sporicidal activity of glutaraldehyde

8/18/2019 Sporicidal Activity of Glutaraldehyde

1/13

Journal of Hospital Infection (1980) 1, 63-75

Sporicidal activity of glutaraldehydes and

hypochlorites and other factors influencing their

selection for the treatment of medical equipment

J. R. Babb, C. R. Bradley and G. A. J. Ayliffe

Hospital Infection Research Laboratory, Dudley Road Hospital,

Birmingham B18 7QH

Summary: Nine glutaraldehyde and 4 hypochlorite solutions were tested

for activity against approximately 10’ spores of B. subtilis var. globigii in

suspension or dried on aluminium foil. All glutaraldehydes were effective

against the spores in suspensions in 3 h or less, but the acid glutaraldehydes

were much less effective than alkaline, particularly against the dried spores.

Although the acid glutaraldehyde preparations were more stable, they also

tended to be more corrosive. The buffered hypochlorite solutions were

highly effective, killing the spores in less than 10 mix-r, but have the dis-

advantages of lack of stability and inactivation by organic matter. The

buffered hypochlorites were relatively non-corrosive to metals, but damaged

rubber and the polyurethane coat of an endoscope after prolonged exposure.

All glutaraldehyde preparations passed the Kelsey-Sykes capacity test

with and without yeast and using Pseudomonas aeruginosa as the test organism.

The hypochlorites failed the test when yeast was added.

Introduction

Glutaraldehyde is commonly used for the disinfection or sterilization of heat labile

medical equipment (Ross, 1966; Lowbury, Ayliffe, Geddes & Williams, 1975). In

recent years, particularly following the expiry of the patent on activated alkaline

glutaraldehyde (‘Cidex’), a number of different glutaraldehydes have been intro-

duced. Although most of these preparations contain 2 per cent glutaraldehyde, some

are alkaline and require an activator whilst others are acid and usually do not

require an activator but have the advantage of greater stability. The sporicidal

activity of glutaraldehydes at room temperature tends to be rather slow, especially

when spores are dried onto surfaces, and results with the A.O.A.C. test indicate

that immersion for 10 hours or more is required for sterilization (A.O.A.C. 1975).

A more rapid sporicidal action has been claimed for buffered hypochlorite solutions

(Death & Coates, 1979).

In this study nine solutions of 2 per cent glutaraldehyde and four hypochlorite

preparations were examined for activity against Pseudomonas aeruginosa and spores

of B. subtilis var. globigii. Since these preparations may be used for treating expensive

medical equipment, some tests on corrosion and damage to materials were made.

The relevance of microbiological and corrosion

tests to practical problems of

disinfection and sterilization will also be discussed.

0105~6701/80/010063 + 13 $01.00/O

63

@ 1980 Academic Press Inc. (London) Limited


2/13

64

J. R. Babb et al.

Materials and methods

Preparations of 2 per cent glutaraldehyde

1. ‘Asep’ (Galen Ltd.) pH 7.6, liquid activated, green, stable for 14 days and con-

tains corrosion inhibitor.

2. Acid glutaraldehyde, experimental formulation (not marketed) pH 5.5, stable

no activation required, colourless.

3. ‘Cidex’ CX 250, (Arbrook Products Ltd.) pH 8.6, powder activated, green,

stable for 14 days and contains corrosion inhibitor.

4. ‘Cidex Long Life’ (Arbrook Products Ltd.) pH 7.6, liquid activated, blue,

stable for 28 days, contains corrosion inhibitor, and a surfactant.

5. Alkaline glutaraldehyde, experimental formulation (not marketed) pH 8.6,

powder activated, blue, stable for 14 days, contains a corrosion inhibitor and

surfactant.

6. ‘Clinicide’ (Bioscan) pH 6.2, powder activated, green, stable for 28 days, contains

a corrosion inhibitor and surfactant. (now marketed with an alkaline buffering

system)

7. 3M Instrument Disinfectant (3M UK Ltd.) pH 58, liquid activated, blue,

stable for 28 days, contains a surfactant.

8. ‘Totacide’ (Tenneco Organics Ltd.) pH 7.5, liquid activated, green, stable for

28 days, contains a corrosion inhibitor and a surfactant.

9. ‘Triocide’ (Vann Medical) pH 5.6-6.3, stable, no activation required, colourless,

contains a surfactant

All glutaraldehydes are supplied at ‘in use’ concentrations with the exception of

‘Clinicide’ which is diluted l/10.

Preparations of hypochlorites

1. ‘Anprosol’ (H. W. Andersen Products Ltd.) is a 0.2 per cent sodium hypochlorite

solution containing buffers and surface active ingredients, giving approximately

2000 parts/lo6 of available chlorine, pH 7.5. All the constituents are contained

in a triple compartment pouch which, when cut open, brings the disinfectant,

activators and corrosion inhibitors together. Additionally in each kit are provided

pouches of conditioning agent (detergent, wetting agents and corrosion in-

hibitors) for instrument preparation before immersion in ‘Anprosol.’ The product

is supplied in a marked container suitable for dilution.

2. Sodium hypochlorite solution ‘Sterite’ (Midland Direct Supplies Ltd.) made up

to give 1800 parts/lo6 available chlorine pH approximately 10.0.

3. Buffered sodium hypochlorite solution 250 parts/lo6 available chlorine pH 7.6

(Death & Coates, 1979).

4. Sodium hypochlorite solution ‘Sterite’ made up to give 250 parts/lo6 available

chlorine pH approximately 9.0.

Titration of available chlorine

The ‘available chlorine’ content of hypochlorites was assessed using the arsenite

method (Coates, 1977). 5 ml of hypochlorite was titrated with O-141

N

or 0.0141

N


3/13

Sporicidal activity o f glutaraldehydes and hypochlorites

65

sodium arsenite and the end point detected by a failure to produce a blue stain on

starch iodide paper. The titration in ml, using 0.141 N, gives the available chlorine

concentration directly in g/l (i.e. 1 mEq to 1000 parts/106).

Measurement of pH

pH was measured before each microbiological test with a model 23A pH meter

(Electronic Instruments Ltd). Glutaraldehyde solutions were measured weekly

over a 2%day period.

Miuobiological tests

Kelsey-Sykes capacity test. Tests were carried out as described by Kelsey &

Maurer (1974) for ‘clean’ and ‘dirty’ conditions. Glutaraldehydes and hypo-

chlorites were tested at ‘in use’ concentrations using Ps. aeruginosa NCTC 6749 as

the test organism. Products were tested when freshly prepared, and the glutaralde-

hydes were also tested 14 days after activation.

Sporicidal activity : suspension test

Preparation of spore suspensions. Blood agar plates (Oxoid Columbia agar base

CM 331 + 7.5 per cent horse blood) were seeded with a freshly prepared

culture of Bacillus subtilis var. globigii (NCTC 10073) and incubated for 18 h at

37°C. The resultant growth was removed with sterile cotton wool swabs and a

heavy aq. suspension prepared. The suspension was washed three times in

sterile distilled water, resuspended and heated to 56°C and held for a period of

6 h. Spores were counted, using a surface dropping technique, and stored

overnight at 4°C to enable the spore challenge to be adjusted to approx.

lOr/ml. Sufficient spore suspension was prepared to test all the hypochlorites, and

glutaraldehydes over a 28 day period. Before each test, spores were heated and

counted by the method described.

Test method. 1 ml of spore suspension was added to 10 ml of the freshly prepared

hypochlorite or glutaraldehyde solution in a universal container (previously

rinsed in the disinfectant under test) and thoroughly mixed. 0.02 ml quantities of

this mixture were removed at specific time intervals-2,5, 10, 30 min and 1, 2, 3,4,

5, 6, 7 and 24 h, and added to each of a set of 5 recovery broths and thoroughly

mixed on a ‘rotamixer’. The disinfectant spore mixture was kept at room tempera-

ture (approximately 20°C) throughout the period of the test and recovery broths

were incubated for at least 14 days and examined for growth of the test organism,

i.e. orange pellicle, granular and later turbid suspension. Doubtful tubes were

sub-cultured to confirm the test organism.

The test was repeated with the glutaraldehyde solutions at weekly intervals, i.e.

7, 14, 21 and 28 days after activation. The recovery medium was double strength

nutrient broth (Oxoid No. 2) with the addition of 10 per cent horse serum for the

glutaraldehyde tests and nutrient broth + 0.5 per cent sodium thiosulphate for the

hypochlorite tests.

Tests were carried out to establish that the recovery broth neutralized glutaralde-

hyde carried over during sampling and was not inhibitory to recovered test spores.


4/13

66

J. R. Babb et al.

To confirm the absence of surviving spores in the suspension test Bacillus

subtilis var. globigii spores were added to 10 ml of the freshly prepared disinfectants.

After 3 h the disinfectant/spore suspension mixture was filtered (Millipore

0.45

PM),

the membrane washed with 100 ml of serum nutrient broth and trans-

ferred to a blood agar plate. Membranes were transferred daily to fresh culture

plates for 14 days and examined for colonies of the test organism.

The suspension tests were repeated, with the addition of 2 ml of a 5 per cent

yeast suspension, on three of the glutaraldehydes (‘Asep’, ‘Cidex’ and ‘Totacide’)

showing good sporicidal activity and on the hypochlorites. The yeast, simulating

dirty conditions, was added before the spore suspension.

Sporicidal activity : carrier tests

Bacillus subtilis var. globigii spores (NCTC 10073) 106, deposited onto rolled alumi-

nium foil from a 90 per cent methanol suspension and prepared by the method of

Beeby & Whitehouse (1965), obtained from ‘Steriseal’ Ltd., Redditch, Worcs.,

were used as the test organism and carriers. These spores are marketed as control

organisms for the ethylene oxide sterilization process.

The spore strips were immersed in 10 ml of freshly prepared glutaraldehyde

or hypochlorite, mixed thoroughly on a ‘rotamixer’ for 5 s and left for periods up

to 24 h at room temperature. At the same time intervals as in the suspension tests,

five strips were transferred with sterile wire hooks to each of a series of five recovery

broths. A set of five strips was immersed in a separate universal container for each

time interval.

The strips were shaken in the recovery broths for 5 s on a ‘rotamixer’, incubated

at 37°C and examined for growth for periods up to 14 days. Tests were carried out

on freshly prepared hypochlorites, and glutaraldehydes at 0, 14 and 28 days after

activation. The pH and available chlorine content of the hypochlorites was

measured at the start of each test. Spores were recovered and counted from un-

treated foils by shaking them with glass beads in 10 ml of quarter strength Ringer’s

solution. Tenfold dilutions were made from these washings and plated out onto

the surface of nutrient agar plates using a dropping technique. Colonies were

counted after 24 h incubation at 37°C.

Corrosion tests

Carbon steel and stainless steel scalpel blades were degreased, washed, dried and

immersed in beakers of freshly prepared glutaraldehyde or hypochlorite solutions

for periods up to 3 days. Each blade was placed in a separate container and supported

with nylon thread so that it was partially immersed in each of the disinfectants

under test. Untreated blades and blades immersed in tap water were used for

comparison with the treated blades.

In a second series of tests, various components of ‘Olympus’ flexible fibreoptic

endoscopes, i.e. insertion tube sheath, nylon channels, guide wires, trumpet valves,

berylium/copper and stainless steel protective coils and bending sections, were

immersed either in the 2 per cent glutaraldehyde solutions, 100 immersions for 3 h,

or in the hypochlorites, ‘Anprosol,’ 100 immersions for 2 m in the conditioner


5/13

Sporicidal activity of glutaraldehydes and hypochlorites 67

followed by 15 m in the hypochlorite, or for other hypochlorites 100 immersions of

20 m. Sections of rubber tubing and square pieces of anaesthetic equipment

(reservoir bags) were separately immersed. All items were washed, dried and

examined for signs of damage, e.g. rusting, tarnish, deposition, or loss of elasticity,

after each treatment or on a daily basis.

Glutaraldehydes

Results

All the glutaraldehydes passed the Kelsey & Sykes test at ‘in use’ dilution under

clean and dirty conditions. No growth was obtained in any of the recovery broths.

Tests with added spores showed that adequate neutralization of the disinfectant

had occurred.

Table I. Sporicidal activity

of

2 per cent activated and 2 per cent stable

glutaraldehydes : Suspension test (inoculum l-4-7.4 x 10’ spores of B.

subtilis var. globigii)

Agent

Recom- No. of tubes showing growth after 14 days

mended

incubation

post-

activation Day of

exposure time

life (days) test 10min 30min lh 2h 3h 4h pH

(a) Sporicidal activity of 2 ner cent activated glutaraldehvdes

‘Asep’

‘Cidex’

0

14 :;:

0

14 :;:

Alkaline

glutaraldehyde

‘Cidex Long Life’

‘Clinicide’

‘Totacide’

14 1:

28

0

28 ;;:

0

28 ii

0

28 14

28

‘3M’

28 104

28

315

515

515

515

515

515

515

515

5/5

315

515

515

5/j

515

515

515

515

515

515

515

515

::

515

275

515

575

515

0

00

:

515

275

415

415

00

0

0

5):

575

515

0

i

:

515

0

45:

$5

315

(b) Sporicidal activity of 2 per cent stable glutaraldehydes

Acid glutaraldehyde 15/15

15/15 10/15

‘Triocide’ 15/15

15/15 s/15

: 0

0 0

0 0

575 175

t 0

515 41:

ii 0

0 :

: :

0 0

0 0

: 0

0

0 0

i 0

4115 0

s/15 0

0077:;

0 7.2

i

t:;:

0 7.6

0

:

;:;

7.8

i

5:;

0 7.4

: 2:;

0 6.0

00 77:;

0 7.1

0 5.8

i 5.8.9

0 5.5

0 5.6

Table I(a) shows the sporicidal activity of the activated glutaraldehydes, and I(b)

the stable glutaraldehydes, when the test organism (Bacillus subtilis uar. globigii)


6/13

68

J. R. Babb et al.

was added in suspension. Table I(b) also indicates the pooled results of sporicidal

activity of the stable products on three occasions over a 1 month period. All

products killed 10’ spores within a three hour period, providing the post-activation

period, recommended by the manufacturer, was not exceeded, i.e. 14 or 28 days.

This apparent kill was substantiated by failure of the spores to grow on the surface

of the cultured membrane filter after daily transfers. Many of the products (‘Asep,’

‘Cidex,’ ‘Cidex Long Life,’ ‘

Totacide,’ and ‘Triocide’) killed the spores in less than

1 h.

Table II. Sporicidal activity of 2 per cent glutaraldehydes : Suspension

test with added yeast

No. of tubes showing growth after 14 days incubation

Time of exposure

Agent

‘Asep’

‘Cidex’

‘Totacide’

10 min

515

515

515

30 min lh

515

515

515

515

515

515

2h

3h

575 i

0 0

Spore challenge 3.0 x 10’

The pH of the alkaline formulations (‘Cidex,’ ‘Asep,’ and ‘Totacide’) dropped

over the 2%day test period and this fall appeared to be associated with their

sporicidal activity. The pH of the acid formulations, including the activated acid

products (‘3M’, and ‘Clinicide’), remained constant over the test period although

the sporicidal activity varied considerably. Table II shows the sporicidal activity of

‘Asep,’ ‘Cidex,’ and ‘Totacide’ when organic material (yeast) was present in the

suspension. 10’ spores were killed within 3 h. These three products were chosen

as they did not appear to damage or corrode immersed materials in preliminary

tests and were reasonably effective in the surface spore tests.

The survival of Bacillus subtilis var. globigii dried on rolled aluminium foil and

immersed in the 2 per cent glutaraldehydes is shown in Table III(a), (b). The

differences between the products are more clearly defined than in the suspension

tests and do not appear to be related to the presence of surfactants. The acid

formulations (acid glutaraldehyde and ‘3M’) required 7 or more hours to kill the

spores. All other products killed the spores in under 3 h although the same loss of

activity was noticed with the 14 day products when this period was extended. The

mean number of spores recovered per foil was 1.9 x 10’ (range 8.0 x 10G-

2.3 x 107) which is approximately 1 log higher than stated by the manufacturer.

Hypochlorites

The sporicidal activity of freshly prepared hypochlorites (Table IV suspension

test, Table V surface test), is more rapid than the glutaraldehydes, especially

when buffered to a pH of approximately 7.6. ‘Anprosol’ (1800 parts/lo6 of available

chlorine, pH 7.5) and buffered sodium hypochlorite (pH 7.6, loo-250 parts/l06 of

available chlorine), as described by Death & Coates (1979) killed 10’ spores in under


7/13

E

9

0

s

s

0

I

4

0

L

S

0

8

S

0

E

L

0

S

L

0

E

L

0

P

9

0

t

9

0

2

9

0

E

L

0

C

L

0

9

L

0

8

L

0

1

8

0

8

8

0

9

L

0

1

8

0

9

8

0

0

L

0

Z

L

0

9

L

0

0

0

0

0

0

0

s

o

S

S

S

S

‘

a

P

~

J

s

o

s

s

s

s

s

s

m

S

S

s

s

S

S

S

S

a

m

%

p

3

s

C

m

n

%

a

q

s

u

a

z

3

L

A

a

+

S

(

q

0

0

s

z

S

S

S

s

s

s

s

s

s

0 0

S

S

s

s

S

S

s

s

s

s

3

s

s

s

s

s

s

s

s

s

i

8

s

s

0

S

S

8

8

S

S

8

8

‘

N

‘

a

P

~

‘

x

P

S

S

w

H

4

P

Y

Y

9

Y

U

9

Y

U

U

U

U

w

(

s

a

3

O

0

3

A

+

J

a

m

a

m

p

D

u

S

+

J

3

M

8

8

U

M

S

S

W

3

‘

O


8/13

70

J. R. Babb et al.

Table IV. Spmicidal activity of hypochlorite solutions : Suspension test

Agent

No. of tubes showing growth after

14 days incubation

Available Time of exposure

chlorine 2

30 1 h 2 h Spore count

(parts 106) min nZn rZn min

w

‘Anprosol’

pH 7-S

Hypochlorite

pH approximately 10

Buffered hypochlorite

pH 7.6

Hypochlorite

pH approximately 9

;;;; w:

215

t : 0 : 5.4.0 x 10’0’

1800 O/S 0 0 8.8 x 106

1800 O/S 0

:

00 : ii 4.4 x lo6

2100 o/5 0 0 0 0 3.1 x 106

1200 s/5 5/s 315 0 0 0 5.4 x 10’

1700 515 515 s/5 415 0 3-o x 10’

1800 515 515 515 5/S 0 : 4.4 x 106

loo s/5 315 415 0 0 4.4 x 106

100 415 0 8 0 0

a0

3.1 x 106

150 o/5 0 0 8-8 x 106

150 o/5 0 0

00

ii

:

5.4 x 10’

250 O/5 0 0 0 0 1.9 x 10’

100 515 515 515 l/5 0 : 3-l x 106

loo s/5 515 515 415 0 4-4 x 106

150 515 s/5 5/s 315 0 5.4 x 10’

150 315 l/5 215 0 0 8-8 x 106

250 s/5 515 515 515 0 0 3.0 x 10’

10 min and often in under 2 min. Unbuffered preparations were less effective, and

times required to kill 10’ spores were comparable with those of the better glutar-

aldehydes.

Table V. Sporidal activity of hypochlorite solutions : Surface test

Agent

Available

No. of tubes showing growth after 14 days

incubation

chlorine

Time of exposure

(parts 103 2min

5 min 10 min 30 min 1 h

‘Anprosol

pH 7.5

Hypochlorite

pH approximately 10

Buffered hypochlorite

pH 7.6

Hypochlorite

pH approximately 9

1800 015 0 0 0 0

1800 515 515 515 0 0

250 015 0 0 0 0

250 515 515 515 315 0

Mean spore count per strip 1.9 x 10’

In the presence of yeast, all hypochlorites failed to kill spores even when the

exposure period was extended to 24 h. These failures were also shown in the

capacity test of Kelsey & Sykes. The activity of the hypochlorites diminishes fairly


9/13

Sporicidal activity of glutaraldehydes and hypochlorites

71

rapidly after preparation, particularly with lower dilutions, as does the available

chlorine concentration, e.g. ‘Anprosol’ showed a fall from 1800 to 1200 parts/l06

in 24 h, and buffered hypochlorites from 250 to 130 parts/lOs.

corrosionests

The alkaline glutaraldehydes ‘Asep, ’ ‘Cidex,’ ‘Cidex Long Life’ and ‘Totacide’ did

not appear to cause damage to carbon steel or stainless steel scalpel blades when

immersed for periods up to 3 days.

‘Triocide’ on the other hand showed some

deterioration of carbon steel (rust and tarnish) after a short period (3 h) of im-

mersion and the stainless steel blade, separately immersed, was rusted within 3

days. Immersion in an acid glutaraldehyde, e.g. ‘Clinicide’ and ‘3M’ also produced

damage to carbon steel blades although there was no damage to stainless steel.

This effect was minimal in 3 h, although considerable at 3 days.

The interpretation of tests on other materials was more difficult. The buffered

alkaline formulations ‘Cidex,’ ‘Cidex Long Life’ and ‘Totacide’ caused no visible

damage to collections of endoscope components after 100 immersions of 3 h. The

acid formulations ‘Clinicide,’

‘

Triocide’ and ‘3M,’ became cloudy and heavy

deposits formed making visual examination of components difficult. Some damage

was certainly caused to collections of dissimilar metals particularly copper alloys.

‘Anprosol’ (pH 7*5),

and buffered hypochlorites (pH 7.6) caused little damage to

immersed scalpel blades and metal endoscope components although non-buffered

hypochlorite, pH 9-10, caused severe corrosion of carbon steel within a few hours.

Two problems were,

however, noticed with the hypochlorites, including the

buffered formulations, especially at higher concentrations. A thick, white deposit

built up on the insertion tube of the ‘Olympus’ flexible fibreoptic endoscopes during

100 immersions and although this could be scraped or wiped off, the polyurethane

coating appeared to be destroyed. Sections of rubber reservoir bags and tubing

were also damaged. The rubber hardened, lost its resilience, and cracks appeared

on the surface when stretched.

Discussion

A reproducible quantitative suspension test measuring the log reduction in numbers

of organisms would have been preferable to the technique used (Reybrouck &

Werner, 1977). However, in view of the large number of tests, this was not possible

and a semi-quantitative method was used to assess sporicidal activity. The results

provide a useful comparison between the agents tested but do not necessarily

indicate exposure times required for sterilization in practice.

Spores vary in their resistance to disinfectants, depending on their method of

preparation, but the spores of B. subtilis var. globigii, as used in these tests, are

likely to be more resistant to glutaraldehydes and hypochlorites than pathogenic

species. The numbers used were also considerably higher than would be found on a

cleaned instrument. The preparation of a reliable spore suspension is often difficult

and the commercial spore strips proved to be consistent in both numbers of spores

and in response to the chemical agents. Glutaraldehyde is not easily neutralized but

dilution in broth containing 10 per cent serum was found to be as reliable as other


10/13

72

J. R. Babb et al.

potential neutralizers and is not toxic to damaged organisms (Russell, Ahonkhai &

Rogers, 1979). Attempts to recover damaged spores using a membrane filter

technique also failed.

All the glutaraldehyde preparations passed the Kelsey-Sykes capacity test, and

killed spores in suspension in 3 h. The alkaline glutaraldehydes were generally

more active than the acid preparations some of which required long periods to kill

spores dried onto metal carriers. The presence of a surfactant did not appear to

improve the effect. Although the A.O.A.C. test suggests a 10 h exposure is neces-

sary to achieve a complete sporicidal effect (A.O.A.C. 1975), 3 h should be sufficient

for practical purposes, particularly as spores are infrequent on clean medical equip-

ment (Spaulding, 1978). Th e immersion, at 20°C of five or more commercial

spore suspensions dried on metal foil could provide a useful laboratory screening

test for glutaraldehydes. This test should be done on activated glutaraldehydes,

stored at 20°C on their expiry date, i.e. at 14 or 28 days after activation.

Opinions on the tuberculocidal activity of 2 per cent glutaraldehyde at room

temperature vary but the immersion times required are considerably shorter than

for spores and 20 m is generally considered adequate (Bergan & Lystad, 1971,

Miner

et al., 1977).

A possible advantage of using acid glutaraldehydes is stability. When heated to

55-60°C their sporicidal activity is greatly enhanced (Sierra & Boucher, 1971),

and in our tests lo6 spores were killed in less than 5 min at this temperature. The

differences between the sporicidal activity of the alkaline and acid glutaraldehydes

diminishes with a corresponding increase in temperature. However, special equip-

ment would be required for this treatment and may have to include equipment for

the removal of toxic vapours. An acid solution of 2 per cent glutaraldehyde (pH

3-4) is stable for several years. However, when alkalinized to produce the optimum

sporicidal activity the corresponding loss in active aldehyde groups, and hence

stability, limits its use to 14 days. By stabilizing the pH to approximately 7.6 the

period of activity of alkaline glutaraldehydes can be extended, with no loss in

sporicidal activity, to 28 days in situations of low dilution (Miner et al., 1977).

This is supported by our findings although the interpretation of these results in the

practical situation should be treated with some caution.

In this study, the acid glutaraldehydes were stable and the activated glutaralde-

hydes showed little loss in activity over 14 days. However, the length of time a

disinfectant is repeatedly used depends on the degree of dilution, the amount of

organic and other materials added to it, and storage conditions as well as its stability.

Although 2 per cent glutaraldehyde is not readily inactivated by organic materials,

repeated use of the same solution over long periods is not good practice. Dis-

infectant solutions should preferably be discarded after each use, but this practice

may be too expensive with glutaraldehyde. If used infrequently and for clean eyuip-

ment only, 7-14 days use would seem to be a useful compromise, but the decision

should be made by the microbiologist depending on the particular use of the

solution.

The interpretation of the corrosion tests presented some problems.’ The acid

formulations usually caused greater damage to immersed metals and as their spori-


11/13


tidal activity is less rapid, longer immersion times may be required. Carbon steel is

rarely incorporated in expensive equipment and corrosion of this alone is of doubtful

practical significance. Nevertheless, it seems reasonable to choose a product which

contains a corrosion inhibitor for disinfection or sterilization of expensive equip-

ment. There is always the possibility of accidental long exposures, e.g. over a week-

end. It is obviously less important when disinfecting inexpensive equipment or

using short exposure times, but it is advisable to carry out in-use tests or to consult

the instrument manufacturer before changing from one preparation to another.

The hypochlorite solutions have several potential advantages. They kill spores

rapidly and instruments could be ‘sterilized’ rather than disinfected between

patients during busy endoscopy sessions. Hypochlorites are cheap and could be

discarded after each use or after an operating session. However, the cost of essential

buffers and corrosion inhibitors is likely to increase considerably this cost. These

solutions have been used for disinfecting infant feeding bottles for many years and

dilute solutions are relatively non-toxic. The main disadvantages of hypochlorites

are inactivation by organic material, instability at low concentration, and possible

damage to instrument components. Two of the buffered preparations examined,

pH 7-S-7.6 (‘Anprosol’ and buffered hypochlorite), showed surprisingly little

damage to metals but did cause some damage to the outer layer of the insertion

tube of an ‘Olympus’ flexible fibre-optic endoscope on prolonged exposure, and to

rubber items, e.g. anaesthetic equipment. We have also received complaints that

endoscope eye piece mounts are likely to bleach during prolonged or frequent

immersions in hypochlorite and that surfactants and conditioning agents, used

prior to or during disinfection, remove essential lubricants and these should be

replaced. It could be argued that only short exposures are required, but accidental

immersion for long periods is always possible particularly if staff are familiarised

with the use of glutaraldehydes. However, if the risk is known, users tend to be

more careful, e.g. alcoholic solutions of chlorhexidine are sometimes used for

disinfection of cystoscopes, and exposure for longer than a few minutes may damage

the lens mounting.

Heat is the most reliable method of disinfection or sterilization, and the use of

chemical solutions of disinfectants or ‘sterilants’ should rarely be required in

hospitals, but autoclaving at high temperature will damage most endoscopes and

destroy flexible fibrescopes. Low temperature steam at 73°C for 10 min is perhaps

the most suitable method for disinfecting cystoscopes between patients, and low

temperature steam with formaldehyde for longer periods for sterilizing laparoscopes

and arthroscopes (Alder, Gingell & Mitchell, 1971). Low temperature steam is also

particularly suitable for the disinfection of respiratory equipment, where hypo-

chlorites cannot be used as rubber is damaged, and inadequate removal of glutar-

aldehyde is always a potential hazard to the patient. However, low temperature

steam machines are not always available and reliable low temperature steam and

formaldehyde machines are still being developed (Cripps, Deverill & Ayliffe, 1976).

Low temperature steam, especially with formaldehyde, will damage most of the

flexible fibre-optic endoscopes in use at the present time. Chemical disinfection is

therefore required and 2 per cent glutaraldehyde is commonly used despite the


12/13

74 J. R. Babb et al.

problems of penetration of narrow tubing and valves and the necessity of thorough

rinsing. Treatment of flexible fibre-optic endoscopes with glutaraldehyde for a

short period, between use on different patients, is a useful compromise (Axon,

Phillips, Cotton & Avery, 1974; Ayliffe & Deverill, 1979; Noy, Harrison, Holmes

& Cockel, 1980). Cleaning according to the manufacturers instructions is more

effective, but takes up to 20 min. Although exposure to glutaraldehydes for 30-

60 min should be adequate following use of an endoscope in a patient with tubercu-

losis, hepatitis or salmonellosis, treatment of the cleaned instrument with ethylene

oxide will further increase the margin of safety.

Treatment with glutaraldehyde for lo-20 min will disinfect but not sterilize, yet

laparoscopes and arthroscopes are often treated for this short time. Despite

inadequate sterilization infection appears to be rare, presumably because there are

few potentially pathogenic spores on the cleaned endoscope. The use of hypo-

chlorites could considerably increase the reliability of this procedure and further

studies are in progress. Endoscopes are expensive, and often complex, and are

difficult to clean, disinfect, or sterilize. It is hoped that manufacturers will produce

instruments that will withstand autoclaving at high temperatures without short-

ening the life of the instrument.

We wish to thank ‘KeyMed’ for supplying the ‘Olympus’ endoscope components used

for disinfectant immersion studies and the disinfectant manufacturers for the products

tested.

A.O.A.C. Official methods of analysis of the Association of Official Analytical Chemists.

Journal of Association of OjjWul and Analytical Chemists. 12th edition. 57-71 (1975).

Alder, V. G., Gingell, J. L. & Mitchell, J. P. Disinfection of cystoscopes by subatmospheric

steam and steam and formaldehyde at 80°C. British MedicalJournal iii: 677-680 (1971).

Axon, A. T. R., Phillips, I., Cotton, P. B. & Avery, S. A. Disinfection of gastrointestinal

fibre endoscopes. Lancet i: 656-658 (1974).

Ayliffe, G. A. J. & Deverill, C. E. A. Decontamination of gastroscopes. Health and Social

ServicesJournal May: 538-540 (1979).

Beeby, M. M. & Whitehouse, C. E. A bacterial spore test piece for the contro l of ethylene

oxide sterilization. Journal of Applied Bacteriology 28: 349-360 (1965).

Bergan, T. & Lystad, A. Antitubercular action of disinfectants. Journal of Applied Bacteri-

ology 34: (4): 751-7.56 (1971).

Coates, D. Kelsey-Sykes capacity test: origin, evolution and current status. Pharmaceutical

&urnizl219: 402-403 (1977).

Cripps, N., Deverill, C. E. A. & Ayliffe, G. A. J. Problems with low-temperature steam and

formaldehyde sterilizers. Hospital Engineering AuglSept International Federation Issue

no. 19 (1976).

Death, Janet E. & Coates, D. Effect o f pH on sporicidal and microbicidal activity of buffered

mixtures of alcohol and sodium hypochlorite. Journal of Clinical Pathology 32: 148-153

(1979).

Kelsey, J. C. & Maurer, I. M. An improved Kelsey-Sykes test for disinfectants. Pharma-

ceutical Journal 213: 528-530 (1974).

Lowbury, E. J. L., Ayliffe, G. A. J., Geddes, A. M. & Williams, J. D. In Control of Hospital

Infection : A Practical Handbook Chapman & Hall: London (1975).

Miner, N. A., McDowell, J. W., Willcockson, G. W., Bruckner, N. I., Stark, R. L. &

Whitmore, E. J. Antimicrobral and other properties of a new stabilised alkahne glutar-

aldehyde disinfectant/sterilizer. American Journal of Hospital Pharmacy 34: 376-382

(1977).


13/13


Noy, M. F., Harrison Lynne, Holmes, G. K. T. & Cockel, R. The significance of bacterial

contamination of fihreoptic endoscopes. Journal of Hospital Infection 1: 53-61 (1980).

Reyhrouck, G. & Werner, H. P. Ausarbeitung eines neuen quantitaven in-vitro-tests fur die

bakteriologische Prufung chemischer Desinfektionsmittel. Zentralbatt fur Bakteriologie,

Parasitenkunde, Infektionskrankheiten und Hygiene, I. Abteilung originale Reihe B 165:

126-137 (1977).

Ross, P. W. A new disinfectant. Journal of Clinical Pathology 19: 318-320 (1966).

Russell, A. D., Ijeoma Ahonkhai & Rogers, D. T. A review: microbiological applications of

the inactivation of antibiotics and other antimicrobial agents. Journal of Applied

Bacteriology 46: 207-245 (1979).

Sierra, G. & Boucher, R. M. G. Ultrasonic synergistic effects in liquid-phase chemical

sterilization. Applied Microbiology 22: 160-164 (1971).

Spaulding, E. H. Fibreoptic endoscopes:

disinfection or sterilization. Microbiological

aspects of the dilemma. Hospital Infection Control 5: 35-39 (1978).

sporicidal activity of glutaraldehyde

Documents