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!