non-sporing bacteria and bacteriophage for as long as 5 min. Experimentally it was shown that organisms may be displaced from the surface of media by falling drops of condensate and/or air/ steam turbulence. It was further shown that the contamination of effluent is greatly reduced if the autoclave chamber is hot at the start of the cycle and infected material is contained so far as is practicable, i.e. buckets should not be overfilled, Petri dishes should be neatly stacked, not tossed haphazardly into the container. However, the load must not be enclosed to the extent that steam penetration is seriously impaired. The effect of some of these factors on the discharge of bacterial spores from horizontal downward displacement autoclaves is shown in Figures 1-3. Conclusion
This paper presents some experimental data by way of explanation for specifications which have been prepared to assist those who need to purchase laboratory autoclaves. Where there is restricted laboratory space available or financial limitation, a multipurpose autoclave capable of sterilizing both discard material and media can be particularly valuable. Fully automatic control goes a long way to ensure predictable and reproducible function of the autoclave and reduces operator errors. Control through sensing temperature from within load simulators ensures firstly that the load is held for an adequate time to achieve sterilization and, secondly, that it has cooled sufficiently to allow the door locking mechanism to be released and the autoclave opened safely. Assisted cooling leads to more efficient usage by reducing the
382
PWLS subcommittee
on laboratory
autoclaves
Figure 1. Effect of containment on displacement of spores from single culture plate* (no baffle at steam inlet; autoclave used previously the same day). (a) Open 250 x 250 mm plate (mean of six experiments); (b) 250 x 250 mm plate in open box (300 x 260 x 210 mm deep) (mean of seven experiments). *Agar surface culture of Bacillus globigii (30-40 x lOlo spores/plate).
cycle time and also reduces heat denaturation of culture media; cooling also contributes to safety. Fully variable time/temperature relationships under the control of the supervisor but not the operator ensure versatility without loss of safety from illadvised modifications to a cycle during the course of a run. Assisted air removal from a discard load promotes safety by hastening heat penetration by steam into the load and reduces the time during which viable organisms may be present in the autoclave e&ent. Air ballasting is a useful option for sterilization of bottle loads of media, contributing to economy by reducing loss from overspill during cooling and lessening the problems of alterations in the ratio of media constituents during processing. Some features, for example prevacuum to the chamber, included in the prototype autoclaves proved of no advantage in practice and so were omitted from the final specifications. A summary of the specifications is appended to this paper. Fuller versions (‘Detailed Specifications’) written in a form and detail necessary for competitive tendering, are available on request. Their use by intending purchasers of new
Figure 2. Discharge of spores in effluent related to hot or cold start. Challenge load: 1.25 1 of liauid culture containing 10’ snores Bacillus glob&ii/ml in enamel tray (240 X 190 X 50 mm): (a) ‘Hot’ start = autoclave used previously iame day (mean of t&o experiments); (b) ‘cold’ start = autoclave not used previously that day (mean of two experiments).
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Figure 3. Arrangement of Petri dishes and presence of spores in effluent (autoclave used previously same day). (a) Filled discard box (300 x 260 x 210 mm deep) containing 78 scattered Petri dishes (90 mm diam.)* (mean of 14 experiments); (b) filled discard box (300 x 260 x 210 mm deep) containing 84 neatly piled Petri dishes (90 mm diam.)* (mean of 21 experiments). *Dishes containing agar surface cultures of Bacillus globigii (30-40 x log spores/dish).
laboratory autoclaves should result in the supply of autoclaves with a performance which is appreciably safer and more efficient than that achieved by many instruments at present in use. The Subcommittee is grateful to Mr P. H. Everall and Mr M. W. Scruton for much of the experimental work undertaken on its behalf; to Mr W. Bruce of the Animal Virus Research Institute, Pirbright for additional experiments on survival of bacteriophage; to Mr S. A. Gibbons of the Trent Regional Health Authority for technical advice; and to Mr K. Oates, Mr D. Hurrell and Mr J. C. T. Williamson of the Scientific and Technical Branch of the Department of Health and Social Security for their cooperation. References Everall, P. H. & Morris, C. A. (1975). Some observations on cooling in laboratory autoclaves. Journal of Clinical Pathology 28, 664-669. Everall, P. H. & Morris, C. A. (1976). Failure to sterilise in plastic bags. Journal of Clinical Pathology 29, 1132. Everal!, P. H., Morris, C. A. & Yarnell, R. (1978). Sterilisation in the laboratory autoclave using direct air displacement by steam. Journal of Chkical Pathology 31, 144-147. Gillespie, E. H. & Gibbons, S. A. (1975). Autoclaves and their dangers and safety in laboratories. Journal of Hygiene, Cambridge 75,475-487. PHLS Subcommittee on Laboratory Autoclaves. (1978). Autoclaving practice in microbiology laboratories: report of a survey. Jourr:al of Clinical Pathology 31,418-422. Appendix Spec@ations The detailed specifications referred to in the text are not appended to this paper but are available on application to the Director, Microbiological -safety Reference Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG. The specifications include the following: Specification 2. Specification for a laboratory &toclave for discard loads. This is for a single cvcle horizontal autoclave designed to sterilize discarded materials and general laboratory wake/equipment, but not culture media, and is for a free standing uni? (but capable of panel mounting) and of at least 285 I(10 cu ft) nominal capacitv. (The size of chamber recommended is Chosen as one suitable for PILLS laboratory nee‘ds but should not be taken to indicate that autoclave chambers of smaller capacity do not have a place in other laboratories.) Details are provided on construction, materials, autoclave housing, thermal
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insulation, the door and its closing and securing mechanism; safety provisions (including illuminated ‘door locked’ signal) for sliding and other doors; general safety requirements, provision of T pieces for testing; thermocouple connections; instrumentation including indicating thermometer sensing from the chamber drain, process control indicators, pressure gauges and temperature recording device independent of the indicating thermometers equipped with two sensing probes, one sensing from the drain and the other from the load; and timers. Means of checking accuracy of thermometers; the method of temperature and process control; the means of air removal from discard loads in a manner which will avoid dispersal of infected material into the working area; performance tests; chamber drain trap bypass; automatic air admission at the last stage of cycle; assisted cooling; fully automatic programming; air ballasting; servicing; commissioning tests and checks to be carried out at works and after installation on site; technical documents and maintenance are also included. Specification B. Specification for a dual purpose laboratory autoclave suitable for the sterilization of media and of discard loads. Construction and general safety provisions are similar to those of Specification A but included is an over-temperature cut out when the load temperature exceeds 5°C of the set working temperature; and fully automatic programming with manual reversion. The method of temperature and process control is also specified in detail. Appendices are included on the following: (1) Standards for materials used in the manufacture of the autoclave chamber and door. (2) Provisional minimum performance tests. (3) Commissioning tests. (4) Summary of tender. The specifications include a number of footnotes giving additional practical advice to assist a purchaser in selecting the most appropriate autoclave offered on the market at the time of choice.