Ballast Water compliance monitoring: a new application for ATP 1

LO CURTO A*a, STEHOUWER P +, GIANOLI C +, SCHNEIDER G +, RAYMOND M*, BONAMIN V +. 2

*Aqua-Tools, France; +SGS international, Switzerland. 3

a Corresponding author address: 26, rue Charles-Édouard Jeanneret, 78300, Poissy, France. Corresponding author email: 4alberto.locurto@aqua-tools.com 5

Abstract 6

The coming in force of USCG ballast water regulations and the IMO convention for ballast water 7

management and the availability of several technologies approved for ballast water treatment requires 8

the development of rapid and robust methods to ensure compliance of these technologies. 9

In collaboration with the SGS Group (Switzerland) and LuminUltra (Canada), aqua-tools (France) has 10

developed an innovative Ballast Water Treatment Monitoring (BWTM) kit for rapid onboard testing. This 11

kit provides results in less than one hour, is easy to use and robust, and ensures that the ballast water 12

treatment system on the ship is fully compliant with the discharge standards upon arrival in port. 13

The heart of this method is a combination of a superior chemistry of reagents (lysis solution and 14

luminase TM enzyme) not inhibited by salinity and a patented homogenizing method for ATP extraction 15

developed for a higher level of ATP recovery from zooplankton and phytoplankton within minutes. 16

BWTM Kit provides fast and accurate results compared to the traditional methods for all three fractions 17

of microorganisms (≥50µm, ≥10 -

from one part of the world to another (GEF-UNDP-IMO GloBallast, 2009). The aquatic organisms that 39

are taken up with the water are thereby introduced to an aquatic ecosystem that is very different from 40

their original environment. Certain exotic, invasive and/or toxic species may find the new conditions 41

favorable for growth (temperature, nutrients, absence of predators, etc.) (Steichen et al., 2014). . The 42

proliferation of these species in their new environment can have a lasting impact on the native flora and 43

fauna. In many cases, there have been irreversible damages to the environment (decreased biodiversity, 44

disappearance of vulnerable species, introduction of an invasive species to an ecosystem, etc.) (Casas-45

Monroy et al., 2015). The introduction of foreign species can also have an economic impact, such as the 46

loss of aquaculture or fishing productivity, or even a health impact due to the consumption of 47

contaminated fish or seafood (infection with Dinophyceae protozoa Chang et al., 1997), cholera 48

epidemics caused by Vibrio cholerae bacteria (Dobbs et al., 2013; McCarthy and Khambaty, 1994). 49

Because of the problems that invasive species can cause, the International Maritime Organization (IMO) 50

adopted the International Convention for the Control and Management of Ships’ Ballast Water and 51

Sediments (2004). This convention contains regulations to reduce the spread of harmful aquatic 52

organisms and pathogens. Part of these regulations is the D-2 Ballast Water Performance Standard 53

which states the discharge limits for organisms in ballast water. These discharge limits are: for the 54

organism class, larger than or equal to 50 microns, less than ten viable organisms per cubic meter. For 55

the organism class larger than or equal to 10 micron but smaller than 50 micron, less than ten viable 56

organisms per millilitre. For the indicator microbe Escherichia coli, less than 250 colony forming units 57

per 100 millilitres. For the indicator microbe Enterococci, less than 100 colony forming units per 100 58

millilitres. For the indicator microbe Vibrio cholerae, less than 1 colony forming unit per 100 millilitres. 59

The United States Coast Guard adopted discharge standards almost identical to those of IMO, except 60

that their limits are for ‘living’ instead of ‘viable’ organisms. 61

These discharge standards prompted the development of so-called ballast water treatment systems 62

(BWTSs), which were designed to remove or kill the organisms in the water. These BWTSs usually use a 63

combination of filtration and disinfection (Gregg et al., 2009; Tsolaki and Diamadopoulos, 2010). The 64

most common disinfection methods are Ultra-Violet (UV) radiation or chlorine (David and Gollasch, 65

2012). 66

To test if these BWTSs are meeting the discharge standards, rapid methods to test compliance of 67

samples on-board are required. One of the methods under consideration for this is Adenosine 68

TriPhosphate (ATP). Compared to other methods such as Pulse Amplitude Modulated fluorometry for 69

instance, ATP has the advantage that it detects all living organisms in the sample. 70

Like DNA (deoxyribonucleic acid), ATP is characteristic of all living organisms. Essentially, ATP acts a key 71

element in energy metabolism by serving as an energy transporter. Determining the amount of this 72

molecule allows quantification of all living organisms present in a liquid environment: bacteria, fungi, 73

yeast, protozoa, algae, diatoms and other planktonic organisms. 74

ATP detection involves enzymatic measurements using an enzyme produced by biotechnological 75

methods: luciferase, copied from a well-known luminescent insect: the firefly Photinus pyralis. 76

As its name indicates, this enzyme produces light in the presence of the chemical luciferin and ATP. The 77

amount of light energy released is directly proportional to the quantity of ATP present and thus the 78

volume of organisms present in the sample (Deininger and Lee, 2001). 79

After removing a sample of the fluid to be analyzed, the ATP is extracted from the cells in the sample 80

via cell lysis (enzymatic digestion of the cell walls of bacteria, fungi, animals or plants). The cell extract 81

is then combined with a solution containing luciferase and luciferin. The bioluminescence intensity is 82

then measured using a luminometer. 83

The first ATP-metry tools appeared in the 1970s (Conn et al., 1975). They became more widely available 84

during the 1980s and 1990s and were relatively successful despite limited applications and practical 85

issues. 86

Differentiating between intra-cellular ATP and extra-cellular ATP is necessary not only to get an accurate 87

measurement of microbial activity, but also to assess microbial health in certain applications. Most 1st 88

Generation ATP tests measure the entire ATP content of a sample via Total ATP, thus providing users 89

with an inflated estimate of the living population. Some ATP test kits provide users with protocols to 90

measure extra-cellular ATP, although extra-cellular ATP can exist in many forms, most of which are only 91

able to react with Luciferase in its ‘free’ form. 92

Extra-cellular ATP is often bound to cellular debris or bound to other components present in a sample, 93

such as heavy metals, cationic treatment polymers, and other inert substances. These bound ATP 94

molecules are unavailable to react with luciferase and as such are not included in an extra-cellular ATP 95

measurement. Therefore, to get a true measurement of extra-cellular ATP, it is important to stabilize all 96

extra-cellular ATP – free and bound – so that it is available to react with luciferase. 97

It is commonly known that the total dissolved solids (TDS) concentration of an assay solution can present 98

problems for enzymatic reactions and other microbiological quantification methods. The ATP assay is 99

severely impacted in terms of measurable signal. Once you get into the 20,000 ppm of TDS range and 100

above, at least 50% loss of light output can be expected, which creates a severe false negative. 101

The second generation of ATP-metry is much more powerful due to key advances in biotechnology, 102

overcoming the limitations of its predecessors: 103

- Microfiltration: inserting a microporous membrane allows viable cells to be retained, while residual 104

compounds that could interfere with the luminescent signal are eliminated. This minimizes interference 105

and background noise. Microfiltration provides two additional major advantages: 106

- concentration of the ATP content of a large sample (up to 100 mL); 107

- separation of free extracellular ATP (released by dead cells) from intracellular ATP (contained in living 108

cells). 109

-- Salinity: high dissolved solids issues are minimized. Test results show that ATP spiked into the diluted 110

extracts of the high salinity samples was completely recovered and results were similar to ATP recovery 111

for the control sample. Salinity do not have any negative effects on Luminase (Technical Whitepaper 112

LuminUltra, 2016). 113

114

These features greatly increase the sensitivity and accuracy of the measurement. The level of stress and 115

cell death in a microbial population within a network, for instance after a disinfection procedure, can 116

also be characterized. 117

- Cell lysis step: the enzyme cocktail (UltraLyse™) has a wide range of action and enhanced activity, and 118

can extract up to 99.99% of the cellular material. The ATP measurement is more representative of the 119

actual biological content (Technical Whitepaper LuminUltra, 2016). 120

- Standardization step: the standard curve is established using a standard range of increasing quantities 121

of ATP (UltraCheck™). Calibration of the device generates a conversion scale between the physical 122

measurement (RLU - Relative Light Units) and a biological measurement (pg ATP/mL or Microbial 123

Equivalents (ME)/mL). The results are therefore more accurate. Most importantly, this conversion into 124

biological units enables comparisons to be made between measurements taken by different 125

luminometers at different sites or different time points (data in RLU can only be compared within a 126

single system). 127

ATP is generally used as a method for detecting bacteria numbers in water, but the ballast water 128

regulations also apply to larger organisms. These larger organisms provide a challenge to the ATP 129

extraction method since the complex structures are more difficult to break down than those of bacteria. 130

From 2012 to 2016 aqua-tools, in collaboration with SGS and LuminUltra, developed an innovative 131

Ballast Water Treatment Monitoring kit for rapid onboard testing using ATP technology. This paper 132

describes the development process of the advances made to this method to make it applicable to larger 133

organisms. 134

135

Materials and Methods 136

Method development 137

Using an existing ATP kit from LuminUltra (Quench Gone Aqueous, QGA), several experiments were 138

carried out in order to adapt this kit (originally used for bacteria in water) with larger sizes planktonic 139

organisms (≥50µm and ≥10 -

2000 C) at 50 times magnification with a Bogorov counting chamber was used to evaluate the 160

homogenization of the sample. 161

Foam formation 162

Since foam formation formed a major difference between the two homogenizers, an additional test was 163

conducted to determine the effect of foam formation on ATP measurements. This time only 1 ml of 164

sample was used instead of 5 ml. Based on the previous test the TD300 used a 5 min. exposure time 165

and the Ultra Turaxx T25 used a 3 min. exposure time. A. salina samples were prepared in the same way 166

as the previous experiment, also to a level of 10 organisms/sample. 167

Lysing agent selection 168

Foam formation did not significantly impact the ATP results, but the foam made handling of the samples 169

more difficult. This foam formation was most likely caused by the surfactant in the Ultralyse. However, 170

the surfactant helps the lysing agent break down the cell walls. Because of this tests were conducted 171

with different formulations of the UltraLyse. Two new UltraLyse formulas were used: UltraLyse 30, 172

which has a higher concentration of lysing agent than the previous UltraLyse 7, and UltraLyse 30 without 173

surfactant. It was also tested if UltraLyse 30 was concentrated enough to extract ATP from organisms 174

without homogenization. 175

Conditions were the same as in the previous experiment with 5 minutes of exposure time, 1 ml of sample 176

containing 10 adult A. salina individuals and 5 ml of lysing agent. 177

Detection of different organism concentrations 178

To evaluate the homogenization protocol established in the previous experiments, tests were 179

conducted with different A. salina concentrations to test if ATP concentrations correlated to organism 180

numbers. The methods were the same as previously: with the TD300, 5 minutes exposure time, 5 ml of 181

UltraLyse 30 without surfactant and 1 ml of sample. A. salina concentrations used were 1, 2, 4, 8, 10, 182

16, 22 and 32 per ml. Organisms concentrations were verified using microscopy. The results are a 183

compilation of tests performed on four separate days. 184

Effect of disinfection methods on ATP concentration 185

Since the goal is to develop a method to detect organism numbers in treated ballast water, the effect 186

of disinfection methods on the ATP analysis was tested. To do this, the samples were exposed to 187

microwave radiation for 30 seconds at 1250 W. After intervals of 5, 10, 30 and 60 minutes the ATP 188

concentration in the samples were measured. The original sample contained 10 A. salina adults per ml. 189

The ATP analysis was executed with identical methods to the previous experiments. 190

Detection of different size classes in a natural sample 191

After using cultured organisms, the ATP protocol was now tested on dilutions of a natural sample from 192

the North Sea. The analysis protocol was kept identical to the previous test, with one change. The 193

exposure time on the homogenization device was split into two periods of 2 minutes and 30 seconds. 194

In between the bead-beating tube was inverted several times to prevent organisms from being trapped 195

in drops on the lid of the tube and escaping homogenization. 196

The size fractions were separated by filtration using nylon meshes with a diagonal of 50 µm and 10 µm. 197

Organisms were flushed off the filter using artificial seawater of the same salinity as the original 198

seawater. All organisms trapped on the 50 µm filter were considered part of the ≥50µm fraction, all 199

organisms trapped on the 10 µm filter after being filtered over the 50 µm filter were considered part of 200

the ≥10 -

Filtration of whole sample 236

In order to separate the different fractions in the ballast water samples, filtration is needed. The 237

preliminary tests used reusable nylon mesh membranes to separate the different fractions. The 238

organisms were then re-suspended in artificial sea water, as described above. There were several issues 239

with this method: there was a risk of losing part of the sample in the procedure, it usually resulted in a 240

larger sample volume of which the analysis only uses 1 ml, the cleaning of the mesh between samplings 241

slows down the procedure and since the filtration relies on gravity this can also slow the process down 242

further. Even with regular cleaning the mesh would lose in performance after several samplings and 243

needed to be replaced. 244

Different kinds of single-use membranes were tested together with a filtration system operated by 245

hand-powered vacuum pump to speed up the process in order to eliminate the risk of errors caused by 246

the filtration process. 247

Determining ATP compliance limits 248

The final step in the development was to determine a correlation between the IMO and USCG 249

compliance limits for the different ballast water fractions and ATP concentrations. Preliminary tests on 250

sea water samples (sampled from the Venice lagoon in Italy) were carried out in November 2015 just 251

before the winter season to be sure to catch different types of microorganisms (zooplankton, 252

phytoplankton and bacteria). The finalized analysis protocol used in these tests is included in Appendix 253

1. 254

Several comparative tests between classical methods (microscopy count for ≥10 -

Chlorine, 2 hour contact time ≥50µm fraction: untreated 10 L

≥10 -

The Ultra Turaxx T25 achieved a good homogenization score already after 3 minutes of exposure time 274

(table 4). The positive aspect of the Ultra Turaxx T25 is the lack of foam development. The negative 275

aspect is the loss of time associated with washing the equipment in between samples. 276

Homogenization and foam formation 277

278

Figure 1. ATP levels of samples homogenized by the TD300 and Ultra Turaxx T25, as well as free ATP 279

levels in the artificial seawater. All ATP values in ng/ml. 280

The results show that foam formation does not negatively affect the luminescence reaction. In fact, the 281

TD300 samples which had persistent foam formation had slightly higher tATP (total ATP) measurements 282

(figure 1), indicating that it homogenized the sample more effectively. 283

Since foaming did not significantly affect the results and the disposable tube principle of the TD300 was 284

considered more practical, the TD300 was chosen for all further experiments. The TD300 was always 285

run at its standard intensity (6000 rpm) in all further experiments. 286

Lysing agent selection 287

288

Figure 2. ATP levels (ng/ml) of samples processed with UltraLyse 30 or UltraLyse 30 without surfactant 289

and homogenized with the TD300. 290

The level of homogenization was observed to be similar to the previous experiments. The UltraLyse 30 291

samples showed abundant and persistent foaming. Slight and temporary foaming was observed in the 292

UltraLyse 30 without surfactant samples. Use of UltraLyse 30 without surfactant resulted in slightly 293

higher ATP levels than using normal UltraLyse 30. 294

UltraLyse 30 without homogenization showed very low ATP levels compared to the samples where 295

homogenization was used (figure 2), showing that even at higher levels of lysing agent the 296

homogenization of organisms is necessary. 297

Detection of different organism concentrations 298

299

Figure 3. Concentration of total ATP (tATP) in samples with different concentrations of Artemia salina. 300

ATP concentrations show a good correlation (R2=0.90) with A. salina numbers (figure 3). The higher the 301

organism concentration, the larger the variation between replicate measurements. This shows that the 302

ATP method accurately detects changes in organism numbers. 303

Effect of disinfection methods on ATP concentration 304

305

Figure 4. Total ATP (tATP) concentration after exposure of sample to microwave radiation. 306

The base line for this experiment is about 20000 ng/mL tATP, based on the experiment performed on 307

the same day shown in figure 3. ATP concentrations dropped after microwave treatment (figure 4). After 308

60 minutes only 10% of the original ATP level is left, indicating that the ATP is degrading quickly in the 309

dead remains of the organisms. 310

Microwave radiation is not a common ballast water treatment; further tests should be made with 311

common ballast water treatments such as UV radiation and chlorine. Other papers have also shown a 312

decrease in ATP levels after treatment (van Slooten et al., 2015). Many ballast water treatment system 313

types, such as chlorine and ozone, treat the ballast water when it goes into the ballast water tank and 314

measurements are only taken at discharge, giving the ATP in the organisms sufficient time to degrade. 315

However, UV based ballast water treatment systems treat both on intake and discharge, which can 316

potentially mean some organisms are only treated seconds before sampling. The results above show 317

that the ATP levels of these organisms will degrade sufficiently in the time between sampling and 318

analysis, as in practical situations the analysis equipment will never be set up directly next to the 319

sampling point. 320

Detection of different size classes in a natural sample 321

322

Figure 5. Dilutions made using the sample of North Sea water. The analysis was split into two size 323

fractions: ≥50µm and ≥10 -

328

Figure 6. ATP values in samples containing microalgae (Tetraselmis) and copepodites (A. salina) after 329

homogenization with 5 mm steel beads and 3 mm steel beads. 330

Microscopic analysis of the Tetraselmis samples showed that intact cells were present after 331

homogenization with both 3 mm and 5 mm steel beads. For A. salina, two out of 39 individuals had 332

survived the homogenization with 5 mm steel beads. tATP levels for both organism types were also 333

higher with 3 mm beads than with 5 mm beads. 334

The experiment showed that the 3 mm steel beads performed better than the 5 mm steel beads on A. 335

salina sub-adults (figure 6). After homogenization with both 5 mm and 3 mm beads there were still 336

intact Tetraselmis cells in the sample. Although the 3 mm beads performed better than the 5 mm, the 337

beads need to be further optimized. 338

Beads mixture test: Tetraselmis 339

340

Figure 7. ATP analysis of Tetraselmis samples homogenized using different bead mixtures. 341

Most bead mixtures release ATP on the same level as just the addition of UltraLyse 30 without 342

homogenization. One bead mixture, the 0.7 mm granite beads, actually had lower ATP levels than just 343

adding UltraLyse without grinding. The only bead mixture which increases the amount of ATP released 344

is the mixture of 2 mm steel beads together with 0.1 mm glass beads (figure 7). 345

Bead mixture test: Phaeodactilum and Artemia salina 346

347

Figure 8. ATP concentrations of samples containing the algae Phaeodactilum sp. after exposure to 348

different homogenization methods. 349

The bead mixture extracted ATP at the same level as exposure to just UltraLyse 30 without 350

homogenization (figure 8). Even without addition of UltraLyse high levels of ATP were measured. For 351

this particular alga no homogenization is needed. 352

353

354

Figure 9. ATP concentrations in samples containing A. salina nauplii, with or without homogenization. 355

356

Newly hatched (nauplia) A. salina were effectively homogenized by the beads mixture. Without 357

homogenization but just with UltraLyse 30 w/o surfactant exposure ATP levels in the sample were much 358

lower (figure 9), showing that even for small A. salina individuals homogenization of the sample is 359

essential. 360

Bead mixture test: natural sample 361

362

363

364

Figure 10. Correlation between ATP concentration and number of organisms in samples of Baltic Sea 365

water. 366

The ATP values measured show a good correlation with the number of organisms in the samples (figure 367

10). This shows the potential for ATP to be used to assess organism concentrations in ballast water. 368

369

Verification experiments 370

371

Figure 11. Dilution experiment using Tetraselmis sp. 372

373

374

Figure 12. Dilution experiment using A. salina nauplia (larvae). 375

376

Figure 13. Dilution experiment using copepods collected from natural sea water. 377

These tests, conducted on both plankton grown in a laboratory and natural plankton show a good 378

correlation between ATP concentrations and organism numbers (figure 11, 12, 13), confirming once 379

again that ATP is a suitable tool to determine organism concentrations in sea water. 380

As part of a project commissioned by the German Federal Maritime and Hydrographic Agency 381

(Bundesamt für Seeschifffahrt und Hydrographie, BSH) these tests were repeated by the Norwegian 382

institute NIVA as an independent test. The report on these tests was submitted to IMO by the BSH 383

Delacroix and Liltved, 2013). This report confirmed the good correlations of ATP with A. salina number 384

(R2=0.96), copepods (R2=0.82) and Tetraselmis sp. (R2=0.98). 385

386

Filtration of whole sample 387

Filtration membranes for both 50µm and 10µm were chosen based on their retention ability, 388

compatibility using the homogenization system and lack of interference with the lysing agent and 389

luminase. Because these filtration membranes are transferred straight from the filtration system to the 390

homogenization system, this guarantees 100% sample extraction. 391

All these tests allowed the creation of protocols for ballast water analysis of all three fractions (≥50µm, 392

≥10 -

For ≥10 -

434

Figure 16. Correlation between ATP 2G/plate count and ATP 2G/mpn. U = untreated, D = dilution, T = 435

treated, UV = UV-radiation, Cl = chlorine. 436

These compliance limits are based only on information from plankton in one location and season. 437

Information from other locations is still being gathered to increase the accuracy of these limits. 438

439

Practical experiences 440

The ATP kit described in this paper is already in use as a so-called compliance monitoring technique 441

during on board tests of vessels. A report on the effectiveness of this technique has been issued to IMO 442

by the government of Singapore (Jow, and Chakravarty et al., 2015). It is also already in regular use by 443

SGS as a ballast water compliance monitoring technique (pers comm. P. Stehouwer). 444

445

Conclusions 446

A second generation ATP method designed for detecting bacterial ATP was successfully adapted to 447

measure samples of larger planktonic organisms. The adaptations consist of: 448

- Homogenization of the sample to eliminate complex structures that the lysing agent cannot 449

break down. 450

- A stronger lysing agent to deal with the more complex cell walls found in phytoplankton and 451

zooplankton. 452

- Using a mixture of large and small beads for homogenization so that both ≥50µm and ≥10 but 453

- Concentrating the entire sample for analysis on a filter, thus both increasing sensitivity of the 455

method and eliminating the risk of losing organisms in re-suspension steps. 456

These adaptations resulted in an ATP method which is able to detect organism down to below the D-2 457

limit values. 458

The test is capable, in less than 1h, to produce results that give a strong indication of the performance 459

of the ballast water treatment system on the ship and, when used on a representative sample, give a 460

clear response in terms of gross exceedance of the discharge standards upon arrival in port. 461

The method has been developed to be able to work on ≥50µm plankton, ≥10 -

GEF-UNDP-IMO GloBallast Partnerships and IOI. Guidelines for National Ballast Water Status 494

Assessments. GloBallast Monographs No. 17. 2009. Available: http://globallast.imo.org/wp-495

content/uploads/2014/11/Mono17_English.pdf 496

Gregg M, Rigby G, Hallegraeff GM (2009) Review of two decades of progress in the development of 497

management options for reducing or eradicating phytoplankton, zooplankton and bacteria in ship's 498

ballast water. Aquatic Invasions 4 :521-565 10.3391/ai.2009.4.3.14 499

Jow, TS, Chakravarty R (2015) Report of voluntarily trial study conducted by Singapore on onboard 500

sampling and analysis of ballast water. Maritime and port authority of Singapore shipping circular no. 5 501

of 2015 502

McCarthy SA, Khambaty FM (1994). International dissemination of epidemic Vibrio cholerae by cargo 503

ship ballast and other nonpotable waters. Appl Environ Microbiol 60:2597–2601. 504

Steichen J, Schulze A, Brinkmeyer R, Quigg A (2014). All aboard! A biological survey of ballast water 505

onboard vessels travelling the North Atlantic Ocean. Bull Mar Poll 87: 201–210. doi: 506

10.1016/j.marpolbul.2014.07.058 507

Technical Whitepaper Comparing 2nd Generation ATP to 1st Generation ATP Technology. 2016 508

www.luminultra.com 509

Tsolaki E, Diamadopoulos E (2010) Technologies for ballast water treatment: a review. Journal of 510

Chemical Technology and Biotechnology 85 :19-32 DOI: 10.1002/jctb.2276 511

van Slooten C., Wijers T., Buma AGJ, Peperzak, L. (2015) Development and testing of a rapid, sensitive 512

ATP assay to detect living organisms in ballast water. J Appl Phycol 27: 2299. doi:10.1007/s10811-014-513

0518-9 514

Appendix 1: ATP analysis protocol 515

The protocol below is based on the ≥50µm size fraction. The ≥10 -

Run the dispersing system 3 times for 2 min each 531

Between each step vigorously shaking the ATPrep tube for at least 10 sec 532

ATP DILUTION STEP 533

Wait at least 5min to allow sedimentation - Don’t move the ATPrep tube after the last shaking step 534

Transfer 100µl of supernatant from ATPrep tube to 5 ml UltraLute™ tube 535

The sample is stable up to 2 hours (verified by manufacturer LuminUltra) and ready for the analysis 536

537

The ≥10 -

Screw the 0.7µm filter to the syringe and add 1 ml UltraLyse 7™ 573

Put the plunger back and push it to filter into 9ml UltraLute™ tube 574

The sample is stable up to 4 hours and ready for the analysis. 575

576

Analysis protocol include different step and it is common for all the three fraction: 577

REHYDRATATION STEP 578

Unscrew the cap from buffer and luminase bottles 579

Remove red rubber cap and throw it away 580

Rehydrate dried luminase by adding buffer 581

Screw white cap and upside down the bottle 582

Wait 15 minute at least before use 583

584

ATP CALIBRATION STEP 585

Add 100 µl of luminase enzyme + 2 drops of Ultracheck 1 into a luminometer tube 586

Insert it to the luminometer and press start 587

Note RLU value after 10 seconds of reading (> 5000 RLU) 588

589

NEGATIVE CONTROLS STEP 590

Insert a luminometer tube it to the luminometer and press start 591

Record the RLU value after 10 seconds of reading as RLUempity tube (