Residue testing of cleaning agents and disinfectants on
process surfacesusing photobacteria
DairyNet - Hygiene Control in Dairies
Satu Salo & Gun Wirtanen, VTT BiotechnologyJuha Lappalainen, Aboatox Oy
Aims of the 1. Synergy task in DairyNET
• Introduction of new method for testing chemical residues
• Comparison of results -> repeatability of the method
• Microbial testing methods for detection of residual cleaning agents and disinfectants -
prevention of ATP bioluminescence measurement errors
Inhibition zone techniqueInhibition zone technique•• Micrococcus luteusMicrococcus luteus was spread over a was spread over a
tryptone soy agar platetryptone soy agar plate–– filter paper discs soaked in detergent filter paper discs soaked in detergent
dilutions were placed on the agardilutions were placed on the agar–– incubation 37 incubation 37 °°C, 18 hC, 18 h–– inhibition ring was measuredinhibition ring was measured
Inhibition zone techniqueInhibition zone technique
Micrococcusluteus1. No inhibition
2. Clear inhibition
Test method
Microbioal Testing Methods for Detection of Residual Cleaning Agents and Disinfectants - Prevention of ATP Bioluminescence Measurement Errors in the
Food IndustryLappalainen et al. 2000
J. Food.Prot. 63, 2000
Vibrio fischeri photobacterium
Copyright SDIX
Vibrio fischeri photobacterium
• Commercial product in kit formate (several producers)
• Sensitive to changes in pH, ionic strength, broad range of chemicals, both organic and inorganic
• Standard test in environmental monitoring• Simple instrumentation
0
20
40
60
80
100
120
0 20 40 60 80 100
concentration/ %
Lum
ines
cenc
eExample of the results
Partners and test plan
Dairynet Partners in Finland, Sweden, Norway, Denmark, Iceland
Partners and test planDairynet Partners in Finland, Sweden, Norway, Denmark,
Iceland
Task 1. Introduce people to the Biotox method for detecting cleaning agents and disinfectants from production surfaces in food factories
Partners and test planDairynet Partners in Finland, Sweden, Norway, Denmark,
Iceland
Task 1. Introduce people to the Biotox method for detecting cleaning agents and disinfectants from production surfaces in food factories
Task 2. compare results obtained in different locations with same chemicals by different operators
Partners and test planDairynet Partners in Finland, Sweden, Norway, Denmark,
Iceland
Task 1. Introduce people to the Biotox method for detecting cleaning agents and disinfectants from production surfaces in food factories
Task 2. Compare results obtained in different locations with same chemicals by different users
Task 3. Use the method in different sites by their operators
The simple instructions were ...
1. Measure luminescence from the unstressed bacteria2. Add sample, incubate for 5 min 3. Measure luminescence4. Calculate results
Incubate
... easy to followDiluting the detergent
Making ready-to-use concentration
Agent 1e.g. Oxonia Aktiv
100% 30% 3% 0.3% = ready-to-use concentration= 1:1
Preparation of thedilution series
2:3 1:2 1:3 1:4 1:6 1:8 1:12 1:16 1:24 1:32 1:48 1:64
1:96 1:128 1:192 1:256 1:384 1:512 1:768 1:10241:1536 1:2048 1:3072 tap water
3 ml3 ml
7 ml tap water 9 ml tap water 9 ml tap water
3 ml 1 ml 1 ml
4 ml sample and2 ml tap water
3 ml sample and3 ml tap water
tap water inthe test tube
3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml
tap water inthe test tube
3 ml3 ml3 ml3 ml3 ml3 ml3 ml3 ml3 ml3 ml
3 ml3 ml3 ml3 ml3 ml3 ml3 ml3 ml3 ml3 ml3 ml
3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml 3 ml
InhibitionConc Avera
ge T0Av 5min
5 min GAMMA LOGC
LOGgamma
0 150 148 0,00 0,00 Y X10 148 5 96,58 28,21 1,0000 1,45035 155 25 83,65 5,12 0,6990 0,7090
2,5 143 55 61,02 1,57 0,3979 0,19461,25 148 120 17,82 0,22 0,0969 -0,6638
0,10
1,00
10,00
100,00
1 10
C/%
Gam
ma
Measured50 % inhib20 % inhibPotenssi (Measured)
1. Awake the photobacteria
Synergy task, coded rinsing water samples
2. Pipet 0.5 ml photobacteria into cuvette.
3. Measure the light output with luminometer.
4. Add 0.5 ml of sample
5. Wait 5 min.
6. Measure the light output.
Task 1. ResultsExample:
Alcohol based disinfectants. Dilution to give 50 % inhibition:
SWE 1:18ICE 1:579NOR 1:6FIN 1:560
Task 1. Results
• after some difficulties this wasaccomplished
• problems related to instruments• results comparable to expected ones• different chemicals have different
properties
Task 2. Results - supplied samples
H=Hypo (hypochlorite based disinfectant) I=Ipa (isopropanol based disinfectant)P=Pesetti (surface active cleaning agent)
Liquid samples vs. swab samples
• Sample volume is five times smaller with swabs compared to the liquid sample
• Swabbing is easier to perform than liquid sampling
• Not too sensitive method
Task 2. Results - supplied samplesSample/ c Inhibition/ %
Liquid Tube SWE FIN 1 NOR DEN ICE FIN 2cf 10 0,562 0,769 0,610 0,685 0,981 0,612
H 0,1 1 99,4 100,0 100,0 - 100,0 100,0H 0,02 8 100,0 99,5 2,5 1,9 12,2 100,0H 0,005 9 19,0 6,4 7,6 10,4 14,9 97,3
H 0,0017(D) 9 29,8 13,3 -3,4 16,6 23,8 -25,0I 10 4 100,0 100,0 100,0 100,0 100,0 100,0I 5 6 100,0 99,7 100,0 100,0 100,0 100,0I 1 2 96,5 92,5 99,8 99,6 94,2 99,6
I 0,3 (D) 2 77,3 73,6 92,5 97,1 63,5 64,9I 0,17 (D) 2 33,0 42,8 47,8 65,0 34,4 12,9P 0,15 5 99,9 99,2 100,0 100,0 99,6 99,9P 0,07 7 99,8 99,2 99,8 100,0 99,1 99,8P 0,015 3 61,4 57,4 82,2 71,5 33,4 50,2
Task 2. Results - supplied samples
Swab Tube SWE FIN 1 NOR DEN ICE FIN 2cf 10 0,340 0,124 0,531 0,660 0,353
H 0,1 1 67,6 87,0 75,5 na 85,3 99,9H 0,02 8 3,9 49,8 -8,5 na -8,9 43,0H 0,005 9 -30,9 93,7 0,3 na -25,4 -7,8
I 10 4 56,6 99,9 94,5 na 20,9 99,9I 5 6 45,5 99,1 64,0 na 71,0 99,4I 1 2 -15,1 58,2 18,2 na 25,2 47,3
P 0,15 5 31,1 99,9 72,2 na 77,5 89,9P 0,07 7 32,8 63,0 47,4 na 55,0 80,6P 0,015 3 4,1 87,9 20,6 na 22,8 24,4
Task 2. Results - comments
h with liquid samples very good correlationh with swab samples good correlation
hdiluted hypochlorite results: highest variation (due to changes in the composition during storage of the diluted sample?)
hswabbing has more variables
Task 3. Results - dairies
<-203 % -20 - 0
10 %
0 - 2021 %
20 - 50 28 %
50 - 10038 %
n = 501
Task 3. Results - DairiesThe distribution of the results, n=502
-150,0
-100,0
-50,0
0,0
50,0
100,0
0 100 200 300 400 500
Inh/
%
Task 3. Same dairy, different day
-40-20
020406080
100
0 10 20 30 40
sample number
Inh/
%
4.9.5.9.12.9.
Task 3. Results - plate count
No correllation between residue test and plate count• chemical residue test represents the situation after the
last action on the surface• TPC test represents the total cleaning
procedure• it can be microbially clean, but there are
chemical residues left on the surface• it can be microbially dirty, but there are no
chemical residues left on the surface
Conclusions
A. Introduce people to the Biotox method for detecting cleaning agents and disinfectants from production surfaces in food factories
Conclusions
A. Introduce people to the Biotox method for detecting cleaning agents and disinfectants from production surfaces in food factories
B. compare results obtained in different locations with same chemicals by different users
Conclusions
A. Introduce people to the Biotox method for detecting cleaning agents and disinfectants from production surfaces in food factories
B. compare results obtained in different locations with same chemicals by different users
C. use the method for real samples from different sites by the user
All participants during the project!