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Assessment of mould spoilage of packaging materials

Bactericidal activity of GAMA Healthcare Ltd.
biocide determined using the European
Standard Test method BS EN 1276:1997
against: Klebsiella pneumoniae NCTC 13368
(ATCC 700603)
March 2007
Author: P. Humphreys
Tests Carried Out By:

University of Huddersfield

Huddersfield Microbiology services
School of Applied Sciences

Microbiological Tests

Test Method
British/European Standard BS EN 1276:1997. Dilution-neutralisation
Test Procedures
Full details of all the test and control procedures used are given in the Test Method
GAMA Healthcare Ltd biocide as used in Clinell
Universal Sanitising Wipes
Batch number: N/A
Appearance product dilutions: colourless, clear product solution.
Interfering Substance (Organic Challenge)

0.3 g l-1 bovine albumin (final concentration) 3.0 g l-1 bovine albumin (final concentration) Temperature

Contact Time Tested

Test Organisms

Culture Medium

Plates were incubated at 37 ºC for 24-48h
Neutraliser, containing 60g/l Tween 80, 60g/l Saponin, 2g/l L-histidine, 2g/l L-cysteine in MRD. General Method
A standard suspension of test organisms containing 1.5 – 5.0 x 108 cells ml-1 of bacteria was prepared. 1 ml of interfering substance was pipetted into a Universal bottle, followed by 1 ml of test organism suspension. The mixture was mixed and left for 2 minutes. After 2 minutes 8 ml of the Gama Health Care Ltd biocide was added. After a contact time of 5 minutes, a 1 ml sample of the reaction mixture was pipetted into 9 ml of neutraliser and left for 5 minutes. A 1 ml sample was then pipetted into 2 Petri dishes and mixed with 15 ml of culture medium tempered at 47 ºC. After setting, the Petri dishes were incubated at 37 ºC. Colony forming units were counted after 2-3 days incubation and the fraction of surviving organisms calculated. Test Organism The test organism Klebsiella pneumoniae NCTC 13368 (ATCC 700603) is a multi-drug resistant strain which: has intermediate resistance to ceftriaxone and gentamicin, is used as a control organism for extended-spectrum beta-lactamase production and produces beta-lactamase SHV-18, is resistant to ampicillin, aztreonam, cefoxitin, cefpodoxime, ceftazidime, chloramphenico, piperacillin and tetracycline.
Requirements of this standard
The product, when tested as stipulated under simulated clean conditions (0.3 g l-1 bovine
albumin) or dirty conditions (3 g l-1 bovine albumin) under the test conditions of ambient
temperature (23 to 25 ºC), 5 minute contact, for Klebsiella pneumoniae NCTC 13368 shall
demonstrate at least a 5 log10 reduction in viable counts.
Results from the test are summarised in Tables 1 and 2, a full set of results can be found in
Table 3.
Test Conditions
Contact Time (minutes)
Log10 Reduction Achieved
Table 1. Log10 reductions in Klebsiella pneumoniae NCTC 13368 viable counts
following a 5 minute exposure to the test material.

Final concentration
Final concentration
CFU ml-1clean 0.3 g l-1
CFU ml-1 dirty 3.0 g l-1
Bovine Albumin
Bovine Albumin
viable count of bacterial colonies, 1 ml sample of 10 viable count of bacterial colonies, 1 ml sample of 10 Table 2. Reductions in Klebsiella pneumoniae NCTC 13368 viable counts following a
5 minute exposure to the test material.

Interpretation of the Results
When tested against Klebsiella pneumoniae NCTC 13368 with a 5 minute contact time
a full strength GAMA Healthcare Ltd biocide met the requirements of the Standard under
simulated clean and dirty conditions.
According to EN 1276:1997, the batch provided of GAMA Healthcare biocide possesses
bactericidal activity in 5 minutes at ambient temperature (23-25oC) under clean conditions
(0.3g/l bovine albumin) and dirty conditions (3g/l bovine albumin) for referenced strain
Klebsiella pneumoniae NCTC 13368.

Dr Paul Humphreys
School of Applied Sciences
University of Huddersfield
Appendix 1
Bacterial Test
Test Procedure Results
Experimental Conditions Validation
Dilution Neutralisation Control
Clean Dirty
Toxicity Control
Clean Dirty
Verification of Methodology
Passed Log10 Reductions/cfu/ml
N is between 1.5E+8 cfu/ml and 5E+8 cfu/ml, N = Nv is between 6E+2 cfu/ml and 3E+3 cfu/ml, Nv = Table 3. Testing of Klebsiella pneumoniae NCTC 13368 the GAMA Healthcare Ltd biocide using
BS EN 1276:1997.


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