Sporicidal activity of sodium dichloroisocyanurate, peroxygen and glutaraldehyde disinfectants against Bacillus subtilis

Journal

of Hospital

Sporicidal peroxygen

Infection

(1996)

32, 283-294

activity of sodium and glutaraldehyde Bacillus

dichloroisocyanurate, disinfectants against

sub tilis

D. Coates

Public Health Laboratory, Royal Preston Hospital, PO Box 202, Sharoe Green Lane North, Preston, PR2 4HG, Lancashire UK Received 18 July 1995; revised manuscript accepted 15 September 1995 Summary:

The activity of sodium dichloroisocyanurate (NaDCC), peroxygen and glutaraldehyde disinfectants against spores of Bacillus subtilis NCTC 10073 was evaluated using suspension tests. The activity of aqueous solutions of NaDCC disinfectants increased with the level -of available chlorine (av.Cl) but was considerablv reduced bv low levels of blood. Five percent Titan Sanitizer (1200 ppm av.Cl) achieved a >lO’-fold reduction in spore count (kill) in 3 h in the absence of blood but no kill in 3 h with 2% blood present. One percent Presept (3180 ppm av.Cl) achieved a >105-fold kill in 1 h in the absence of blood and in 2 h with 2% blood present. One percent Haz-Tab (5750ppm av.Cl) achieved a >lO’-fold kill in 5 min in the absence of blood and in 30 min with 2% blood present. One percent Virkon (peroxygen) achieved a lo’-fold kill in 2-3 h in the absence of blood but little kill in 3 h with 2% blood present. Nu-Cidex (peracetic acid) was rapid in action and tolerant of organic matter. A 24 h old solution achieved a >lO”fold kill in 5 min with 10% serum present. Cidex Long-Life (glutaraldehyde) worked much slower: a 28-dav-old solution achieved a >lO’-fold kill in 2 h with 4% blood present. Neat sporicidin (glutaraldehyde-phenate) was slightly superior to Cidex Long-Life but in a 1 in 8 dilution exhibited markedly reduced activity; a 30-day-old solution achieved a lo”-fold kill in 10 h in the absence of blood but only a IO’-fold kill in 10 h with 2% blood present. Keywords: Bacillus chloroisocyanurate;

subtilis; peroxygen;

sporicidal activity; glutaraldehyde.

disinfectants;

sodium

di-

Introduction

Usually, bacterial spores are hundreds or thousands of times more resistant to chemical disinfectants than are vegetative bacteria.’ Consequently, it is generally assumed that a disinfectant that is capable of killing bacterial spores will also kill all vegetative organisms. Hence sporicidal disinfectant products are usually promoted as sterilants although they may only achieve sterilization under certain conditions. Only a few classes of liquid chemical disinfectant possess useful sporicidal activity2-7 including iodinebased,i,k8-lt chlorine-based’h~“-I~‘~~*~‘~ and peroxygen disinfectants,5-7,‘c’7 Correspondence

to: Dr

D.

Coates.

284

D. Coates

which are strong oxidizing agents, and aldehyde disinfectants,‘-7”8-25 which are strong alkylating agents. Halogen, peroxygen and aldehyde disinfectants all have long histories. Over the years new classes of liquid sporicidal agent have been sought but practical alternatives to those above have not been found. Consequently, recent research has been directed at evolving new products from established sporicides. One approach has been to investigate the possible benefits of mixing a bactericide with a sporicide and it was found that mixing alcohol with sodium hypochlorite3,‘2v’3 or phenol with glutaraldehyde21 boosted the sporicidal activity. Another approach has been to develop specialized products for hospitals from sporicides previously used mainly by industry e.g. stabilized solutions of peracetic acid (PAA) and chlorine dioxide have recently been developed for instrument sterilization. Sodium hypochlorite (Milton: Procter & Gamble, Egham, Surrey, UK) has been used to ‘sterilize’ babies’ bottles for almost 50 years. The sporicidal activity of sodium hypochlorite is greatly affected by pH, increasing with acidity.s,‘3,26 In recent years products based on the solid organo-chlorine compound sodium dichloroisocyanurate (NaDCC) have become increasingly popular because of their many advantages over bleach.26-35 One application is the use of NaDCC granules for spills of body fluids. Laboratory tests and hospital trials33,35 have shown that granules containing appropriate levels of available chlorine (av.Cl) rapidly absorb and disinfect body fluids heavily contaminated with vegetative bacteria, and the use of these granules as an alternative to bleach has been endorsed.“b’37 Fundamental differences exist between the mode of action and properties of sodium hypochlorite and NaDCC systems2’ with NaDCC showing superior bactericidal yet inferior sporicidal activity.2326 Specific data on the sporicidal activity of granular NaDCC products marketed for spills of body fluids is lacking and such activity is required, for example, when dealing with faecal material containing Clostridium difficile. Inorganic peroxides such as hydrogen peroxide and organic peracids such as PAA are well-established industrial disinfectants/sterilants but, until recently, have been little used in hospitals. The use of nebulized hydrogen peroxide for disinfecting respiratory ventilators was suggested many years ago3’ and has proved useful for certain types of ventilator39 whilst recently PAA products such as Steris (DiagMed, Thirsk, Yorkshire, UK) and Nu-Cidex (Johnson & Johnson Medical, Ascot, Berkshire, UK) have been introduced for the sterilization of endoscopes.40-42 A general purpose peroxygen disinfectant, Virkon (Antec International, Sudbury, Suffolk, UK), was first marketed for veterinary use and now also for hospital use. It is a powder composed of a balanced, stabilized blend of peroxygen compounds, surfactant, organic acids and an inorganic buffer system. The main oxidizing agent present is potassium monopersulphate. One application is the use of the powder for dealing with spills of body fluids. Laboratory tests and hospital trials35 have shown that the application

Sporicidal

activity

of disinfectants

285

of this non-absorbent powder to spills of body fluids heavily contaminated with vegetative bacteria rapidly disinfects the spilled material without any release of chlorine fumes which can occur when NaDCC granules are applied to urine and acidic fluids.33s43As yet, no data has been published on sporicidal activity. Glutaraldehyde is currently used in the majority of endoscopy units despite its well-documented toxocity. Both alkaline and acidic formulations generally contain 2% glutaraldehyde. Lowering the concentration of glutaraldehyde reduces the toxicity but concomitantly the sporicidal activity. One possible way of lowering the glutaraldehyde concentration whilst maintaining the activity is to add phenol/sodium phenate which has been claimed to have a synergistic effect.” Sporicidin (Sporicidin International, Rockville, MD, USA) used this principle and was a mixture of 2% glutaraldehyde, 7.05% phenol, 1.2% sodium phenate and inert ingredients. A 1:8 dilution of an ‘improved formula’ (investigated in the present study) was claimed to sterilize in 8 h during the first 14 days after activation and in 10 h for the next 16 days. A 1: 16 dilution was claimed to achieve hospital level disinfection in 10 min. In 1991, the USA Environmental Protection Agency (EPA) started testing products registered with them as sterilants using the Association of Official Analytical Chemists (AOAC) sporidical test which is a carrier test using porcelain penicylinders inoculated with spores of B. subtilis or Clostridium sporogenes. Sporicidin failed this test,7,44 had its EPA registration cancelled,44,4” and consequently was withdrawn from the market. The need for sterilization or high-level disinfection of the many different types of endoscopes has been the subject of much debate. Sterilization is safer but usually takes much longer to achieve. In practice, infections with spore-forming bacteria following endoscopy are rare and current attention is focussed on mycobacteria and viruses. In general, sterilization is regarded as necessary (although not always carried out) for endoscopes, which enter sterile tissue or body sites such as arthroscopes and laparoscopes, whereas high-level disinfection is considered adequate for other types. The present paper gives sporicidal data obtained with a number of products marketed for hospital use over recent years. Materials

and

methods

Disinfectants Titan Sanitizer disinfectant detergent. (Lever Industrial, Runcorn, Cheshire, UK) is a powdered blend of anionic detergent, mild alkali, polyphosphate and NaDCC with a nominal av.Cl content of 2.2% which can be used in different ways for different purposes. For spills of body fluids, the manufacturer’s instructions are to apply the powder liberally over the spillage and leave for at least 5 min before mopping up with paper towels.

286

D. Coates

For routine cleaning and disinfection a 1% aqueous solution is recommended and for specific disinfection a 5% aqueous solution with a 5 min contact time. According to the manufacturer, the product is completely effective against all bacteria, spores, yeasts, moulds and viruses when used as recommended. Presept disinfectant granules. (Johnson & Johnson Medical) are based on NaDCC, have an av.Cl content of around 30%, and are designed for use on spills of body fluids. Haz- Tab disinfectant granules. (Guest Medical, Edenbridge, Kent, UK) are based on NaDCC, have an av.Cl content of around 60% (one of two formulations available), and are designed for use on spills of body fluids. Vi&on. (Antec International) is a balanced, stabilized blend of peroxygen compounds, surfactant, organic acids and an inorganic buffer system which, according to the manufacturer, contains approximately 10% available oxygen. It is a general purpose disinfectant which can be used directly as a powder on spills of body fluids or as an aqueous solution with a standard use-dilution of 1%. Two different batches of Virkon were tested three years apart using two different batches of I?. subtilis spore suspension. The first batch (A) of Virkon tested came supplied in a 5 kg drum. A 20 g sample was taken at random from the top to prepare a test solution. Subsequently, the manufacturer recommended that the entire contents of a 5Og sachet should be used to prepare a test solution. It had been postulated that, in a large container, different components of complex powder formulations might settle out at different rates so a small sample taken from the top might not be representative. Hence, the second batch (B) of Virkon tested came supplied in a 50 g sachet and the entire contents were used to prepare the test solution. is a stabilized buffered solution Nu-Cidex. (J o h nson & Johnson Medical) of PAA which is prepared (activated) by mixing a 5% PAA concentrate with a buffered stabilizer/corrosion inhibitor system to produce a solution containing 3500 ppm PAA which has a 24 h shelf life. The solution can be re-used over this period provided the PAA concentration stays above 2500 ppm. Nu-Cidex was designed to replace glutaraldehyde in endoscopy units where staff sensitivity is a problem or concern. According to the manufacturer, Nu-Cidex used as instructed will disinfect in 5 min and sterilize in 10 min. Cidex Long-Life. (Johnson & Johnson Medical) is a buffered, alkaline solution of 2% glutaraldehyde with a shelf life of 28 days after activation. It is used for disinfection and ‘sterilization’ of endoscopes. Sporicidin. (Sp oricidin International) 7.05% phenol, 1.2% sodium phenate of 30 days after activation intended

was a mixture of 2% glutaraldehyde, and inert ingredients with a shelf life for disinfection and ‘sterilization’ of

Sporicidal

activity

287

of disinfectants

endoscopes. According to the label of the ‘improved formula’ tested, a 1:8 dilution would sterilize if articles were completely immersed for 8 h during the first 14 days of re-use and for 10 h thereafter. Also, a I:16 dilution would disinfect in 10 min.

Available chlorine assays Assays of available chlorine in NaDCC disinfectant using a sodium arsenite titration method.“6

solutions

were

made

Test organism The test organism was Bacillus subtilis NCTC 10073 which is used in the UK for making spore test pieces for controlling ethylene oxide sterilization. Spore suspensions in deionized water containing approximately lo9 spores/ mL were prepared by the method of Beeby and Whitehouse” and kept in the refrigerator until required.

Suspension tests Sporicidal tests were carried out at room temperature. Test mixtures (10 mL) were made by mixing appropriate volumes of disinfectant (freshly prepared in distilled water), defibrinated horse blood (organic matter challenge) distilled water and spore suspension and then vortexing. Blood was replaced by inactivated horse serum for testing Nu-Cidex because mixing blood with Nu-Cidex produced copious volumes of froth. For experiments with blood or serum the spores were added 2 min after the addition of blood or serum to the disinfectant and vortexing to promote neutralization. After set time intervals, 1 mL samples were pipetted into 9 mL volumes of neutralizer solution (see below). Serial 1 O-fold dilutions in distilled water were prepared and seven drops (0.14mL) from each dilution and the neutralizer solution were pipetted on to blood agar plates with pipettes delivering 50 drops/ml. The plates were incubated at 37°C for up to seven days and colony counts made. Counts from the seven drops were meaned. Reductions in count were calculated from the difference between counts obtained in the presence of disinfectant and control counts (distilled water substituted for disinfectant). The neutralizer solutions employed were distilled water containing 1% sodium thiosulphate and 10% inactivated horse serum for NaDCC disinfectants and Virkon, nutrient broth (Oxoid No. 2, Basingstoke, Hants, UK) containing 5% sodium thiosulphate and 0.025% catalase (Sigma Chemical, Poole, Dorset, UK) for Nu-Cidex, and nutrient broth containing 3% Tween 80 and 10% inactivated horse serum for glutaraldehyde disinfectants, which were shown to be effective under the experimental conditions by the method of Maurer.”

D. Coates

288 Table

I.

Activity

aqueous solutions of Titan Sanitizer, Presept and Haz-Tab of B. subtilis at room temperature (18”-24°C)

of

Product

%

Titan Sanitizer

1 1 5

ppm

1 1 1 1 1 1

Haz-Tab

: 1 1 Av.Cl,

available

chlorine.

Blood (%)

* Log,,

reduction

Reaction time

0

240 240 1200 1200 1200 1200 3180 3180 3180 3180 5750 5750 5750 5750 5750 5750 5750

5 5 5 1

Presept

av.Cl

3h 3h lh 2h 3h 3h lh lh 2h 3h 2.5 min 5.0 min 15 min 30 min 15 min 30 min 45 min

2 0

: 2 0 2 2 4 : ; % 4 factor=log,,

inoculum survivor

against spores h&o reduction factor*

Cl 0 d 3 >5 0 >5 3-4 >5 0 4 >5 4 >5 Cl 2 >5

count count

Results

The sporicidal activity of aqueous solutions of the NaDCC-based products tested increased with the level of av.Cl present but decreased markedly in the presence of blood (Table I). One percent Titan (240 ppm av.Cl) showed negligible activity in 3 h in the absence of blood. Five percent Titan (1200 ppm av.Cl) achieved a >lO’-fold reduction in spore count (kill) in 3 h in the absence of blood but no kill in 3 h with just 2% blood present. One percent Presept (3180 ppm av.Cl) achieved a >lO’-fold kill in 1 h in the absence of blood, in 2 h with 2% blood present but no kill in 3 h with 4% blood present. One percent Haz-Tab (5750 ppm av.Cl) achieved a >lO’fold kill in 5 min in the absence of blood, in 30 min with 2% blood present, and in 45 min with 4% blood present. The sporicidal activity of Virkon is shown in Table II. Considering the time interval between tests and that different batches of Virkon and spore suspensions were used, the results obtained for batches A and B are reasonably consistent. One percent aqueous Virkon achieved a lo’-fold kill in 2-3 h in the absence of blood but showed little activity in 3 h with 2% blood present. Two percent Virkon achieved a 104-lo’-fold kill in 30-60 min in the absence of blood and moderate activity in 3 h with 2% blood present. Three percent Virkon achieved a 104-1 OS-fold kill in 15 min in the absence of blood and a 103-104-fold kill in 3 h with 2% blood present.

Sporicidal Table

II.

Activity

of

Batch

aqueous

activity

solutions temperature

Virkon @I

of

of disinfectants

Virkon against (18.5”-22.5”C)

Blood (%I

289 spores

Reaction time

A*

0 0 0 0 0 2 :: 2 2 4 0 2 4

2

* See Materials

and methods.

Table

III.

t See Table

0

1

* See Table

against spores (2O.O”C)

Serum (%I

Reaction (min)

4 4 10 10 4 4

:

I:, 10 10

at

room

l-2 4 5 Cl l-2 5 5 2-3 1 4-s 3-4 <1

I

Activity of Nu-Cidex at room temperature

Days since activation

subtilis

LO&” reduction factor?

lh 2h 3h lh 3h 10 min 20 min lh lh 2h 3h 15 min 30 min lh 3h 3h 15 min 3h 3h

1

B*

of B.

1 2 1 2 5 1 2 5

time

of B. subtilis

hhl reduction factor* 3-4 >5 2-3 >5 2 4-5 >5 2 +5 ZS

I

The sporicidal activity of Nu-Cidex in the presence of 4% and 10% horse serum is shown in Table III. Reaction mixtures were prepared by mixing 1 mL of serum, 1 mL of spore suspension and 8 mL of Nu-Cidex so the level of PAA present in the reaction mixture was 80% of that in the NuCidex. Hence, if the Nu-Cidex contained 3500 ppm initially then the

D. Coates

290 Table

IV.

Activity

Product

of Cidex

Dilution

Cidex Long-Life

against

Blood (%)

0 “2:

1Neat in 8

1 in

Sporicidin (19.5”-2Z.O”C)

Days since activation

Neat

Sporicidin

* See Table

Long-Life and temperature

16

spores

of B. subtilis

Reaction time (h)

at room

~%?o reduction factor*

28 28

0 4 4

1 1 2 1 2

>5 3 >5 3 >5

300 0

i 0

41 8

2z >6

0 ii 0

2 22 4

1: 24 10

425 ~6 2

3: 30

i 0

248 ::

z 4

2 30 30 0

02 2 4 0

10 24 24 24

>62 4-5 l-2 l-2

I.

reaction mixture contained 2800 ppm. Sporicidal activity in the presence of 4% or 10% serum was almost the same and very rapid. Nu-Cidex with 10% serum present achieved a >lO’-fold kill in 2 min when first activated and in 5 min when 24 h old. The sporicidal activity of Cidex Long-Life and Sporicidin are shown in Table IV. Reaction mixtures were prepared by mixing 1 mL blood, 1 mL spore suspension and 8 mL disinfectant so the level of glutaraldehyde present in the reaction mixtures was around 1.6%. Neat sporicidin (30 days old) showed greater activity than Cidex Long-Life (28 days old) achieving a >lO’-fold kill in 1 h with 4% blood present whilst Cidex Long-Life took 2 h. However, the activity of a 1 in 8 dilution of Sporicidin was considerably reduced. The manufacturer claimed sterilization in 8 h during the first 14 days after activation and in 10 h for the next 16 days. In the present study, a freshly activated solution achieved >106-fold kill in 8 h in the absence of blood but only 103-fold kill with 2% blood present. A solution 30 days old achieved a 104-fold kill in 10 h in the absence of blood but only loo-fold kill with 2% blood present. Discussion

The present study was carried out over several years using three batches of B. subtilis spore suspension so it is not possible to directly compare all

Sporicidal

activity

of disinfectants

291

the results obtained for all the different products. The NaDCC products were tested together using one batch of spores so direct comparison is valid. Likewise the glutaraldehyde products were tested together using another batch of spores. Furthermore, the results obtained for Virkon involving tests carried out three years apart on two different batches of Virkon using two different batches of spores were reasonably consistent. B. subtilis NTCT 10073 was chosen as the test organism because the spores are used on test pieces for controlling sterilization cycles and there is a well-established method of producing a standardized spore suspension.” The use of these spores for evaluating sporicides provides a good safety margin because they are more resistant than the spores of bacterial species usually encountered in clinical practice.7*22 The results obtained with the NaDCC products show that the sporicidal activity of aqueous solutions increases with the level of av.Cl present but is markedly reduced in the presence of low levels of blood. Five percent Titan (1200 ppm av.Cl) achieved a >lO’-fold kill in 3 h in the absence of blood but no measurable kill with 2% blood present. This result does not support the manufacturer’s claim that 5% Titan applied for a contact period of 5 min is completely effective against all spores. However, Titan contains a detergent and the cleaning properties of such products are not taken into account in suspension tests. One percent Presept (3180 ppm av.Cl) achieved a >105-fold kill in 1 h in the absence of blood and in 2 h with 2% blood present. One percent Haz-Tab (5750 ppm av.Cl) achieved a >lO’-fold kill in 5 min in the absence of blood, in 30 min with 2% blood present and in 45 min with 4% blood present. Titan, Presept and Haz-Tab granules are all promoted for use on spills of body fluids. Usually, they are applied over a spill in a ratio of approximately one part granules to one part spill material and left for a contact period of just a few minutes before wiping up. The above results suggest that spills containing large numbers of bacterial spores surrounded by organic material are unlikely to be sterilized under these circumstances. However, the fluid component of the spill should become saturated with NaDCC which is present in excess and the spores of pathogens which may be present such as C. dif$ciZe in faecal material are less resistant than those of B. subtiZis.22 Virkon powder is also promoted for use on spills of body fluids. A 3% aqueous solution achieved a 101-lO’-fold kill in 15 min in the absence of blood but a lO’-fold kill in just 5 min with 10% serum present. Other workers have obtained similar results.‘” The time interval available to decontaminate endoscopes between

292

D. Coates

patients on a list is usually very limited. Consequently, cleaning followed by disinfection, e.g., 4-20 min in Cidex Long-Life is generally practised whereas cleaning followed by sterilization, e.g., 3 h in Cidex Long-Life (accepted in the UK) might be preferred. The use of Nu-Cidex seems to offer a real chance of carrying out cleaning and sterilization of endoscopes between patients on a list. Sporicidin offered the prospect of a glutaraldehyde solution with the same activity as 2% glutaraldehyde but containing a lower level of glutaraldehyde due to the enhancement in activity produced by the phenol/ phenate. The present results (Table IV) confirm that neat Sporicidin (30 days old) is more sporicidal than Cidex Long-Life (28 days old). The manufacturer claimed that a 1 in 8 dilution would sterilize in 8 h during the first 14 days after activation and in 10 h for the next 16 days. The present results demonstrate that whereas 2% glutaraldehyde is tolerant of organic matter, a 1 in 8 dilution of Sporicidin is not. In the absence of blood, a 1 in 8 dilution achieved a >106-fold kill in 8 h when freshly activated but only a 104-fold kill in 10 h when 30 days old. In the presence of 2% blood, a 1 in 8 dilution achieved a 103-fold kill in 8 h when fresh and only a loo-fold kill in 10 h when 30 days old. The present results endorse those of other workers.7,23,50A 1 in 8 dilution failed the AOAC sporicidal test using contact periods of both 6.75 h and 12 h’ and a 1 in 16 dilution showed no useful sporicidal activity.7,23,50 Sporicidin had its EPA registration cancelled44’45and was withdrawn from the market. Other formulations have been tried in the USA but diluting glutaraldehyde-based sterilants below 2% glutaraldehyde has resulted in failure to pass the AOAC sporidical test.‘xz4 However, this test has often been criticized and there have been many reports suggesting that it is inaccurate, inconsistent, and not repeatable.‘l References

5.

6. 7. 8. 9. 10.

Phillips CR. Relative resistance of bacterial spores and vegetative bacteria to disinfectants. Bacterial Rev 1952; 16: 135-143. Sykes G. The sporicidal properties of chemical disinfectants. r Appl Bact 1970; 33: 147-156. Kelsey JC, MacKinnon IH, Maurer IM. Sporicidal activity of hospital disinfectants. J Clin Path 1974; 27: 632-638. Babb JR, Bradley CR, Ayliffe GAJ. Sporicidal activity of glutaraldehydes and hypochlorites and other factors influencing their selection for the treatment of medical equipment. J Hosp Infect 1980; 1: 63-75. Waites WM. Resistance of bacterial spores. In: Russell AD, Hugo WB, Ayliffe GAJ, Eds. Principles and Practice of Disinfection, Preservation and Sterilization. Oxford: Blackwell 1982. Russell AD. Bacterial spores and chemical sporicidal agents. Clin Microbial Rev 1990; 3: 99-119. Rutala WA, Gergen MF, Weber DJ. Sporicidal activity of chemical sterilants used in hospitals. Infect Control Hosp Epidemiol 1993; 14: 713-718. Sykes G. Disinfection and Sterilization. 2nd end. London: Spon 1965. Trueman JR. The halogens. In: Hugo WB, Ed. Inhibition and Destruction of the Microbial Cell. New York: Academic Press 1971. Russell AD. The Destruction of Bacterial Spores. New York: Academic Press 1982.

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D. Coates Ayliffe GAJ, Lowbury EJL, Geddes AM, Williams JD, Eds. Control of Hospital Infection: A practical Handbook. 3rd edn. London: Chapman & Hall Medical 1992, 111-112. Crow S. Peracetic acid sterilization: a timely development for a busy healthcare industry. Infect Control Hosp Epidemiol 1992; 13: 111-113. Bradley CR, Babb JR, Ayliffe GAJ. Evaluation of the Steris System 1 Peracetic Acid Endoscope Processor. J Hosp Infect 1995; 29: 143-l 5 1. Mannion PT. The use of peracetic acid for the reprocessing of flexible endoscopes and rigid cystoscopes and laparoscopes. J Hosp Infect 1995; 29: 313-3 15. Department of Health. Spills of urine: potential risk of misuse of chlorine-releasing disinfecting agents. SAB No 59 (90) 41. Centers for Disease Control. Federal regulatory action against Sporicidin cold sterilizing solution. MMWR 1991; 40: 880-881. Anonymous. EPA cites sterilant manufacturer, stops sales of three products. Hosp Infect Control 1994; 21: 35-37. Coates D. Kelsey-Sykes capacity test: origin, evolution and current status. Pkarmaceut J 1977; 219: 402-403. Beeby MM, Whitehouse CE. A bacterial spore test piece for the control of ethylene oxide sterilization. J Appl Bact 1965; 28: 349-360. Maurer IM. Hospital Hygiene. 3rd edn. London: Edward Arnold 1985; 99-101. Babb JR, Bradley CR. Endoscope decontamination: where do we go from here? J Hasp Infect 1995; 30 (Suppl): 543-551. Ayliffe GAJ, Babb JR, Bradley CR. Disinfection of endoscopes. J Hosp Infect 1986; 7: 296-299. Miner NA, Mulberry GK, Starks AN, et al. Identification of possible artifacts in the Association of Official Analytical Chemists Sporicidal Test. Appl Environ Microbial 1995; 61: 1658-1660.