OBJECTIVE
To assess the tuberculocidal activity of D-Stroy/Activ8 disinfectant
using a suspension test. In the presence and absence of an organic
load. Tests were carried out when freshly prepared.
MATERIALS AND METHODS
The European test method to establish tuberculocidal activity
has not yet been formulated and ratified. The test method used
is that of Griffiths et al. Journal of Hospital Infection (1998)
38.183-192.
DISINFECTANT
The disinfectant was supplied by the manufacturer in liquid form.
Dilutions were prepared in sterile hard water – 1 part plus
4 parts.
TEST ORGANISM
Myhcobacterium terrae NCTC 10856 (ATCC 15755) was used
as the test organism to represent tuberculocidal activity. It
is now recognised that this organism is a suitable surrogate test
organism for Mycobacterium tuberculosis and can be used
to establish tuberculocidal activity (CEN TC216).
The type strain of Mycobacterium terrae was obtained
freeze dried from the National Collection of Type Cultures, Central
Public Health Laboratory, Colindale, London. The glass vial was
carefully broken and a small aliquot of 7HO broth was added to
the tube to rehydrate the organism. One hundred µl of this
suspension was spread on Middlebrook 7H11 agar with OADC supplement
(Becton Dickinson Ltd). And incubated at 37°C for 14 days.
One colony of the test organism was taken from the plate after
14 days incubation and inoculated into 100ml 7H9 broth and incubated
at 37°C for 21 days. The suspension was subjected to ultrasonics
(50 Hz) for 10 minutes every second day and inverted several times
to minimize clumping. Ten per cent glycerol was added as a preservative
and this also helped to maintain a homogeneous suspension. One
ml aliquots of the suspension were decanted into 1.5ml microcentrifuge
tubes and held at –70°C until required.
NEUTRALIZATION AND RECOVERY
Preliminary tests were performed to establish the most effective
recovery/neutralization broth. It was established that a mixture
of Lecithin, Tween, Saponin, Sodium thiosulphate and histidine
described in the proposed CEN method for mycobacterial suspension
test was suitable in recovering small numbers of mycobacteria
in the presence of disinfectant residues without being inhibitory.
Dilution also had a function in the recovery system.
PREPARATION OF TEST SUSPENSION
Prior to testing, one of the suspensions was removed from the
freezer, thawed at room temperature, centrifuged, washed twice
and spread onto a 7H11 plate for confluent growth using a sterile
swab. One loopful was also spread on a 7H11 plate for single colonies
to confirm purity of the suspension. After 14 days incubation
at 37°C, the growth was harvested from the plate and mixed
with moistened (sterile distilled water) glass beads in a sterile
bottle for five minutes. Ten ml of sterile distilled water was
added to the beads, shaken and the mixture left to settle for
30 minutes. The supernatant was removed to a second sterile bottle
and left to settle, by gravity, for a further 2 hours. The supernatant
from this suspension was used as the test suspension for disinfectant
tests.
SUSPENSION TEST
One hundred µl of the test suspension was added to 900µl
of the disinfectant in a microcentrifuge tube. After contact times
of 1, 3, 5, 10 and 20 minutes. 10µl were removed and added
to 990µl of neutralization/recovery medium. This was then
serially diluted to 
in Ringers solution. One hundred µl of the neat and subsequent
dilutions were spread onto 7H11 agar in duplicate using sterile
spreaders. Plates were incubated at 37°C and checked for growth
after 2 weeks incubation and thereafter every week for up to 4
weeks. The test was also carried out in the presence of 1% horse
serum (dirty conditions).
RESULTS
A >5 
reduction in Mycobacterium terrae is used as an indication
of high/intermediate level disinfection and tuber culocidal activity.
D-Stroy/Activ8 gave a >5
reduction in 10 mins under clean and dirty conditions when freshly
prepared.
MYCOBACTERIAL ACTIVITY OF D-Stroy/Activ8 
REDUCTIONS OBTAINED AFTER SPECIFIED CONTACT TIMES.
| Contact time |
Clean conditions |
Dirty conditions |
| 0 mins (challenge) |
8.23 |
8.23 |
| 1 mins |
0.88 |
0.89 |
| 2 mins |
1.99 |
1.75 |
| 5 mins |
3.17 |
3.08 |
| 10 mins |
5.23 |
5.05 |
| 20 mins |
>5.23 |
>5.23 |
30 mins |
>5.23 |
>5.25 |
Figures in bold represent a >5
reduction
CONCLUSION
A >5
reduction in Mycobacterium terrace is used as an indication of
high/intermediate level disinfection and tuberculocidal activity.
D-Stroy/Activ8 when tested freshly prepared at a dilution of 1
part product plus 4 parts water gave a >5
reduction in 10 mins under clean and dirty conditions.
Miss Christine Bradley
Laboratory Manager
Dr Adam P Fraise
Director
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