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Testing

What’s in Your Water?®

Special Pathogens Laboratory offers healthcare, hospitality, water treatment industries and other commercial and industrial sectors reliable high-quality microbiology services. Our team of microbiologists possess specialized knowledge required for Legionella detection. We the first, and one of the few labs to be accredited by the A2LA  for Legionella testing. What’s more, our testing protocol is the only method validated in prospective studies that correlates disease risk with Legionella culture results and the extent of environmental contamination.

Legionella Culture

Legionella colonies on culture plate.

Significance
Legionnaires’ disease is an acute lobar pneumonia with multi-system manifestations.

Legionnaires’ disease results from exposures to water sources containing the organism. Predisposing factors in a susceptible host include a compromised immune system, underlying disease, recent surgery, increased age, and heavy smoking.

It is important to be able to culture Legionella from environmental specimens. Analysis of water suspected of being contaminated with Legionella identifies potential sources of the disease. Commonly sampled sources include non-potable water from cooling towers, condensers, whirlpools, etc. and potable water from water distribution systems, water heaters and tanks, drinking fountains and ice machines.

The organisms are nutritionally fastidious, non-spore forming, aerobic, gram-negative, slender rods. Media containing cysteine, yeast extract, α-ketoglutarate, and iron (BCYE) are required for isolation of Legionella. Selective BCYE (medium containing antibiotics), is recommended for specimens likely to be contaminated with other bacteria. Colonies gray-white to blue-green typically grow five days after incubation at 36ºC

The most common disease-causing member of the Legionellaceae family is Legionella pneumophila. This member has 16 named serogroups, all of which we can detect. Approximately half of the more than 60 named species of Legionella have been implicated in human disease. The methods and reagents used by Special Pathogens Laboratory can detect the primary disease-causing species and serogroups of Legionella. These methods include primary culture followed by latex agglutination and fluorescent antibody staining methods. If these classical identification methods are indeterminate, molecular sequencing methods can be used for further identification to the species level. This technology would identify any member of the family for which a nucleotide sequence is in GenBank.

The list of Legionella species implicated in disease and tested for using SPL methods and reagents:

  • L. pneumophila (serogroups 1–16)
  • L. dumoffii
  • L. micdadei
  • L. bozemanii (serogroup 1)
  • L. longbeachae (serogroups 1, 2)
  • L. jordanis L. feeleii (serogroup 1)
  • L. gormanii
  • L. wadsworthii
  • L. hackeliae
  • L. maceachernii
  • L. oakridgensis
  • L. cherrii
  • L. sainthelensi
  • L. anisa
  • L. parisiensis
  • L. spiritensis
  • L. jamestownensis
  • L. steigerwaltii
  • L. rubrilucens
  • L. erythra

Test Method
SPL modified ISO standards: 11731: 2017. Water Quality: Enumeration of Legionella

Turn Around Time
7–10 days

Test Code
101

Sample Transport
Overnight delivery is recommended. Please see Legionella Sampling and Shipping or consult with the laboratory for sample transport requirements.


 

QuickCheck and Case Investigation

QuickCheck
QuickCheck, gives you the first look at Legionella culture results. Now, you can receive a preliminary report within four business days of submission. No more waiting, wondering, or calling about the trend of Legionella growth.

To add QuickCheck to your routinely processed samples, just check the box for QuickCheck on your chain of custody. Perfect for samples received on Monday, Wednesday, Thursday, Friday, or Saturday and only $75 per submission. Samples received Tuesdays are reported the following Monday.

Case Investigation
We understand the stress and anxiety that comes from having just one case of Legionnaires’ disease, so we’ve bundled everything you need from results to communication and support. Our case investigation processing and reporting includes the extra steps to expedite Legionella isolation. What’s more you get preliminary reporting, phone notification, results interpretation, and isolate storage. For a case investigation just check Case Investigation on your chain of custody. Cost: 15% + per sample.
If you have an outbreak, see our Outbreak Response services

Legionella qPCR

Significance
Quantitative polymerase chain reaction (qPCR) is a rapid molecular method that can be used to detect and quantitate Legionella pneumophila and Legionella pneumophila serogroup 1 in potable or non-potable water samples. This is achieved by amplification of a target gene sequence that is unique to the genome DNA of L. pneumophila or L. pneumophila serogroup 1. The amplified sequence is detected using a fluorescent signal. Approximations of amount of target gene detected can be determined as low, moderate, or high by comparing the amount of fluorescence of the target gene to the amount of fluorescence of a known quantity of DNA (i.e. compared to a standard curve). Testing can be performed in approximately 2 to 4 days as compared to 7 to 10 days for culture. The procedure follows International Standard Organization (ISO) standard 12869 with minor modifications.

The qPCR assay cannot discriminate the genome of live vs dead/impaired L. pneumophila and may also detect L. pneumophila that are not culturable. Because of this, genome copies (genomic units) cannot be directly correlated to CFU.

There are more than 50 Legionella species, however L. pneumophila (specifically L. pneumophila serogroup 1), are responsible for a large majority of illness. Therefore the qPCR test is designed to identify or rule out only the most clinically relevant strains. If requested, or when species identification is required such as in investigations of clinical cases, culture identification and DNA sequencing can be performed for identification of species other than L. pneumophila.

Test Method

  • Quantitative Polymerase Chain Reaction (qPCR) For Detection of L. pneumophila and L. pneumophila, serogroup 1.
  • Water samples are concentrated by filtration.
  • DNA is extracted using the Qiagen DNeasy Power Water Kit.
  • Taqman qPCR is performed on the extracted DNA with two primer/probe sets: 1) Legionella pneumophila specific for a sequence within the 16s rRNA gene; 2) Legionella pneumophila serogroup 1 specific for a sequence within the LPS gene.
  • Amount of L. pneumophila genome present is extrapolated using a standard curve, and reported as not detected, low, moderate, or high level present (serogroup 1 reported as detected or not).

Turn Around Time
2–4 days

Test Code
124

Sample Collection and Shipping

See Legionella Sampling and Shipping for sampling guidelines.

References

qPCR Application Guide – Experimental Overview, Protocol, Troubleshooting. 3rd Edition. 2011-2012. Integrated DNA Technologies.

Qiagen DNeasy PowerWater DNA Isolation Kit Instruction Manual.

Applied Biosystems. Quantstudio 6 and 7 Flex Real-Time PCR System Software. 2013. Booklets 2 and 5.

Donohue MJ, O’Connell K, Vesper SJ, Mistry JH, King D, Kostich M, Pfaller S. Widespread molecule detection of Legionella pneumophila Serogroup 1 in cold water taps across the United States. Environ Sci Technol. 2014 Mar 18; 48(6):3145-52.

ISO Standard 12869: 2019. Water Quality – Detection and quantification of Legionella spp. by concentration and genic amplification by quantitative polymerase chain reaction (qPCR)

Legionella Serotyping of Isolates (DFA)
Legionella qPCR

Legionella qPCR

Significance
The direct fluorescent antibody (DFA) test is a rapid microscopic procedure used to identify Legionella pneumophila and other Legionella species. Antibodies specific for Legionella species and serogroups are tagged with a fluorescent dye. When Legionella is isolated by culture from an environmental source or a patient, the bacteria are placed on a slide and exposed to a panel of DFA reagents. A positive reaction confirms the identification. DFA is sensitive and highly specific when used for species/serotype confirmation.

DFA staining is not recommended as a method to be applied directly on an environmental sample due to false positive and false negative results. DFA staining will stain both viable (living) and non-viable (dead) Legionella cells.

There are more than 60 Legionella species and 16 serogroups of L. pneumophila, however only a select few species and serogroups are responsible for most illness. Therefore DFA testing is performed to identify or rule out only the most clinically relevant strains. If requested or when species identification is required such as in investigations of clinical cases, DNA sequencing can be performed for identification of unusual species or those not included in the DFA panels.

Test Method

  • Legionella Identification by direct fluorescent antibody staining.
  • Legionella pneumophila confirmed using specific species/serogroup reagents.
  • Legionella species, not pneumophila, are confirmed using specific reagents to include the following: L. anisa, L. bozemanii serogroups 1 and 2, L. gormanii, L. longbeachae serogroups 1 and 2, L. dumoffii, L. micdadei, L. wadsworthii, L. oakridgensis, L. feeleii serogroups 1 and 2, L. sainthelensi, L. jordanis, L. hackeliae serogroups 1 and 2, L. maceachernii, L. santicrucis, L. spirtensis, L. jamestowniensis, L. cherrii, L. steigerwaltii, L. rubrilucens /L. erythra.

Turn Around Time
2–3 days

Test Code
401

Transport
Submit isolate on a slant or agar plate of Buffered Charcoal Yeast Extract (BCYE) in double containment packaging according to IATA regulations for shipping Biological Substances, Category B (UN3373). Contact SPL if you have questions.

See Sampling and Shipping for sampling guidelines.

References

  1. Procedures for the Recovery of Legionella from the Environment. Centers for Disease Control publication. January 2005.
  2. DFA manufacturers’ instructions: Remel, Lenexa, KS;  Bio-Rad, Redmond, WA and m-Technologies, Alpharetta, Ga.
Heterotrophic Plate Count

Significance
The Heterotrophic Plate Count (HPC) is used to count total bacteria in water under certain growth conditions (media and temperature).

HPC can be used to monitor changes in the bacteriological quality of finished water throughout a distribution system, thus giving an indication of the effectiveness of chlorination or other disinfection in the system, as well as the possible existence of cross-connections, sediment accumulations and other problems within the distribution lines.

HPC is also used to evaluate the effectiveness of biocides in cooling water systems. Types of water that may be tested include potable water, swimming pools, spas, cooling towers, and reclaimed water.

Test Method

  • Enzyme Substrate Method, incubation 35°C ± 0.5°C for 2–3 days in humidified air.
  • Standard Methods for the Examination of Water and Wastewater: Method 9215E.
  • Measure of uncertainty available upon request.

Turn Around Time
2–7 days

Test Code
103

Sample Instructions and Shipping
HPC sample collection for water systems and cooling towers and compliance testing for drinking water require different procedures. For instructions please see Sampling and Shipping.

NELAP ACCREDITATION

SPL is accredited by the National Environmental Laboratory Accreditation Program (NELAP) and the American Association for Laboratory Accreditation (A2LA) to perform this test on drinking water. DEP Lab ID: 02-04660. See Accreditations and Proficiency..

Pseudomonas Aeruginosa

Pseudo on MPAC

 

Significance

Pseudomonas aeruginosa is a bacterium that can be found in various types of moist environments. This opportunistic pathogen is commonly associated with hospital-acquired infection, most notably in immunocompromised individuals. Pseudomonas aeruginosa accounts for 10% of all hospital-acquired infections and is the second most frequently recovered pathogen from intensive care units. Healthy individuals can acquire eye, ear and skin infections as well, through contaminated recreational waters. Types of water that may be tested include: potable water where high risk patients are found, swimming pools, spas and hot tubs.

Test Method
Filter concentration method using M-PA-C selective media. Modification of the ASTM International Standard Test Method for the Isolation and Enumeration of Pseudomonas aeruginosa from water (D5246) with the addition of direct plating in the event of high concentrations of the organism.

Turn Around Time
2–10 days

Test Code
102

Sampling Instructions
Prior to collecting samples for potable water or for swimming pools, spas or hot tubs:

  1. Complete SPL Chain of Custody Test Request form.
  2. Use waterproof pen to label bottle with sample location, description, and date.

The sample volume is ≥120 mL and should be collected as follows:

Potable Water
Turn on hot water and immediately fill the bottle. Close bottle and invert to mix neutralizer.

Swimming Pools, Spas, Whirlpools or Hot Tubs

  1. Collect sample by removing cap and holding at an angle of 45°. Fill in one slow sweep down through the water with the mouth of the bottle always ahead of the hand.
  2. Fill the bottle (≥120 mL). Be careful to retain the sodium thiosulfate neutralizer in bottle.
  3. Replace cap and mix.

Sample Transport

Ship the same day samples are collected for receipt in laboratory. The time from sample collection to analysis should not exceed 30 hours. See Sampling and Shipping for sampling guidelines.

SPL is accredited by the American Association for Laboratory Accreditation (A2LA) to perform this test. See Accreditations and Proficiency.

Acinetobacter

Significance

Acinetobacter is a group of bacteria generally considered nonpathogenic to healthy individuals. However, at least 23 species have been associated with human disease. Acinetobacter infections have increased and gained attention because of the organism’s prolonged environmental survival and propensity to develop antimicrobial drug resistance. The settings for infection are usually medical or surgical intensive care units involving patients with preexisting conditions. Common routes of bacterial transmission include contaminated medical devices such as ventilation devices and catheters.

Test Method Filter concentration method using Leeds Acinetobacter medium. Direct plating is also included in the event of high concentrations of the organism. Modification of the ASTM International Standard Test Method for the Isolation and Enumeration of Pseudomonas aeruginosa from water (D5246).

Turn Around Time

2–7 days

Test Code

105

Sampling

The sample volume is ≥120 ml and should be collected as follows:

  1. Complete the Chain of Custody Test Request form.
  2. Use waterproof pen to label bottle with sample location, description, and date.
  3. Turn on hot water and immediately fill the bottle. Close bottle and invert to mix neutralizer.

Sample Transport

Ship the same day samples are collected for receipt in laboratory. The time from sample collection to analysis should not exceed 30 hours. See Sampling and Shipping for sampling guidelines.

Nontuberculous Mycobacteria
Nontuberculous Mycobacteria

Nontuberculous Mycobacteria

Significance
Water is recognized as a major source of Nontuberculous Mycobacteria (NTM) species responsible for opportunistic infections in humans. These bacteria usually affect individuals with pre-existing conditions, specifically, lung disease, immunosuppression, transplantation and HIV infection. The most common infections are pneumonia and gastrointestinal disease caused by M. avium and M. intracellulare. Non-immunosuppressed individuals can also acquire NTM infections, most commonly, catheter sepsis, surgical and posttraumatic wound infections and cervical lymphadenitis in children. Types of water that may be tested include potable hot water systems where high risk patients are housed, spas and hot tubs.

Test Method
Filter concentration and decontamination with 0.2 M KCL-HCL, pH 2.2. Cultivation using Middlebrook 7H10, Mitchison 7H11, and Rapid Growth Medium (RGM), incubation at 30°C for 6 weeks.

Turn Around Time
6–8 weeks

Test Code
108

Sampling
The sample volume is ≥220 mL (except for ice—see below) and should be collected as follows:

  1. Complete Chain of Custody Test Request form.
  2. Use waterproof pen to label bottle with sample location, description, and date.
  3. Turn on hot water and immediately fill the bottle. Close bottle and invert to mix neutralizer.
  4. When sampling ice, completely fill one 500 mL bottle with ice.

Sample Transport

Ship the same day samples are collected. The time from sample collection to analysis should not exceed 48 hours. See Sampling and Shipping for sampling guidelines.

Stenotrophomonas maltophilia

Significance
Stenotrophomonas maltophilia is an opportunistic bacterial pathogen previously known as Pseudomonas maltophilia and Xanthomonas maltophilia. Stenotrophomonas is of most concern in the hospital setting. This organism is inherently resistant to multiple antimicrobial agents that are used to treat gram-negative infections. Contaminated water can transmit the organism through indwelling catheters and respiratory therapy equipment to susceptible patients causing infection. Stenotrophomonas infection in intensive care unit (ICU) patients has been identified as an independent risk factor for mortality.

Test Method
Filter concentration method using Trypticase Soy Agar with imipenem selective media. Direct plating is also included in the event of high concentrations of the organism. Modification of the ASTM International Standard Test Method for the Isolation and Enumeration of Pseudomonas aeruginosa from water (D5246).

Turn Around Time
2–10 days

Test Code
104

Sampling
The sample volume is ≥120 mL and should be collected as follows:

  1. Complete the Chain of Custody Test Request form.
  2. Use waterproof pen to label bottle with sample location, description, and date.
  3. Turn on hot water and immediately fill the bottle. Close bottle and invert to mix neutralizer.

Sample Transport

Ship the same day samples are collected for receipt in laboratory. The time from sample collection to analysis should not exceed. See Sampling and Shipping for sampling guidelines.

Burkholderia cepacia
Burkholderia on selective media

Burkholderia on selective media

Significance
Burkholderia cepacia, formerly Pseudomonas cepacia, is a bacterium that can be found in soil and water. Ordinarily it is not a pathogen in the ambulatory setting but may colonize and or infect the respiratory tract of patients with cystic fibrosis or bronchiectasis. Skin and soft tissue infections, surgical wound infections, central venous catheter and urinary tract infections have all been reported in healthcare settings. This bacterium is often naturally resistant to common antibiotics.

Transmission of Burkholderia cepacia has been reported from contaminated medicines and medical devices as well as person-to-person contact and contact with contaminated surfaces. Environmental monitoring for contamination of showers and sinks has been suggested.

Test Method
Filter concentration method using Burkholderia selective media. Direct plating is also included in the event of high concentrations of the organism. Modification of the ASTM International Standard Test Method for the Isolation and Enumeration of Pseudomonas aeruginosa from water (D5246).

Turn Around Time
3–10 days

Test Code
123

Sampling
The sample volume is ≥120 mL and should be collected as follows:

  1. Complete the Chain of Custody Test Request form.
  2. Use waterproof pen to label bottle with sample location, description, and date.
  3. Turn on hot water and immediately fill the bottle. Close bottle and invert to mix neutralizer.

Sample Transport

Ship the same day samples are collected for receipt in laboratory. The time from sample collection to analysis should not exceed 30 hours. See Sampling and Shipping for sampling guidelines.

CMS Waterborne Pathogens Panel

Significance
There are a variety of microorganisms found in the water of healthcare facilities that are not generally pathogenic for healthy individuals but may be pathogenic for the immunocompromised patient. The conditions associated with increased risk of acquiring an infection from a waterborne pathogen include: hematological malignancy, immunosuppression, transplantation and burns. These infections can result in significant morbidity and mortality. The CMS pathogens panel is designed for use in healthcare facilities to evaluate the potential for exposure to six waterborne pathogens Legionella, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter species, Burkholderia cepacia, and nontuberculous mycobacteria.

Test Method:
Legionella spp.
Special Pathogens Laboratory follows a modified version of the ISO Standard 11731:2017, Water Quality-Enumeration of Legionella. Special Pathogens Laboratory is accredited by the American Association for Laboratory Accreditation (A2LA), New York ELAP, and the State of Connecticut Department of Health to perform this test.
LEARN MORE ABOUT LEGIONELLA

Pseudomonas aeruginosa
Filter concentration method using M-PA-C selective media. Modification of the ASTM International Standard Test Method for the Isolation and Enumeration of Pseudomonas aeruginosa from water (D5246) with the addition of direct plating in the event of high concentrations of the organism. Special Pathogens Laboratory is accredited by the American Association for Laboratory Accreditation (A2LA) to perform this test.
LEARN MORE ABOUT PSEUDOMONAS

Stenotrophomonas maltophilia
Filter concentration method using Trypticase Soy Agar with imipenem selective media. Direct plating is also included in the event of high concentrations of the organism. Modification of the ASTM International Standard Test Method for the Isolation and Enumeration of Pseudomonas aeruginosa from water (D5246).
LEARN MORE ABOUT STENOTROPHOMONAS

Acinetobacter species
Filter concentration method using Leeds Acinetobacter medium. Direct plating is also included in the event of high concentrations of the organism. Modification of the ASTM International Standard Test Method for the Isolation and Enumeration of Pseudomonas aeruginosa from water (D5246).
LEARN MORE ABOUT ACINETOBACTER

Burkholderia cepacia
Filter concentration method using Burkholderia selective agar. Direct plating is also included in the event of high concentrations of the organism. Modification of the ASTM International Standard Test Method for the Isolation and Enumeration of Pseudomonas aeruginosa from water (D5246).
LEARN MORE BURKHOLDERIA.

Nontuberculous Mycobacterium (NTM)
Filter concentration and decontamination with 0.2 M KCL-HCL, pH 2.2. Cultivation using Middlebrook 7H10, Mitchison 7H11, and Rapid Growth Medium (RGM), incubation at 30°C for 6 weeks.
LEARN MORE ABOUT NTM

Test Code
130

Turn Around Time
6 to 8 weeks

Sampling Instructions
The sample volume is 1 liter and should be collected as follows:

  1. Complete the Chain of Custody Test Request form.
  2. Use waterproof pen to label bottle with sample location, description, and date.
  3. Turn on hot water and immediately fill the 1 liter bottle. Close bottle and invert to mix neutralizer.

Sample Transport

Ship the same day samples are collected for receipt in laboratory. The time from sample collection to analysis should not exceed 30 hours. See Sampling and Shipping for sampling guidelines.

Coliforms (E.Coli and Total)

Significance
Drinking water should be free of disease-causing organisms. Coliform bacteria are organisms that are present in the environment and in the feces of all warm-blooded animals and humans. Therefore, water is analyzed for coliforms because their presence is an indication there may be disease-causing bacteria, parasites, and viruses in the water.

Types of water that may be tested include drinking (potable) water, spa, swimming pool water, and reuse/reclaimed water.

Test Method

  • The Colilert®-18 (IDEXX)
    Reference: Standards Methods for the Examination of Water and Wastewater, 9223B, an enzyme substrate test.  Colilert®-18 is a US EPA approved method.
  • Measurement uncertainty is available for Coliforms: Total and E.coli test upon request.

Turn Around Time
1-3 days

Test Code
106 (presence-absence), 107 (quantitative)

Sampling
See Coliforms (Total and E.coli) Sampling

Sample Transport
Coliform bacteria can grow rapidly in some water samples therefore the sample should be kept cold in transit.

SPL is NELAP certified for testing coliforms and E.coli in drinking water. See Accreditations and Proficiency.

Microbiologically Influenced Corrosion

Microbiologically Influenced Corrosion (MIC) refers to corrosion brought about by the presence and activities of microorganisms in biofilms, on the surface of pure metals or metal containing materials. Since the organisms involved, bacteria (aerobic and anaerobic), algae, fungi, and others, are generally ubiquitous, MIC can occur in almost every environment. Each of these groups can cause different problems, not just corrosion. For example, slime forming bacteria can cause plugging, loss in efficiency of heat exchangers, clouding, taste and odor problems. Iron related bacteria, including iron oxidizing and reducing, can cause plugging, corrosion, cloudiness and color change.

If these nuisance microorganisms are present, MIC can occur on these surfaces if a nutrient source and water are also present. Their impact can make the water unsafe, unacceptable or unavailable due to losses in flow through plugging or equipment failure due to corrosion. Generally, testing for more than one group of bacteria with a combination of tests should be used to determine which ones are present and causing problems.


Acid Producing Bacteria

Significance 

Acid producing bacteria are capable of producing organic and inorganic acids which can significantly drop the pH beneath the biofilm into the acid range.  Under these conditions, an acid-driven form of corrosion can occur causing metals to dissolve and concrete structures to lose integrity. These acidic metabolic by-products are produced under very reductive (oxygen free) environments. The sulfate reducing bacteria are often found within this nutrient rich, oxygen free environment.

Telltale signs of acid producing bacteria corrosion include, but are not limited to, pitting   corrosion and pinhole leaks.

Types of water/sources that may be tested include: hot and cold water distribution systems, open and closed recirculating heating and cooling systems, water-based fire protection sprinkler systems, and water-based fracturing fluids in gas and oil industry.

Test Method

APB-BART™ Biological Activity Reaction Test for Acid Producing Bacteria. Manufacturer: Droycon Bioconcepts Inc. Saskatchewan, Canada

Turn Around Time 

10–12 days

Test Code

113


Negative (left) Positive (right)


Iron Related Bacteria

Significance 

Iron related bacteria include both iron oxidizing and iron reducing bacteria. These bacteria function under different reduction-oxidation (redox) conditions and utilize a variety of substrates for growth.  This consortium is complex and includes stalked and sheathed bacteria as well as heterotrophic and slime forming bacteria. Examples of this group include: Gallionella, Crenothrix, Sphaerotilus, Siderocapsa, Thiobacillus and Pseudomonas.  

The iron utilizing bacteria can be problematic in that they can cause corrosion of iron and steel.  This process can cause pitting of the metal surface, resulting in pin hole leaks. These bacteria can also accumulate iron to the point where the growth becomes a hard encrustation or tubercle. Over time these deposits can reduce water flow and affect system performance.  

Types of water that can be tested include: potable water plumbing systems, cooling towers, heat exchangers, water heaters, and water based fire protection sprinkler systems.

Test Method

IRB-BART™ Biological Activity Reaction Test for Iron Related Bacteria. Manufacturer: Droycon Bioconcepts Inc. Saskatchewan, Canada

Turn Around Time

10–12 days

Test Code

 109


Negative (left) Positive (right)


Slime Forming Bacteria

Significance 

The slime forming bacteria is the name given to a group of bacteria that are capable of producing a variety of extracellular polysaccharide polymers.  It is these long chain molecules which act as the foundation and cement for the formation of biofilm. The slime-like growth coating the inside of pipes and fixtures is called the biofilm.  The purpose of this slime layer appears to be protective. Under harsh environments (e.g., temperature changes, chemicals, shortage of nutrients) slime layers can get thicker. As the biofilm matures, aerobic bacteria creating the biomass produce metabolic by-products all the while consuming oxygen.  This facilitates micro-environments underneath the biofilm which can then support the growth of anaerobic bacteria. The slime forming bacteria are an important part of the microbial influenced corrosion process in that they can function under different reduction-oxidation conditions. This transition of aerobic to anaerobic environments within the biofilm supports the growth of iron related, sulfate reducing and acid producing bacteria.

The slime producing bacteria can cause engineering problems related to the reduction of hydraulic or thermal conductivity as well as clogging, taste, odor and color changes.

Types of water sources that may be tested include open-evaporative cooling systems, water-based fire protection sprinkler systems, condensers, and well water.

Test Method

SLYM-BART™ Biological Activity Reaction Test for Slime Producing Bacteria. Manufacturer: Droycon Bioconcepts Inc. Saskatchewan, Canada

Turn Around Time

10–12 days

Test Code 

111


Negative (left) Positive (right)


Sulfate Reducing Bacteria

Significance 

This test is specific for sulfate reducing bacteria such as Desulfovibrio, Desulfomonas and Desulfotomaculum. These bacteria require metal ions as a source of energy and produce hydrogen sulfide as an end result. They are probably the most destructive group of bacteria causing microbial influenced corrosion. Detection of these bacteria can be problematic.  They are strict anaerobes and grow deep within biofilms. In fact, sulfate reducing bacteria may not be present in the free-flowing water over the site of the fouling. Their presence relates to corrosion problems, taste and odor problems (“rotten egg” odors), and blackened waters. Slimes rich in sulfate reducing bacteria tend to also be black in color.

Types of water sources that may be tested include: oxygen-deficient environments such as deep wells, plumbing systems, water softeners and water heaters; condensers.

Test Method

SRB-BART™ Biological Activity Reaction Test for Sulfate Reducing Bacteria. Manufacturer: Droycon Bioconcepts Inc. Saskatchewan, Canada.

Turn Around Time

10–12 days

Test Code

110

Negative (left) Positive (right)


Sampling Instructions

  1. Complete SPL Chain of Custody Test Request form. 
  2. Use waterproof pen to label bottle with sample location, description, and date. 
  3. Samples for more than one MIC test may be collected in a single 250 mL collection bottle as long as there is  ≥30 mL for each type of test requested.
  • Cooling Tower and Non-potable Water
    1. Submerge open bottle just under the surface of water to obtain ≥30 mL of sample. Avoid sediment. 
    2. Close bottle and invert to mix the sodium thiosulfate neutralizer.
  • Potable Water
    1. Turn on water (hot or cold) and immediately fill bottle to ≥30mL.
    2. Close bottle and then invert to mix the sodium thiosulfate neutralizer.

Sample Transport

Deliver or ship samples the same day they are collected. The time from sample collection to analysis should not exceed 30 hours. See Sampling and Shipping for sampling guidelines.

Nitrifying Bacteria

Significance
Nitrifying bacteria include the genera Nitrosomonas, Nitrobacter and Nitrosococcus. They are an important group of bacteria that can be used as an indicator for the presence of organic nitrogenous material in water. During the nitrification process, bacteria aerobically oxidize ammonium to nitrite and then further to nitrate. Aggressive nitrifying bacteria can produce high concentrations of nitrates. This could pose a potential health risk, particularly to infants who haven’t developed a tolerance to nitrates. The presence of aggressive nitrifying bacteria in water may indicate contamination from a sewage system, septic tank or industrial waste.

Types of water to be tested include potable water plumbing systems, well water.

Test Method
N-BART™ Biological Activity Reaction Test for Nitrifying Bacteria. Manufacturer: Droycon Bioconcepts Inc. Saskatchewan, Canada

Turn Around Time
5 days

Test Code
112

Sampling Instructions

  1. Complete SPL Chain of Custody Test Request form provided.
  2. Use waterproof pen to label bottle with sample location, description, and date.
  • Cooling Tower and Non-potable Water
    1. Submerge open bottle just under the surface of water to obtain ≥30 mL of sample. Avoid sediment.
    2. Close bottle and invert to mix the sodium thiosulfate neutralizer.
  • Potable Water
    1. Turn on water (hot or cold) and immediately fill bottle to ≥30 mL.
    2. Close bottle and then invert to mix the sodium thiosulfate neutralizer.

Sample Transport

Deliver or ship samples the same day they are collected. The time from sample collection to analysis should not exceed 30 hours. See Sampling and Shipping for sampling guidelines.

Molecular Typing

Significance
Pulsed-Field Gel Electrophoresis (PFGE) is a powerful molecular tool with high discriminatory power. Reliability and reproducibility make PFGE a “gold standard” in epidemiological investigations seeking to identify the source of illness.

Using PFGE, the organism’s DNA is digested and separated to produce a banding pattern of DNA fragments. This pattern is known as the genetic fingerprint. The “fingerprint” of the bacteria from the patient is compared to the “fingerprint” of the bacteria from the environment. If they match, the results support a link between the environmental source and the patient.

PFGE can also be used to compare bacterial strains from different patients. This information will help determine if there is an outbreak situation, or if the patients acquired the infection from different sources. The answer to these questions may have infection control implications.

Test Method
A modified version of the CDC’s PulseNet protocol for E.coli O157:H7, Salmonella, and Shigella.

Test Code
301

Turn Around Time
3–4 weeks

Sample Transport
Submit isolate on a slant or agar plate of Buffered Charcoal Yeast Extract (BCYE) in double containment packaging according to IATA regulations for shipping Biological Substances, Category B (UN3373).


Banding DNA fragments after PFGE for 15 bacterial isolates.

Making the epidemiological link between cases of Legionnaires’ disease and a suspected environmental source can be difficult due to the sporadic nature of the disease and the variety of sources from which the organism can be isolated.

Both phenotypic and genotypic methods have been used to demonstrate identity among strains of Legionella pneumophila in epidemiological investigations. Monoclonal antibody (MAB) subtyping is a useful phenotypic method for subdividing strains of L. pneumophila, serogroup 1.

Genotypic methods, such as pulsed-field gel electrophoresis (PFGE), have become increasingly valuable for epidemiological investigation due to their high discriminatory power, broad application and speed of results.

See Sampling and Shipping for sampling guidelines.

Copper Silver Analysis

Significance
Copper-silver ionization is a disinfection method that is used to eradicate Legionella from hot water recirculating systems. Disinfection with ionization occurs when the positively charged copper and silver ions released from the ionization system bind to the negatively charged cell wall of Legionella causing cell death. Ion concentration should be monitored to document that effective levels have been achieved and to avoid excess ion levels.

Target Range
Suggested target ion range for Legionella control: Copper: 0.20–0.80 mg/L; Silver: 0.01–0.08 mg/L

Choosing the right sampling sites is important to ensure that the proper levels of copper and silver ions are being distributed to the entire water system so that maximum disinfection will be achieved.

Test Method
EPA 200.8; Determination of Trace Elements in Waters and Wastes by Inductively Coupled Plasma-Mass Spectrometry

Turn Around Time
7–10 days

Test Code
201

Suggested Sampling Sites

  • The immediate water after it passes through the electrode cell on the supply side of the heating element or first distal outlet in the hot water system.
  • Return line or last distal outlet before the water passes through the electrode cell.
  • Distal outlets representing the midpoint of disinfection loop.

Sampling

  1. Complete the Chain of Custody Test Request form.
  2. Use waterproof pen to label bottles with sample location, description, and date.
  3. Turn on hot water and wait until the hot water temperature reaches its maximum and fill the bottle (≥120 mL).  Sample should be clear, avoid sediment.
  4. Place bottles in box with insulated liner.

Sample Transport
Overnight delivery is recommended but not required. Ship the same day samples are collected. See Sampling and Shipping for sampling guidelines.

Analytical Water Chemistry

Significance
The analytical water chemistry panel includes the following metals: lead (Pb), zinc (Zn), iron (Fe), manganese (Mn), copper (Cu), calcium (Ca), and magnesium (Mg). These commonly found metals are used to assess the general inorganic chemistry of building water systems, evaluation of corrosion, metals leaching, and scaling potential, and assist in secondary disinfection selection.

Test Method:
EPA 200.8; Determination of Trace Elements in Waters and Wastes by Inductively Coupled Plasma- Mass Spectrometry

Turn Around
Time 7–10 days

Test code
241

Sampling

  1. Complete the Chain of Custody Test Request form provided.
  2. Use waterproof pen to label bottles with sample location, description, and date.
  3. Open the sampling valve and flush for 60 seconds.
  4. Collect cold sample for chemistry in the unpreserved sample bottle provided.
    • If sampling for other biological tests (e.g. Legionella), use the correct sample bottles provided and follow appropriate sampling instructions.
    • Sample bottles are sealed. If the bottle has been previously opened or used, discard the bottle and collect the sample in a new bottle.
    • To prevent contamination, avoid touching the inside of the bottle, the lid, sample valve or outlet with hands.

These procedures may not meet EPA sampling procedures for drinking water compliance and are provided only to assist with water management plan needs.

Sample transport

Ship samples the same day they are collected for receipt in laboratory. See Sampling and Shipping for sampling guidelines.

Accreditations and Proficiency

Accreditations and Proficiency

Special Pathogens Laboratory is accredited in accordance with recognized International Standard ISO/IEC 17025:2005 General Requirements for the Competence of Testing and Calibration Laboratories by two accrediting bodies: American Association of Laboratory Accreditations (A2LA) and by Pennsylvania Department of Environmental Protection Laboratory Accreditation Program (PA DEP). PA DEP accredits the laboratory for meeting requirements of the National Environmental Laboratory Accreditation Program (NELAP). The accreditations by A2LA and PA DEP/NELAP demonstrate the operation of a laboratory quality management system and technical competence for defined scopes of testing water.

For the State of New York, SPL holds New York State Department of Health(NY Lab ID No: 12024)  ELAP certification in Legionella testing in Nonpotable and Potable Water categories and secondary ELAP accreditation for HPC and Total Coliforms and E. coli. SPL is also registered in the State of Connecticut for Legionella testing in the Drinking Water, Non-Potable Water/Wastewater category (PH 0125).

SPL is also proficiency certified through the CDC Environmental Legionella Isolation Techniques Evaluation Program (ELITE) for testing all water types for Legionella by culture and participates in an international proficiency program through Public Health of England (PHE).

A2LA
The American Association for Laboratory Accreditation (A2LA) is a nonprofit, non-governmental, public service, membership society. A2LA provides comprehensive services in laboratory accreditation and laboratory-related training. In completing the A2LA evaluation process, SPL is accredited to perform the following tests (certificate No. 2847.01) on potable and non-potable environmental water and swabs:

TEST REFERENCE METHOD TEST METHOD
Environmental Culture Test: Legionella ISO 11731 SPL M01, SPL M04
Heterotrophic Plate Count Standard Methods for the Examination
of Water and Wastewater
9215E SPL M06
Environmental Culture:  Pseudomonas aeruginosa  ASTM International D 5246 SPL M02

NELAP
SPL is accredited by NELAP through the Pennsylvania Department of Environmental Protection (DEP Laboratory ID: 02-04660) to test drinking water for the following:

TEST METHOD PRIMARY
Heterotrophic Bacteria (Enumeration) SM 9215E Pennsylvania
Total Coliform and E. coli SM 9223 (Colilert) Pennsylvania

Connecticut

      • Certified for Legionella testing in Drinking Water/Non-Potable/Wastewater testing by the Connecticut Department of Health, (PH 0125)

New York

Legionella Proficiency Programs
Legionella culture and identification requires special knowledge and expertise not found in most microbiology laboratories. Our skill and experience give you absolute confidence that the results you receive from SPL are reliable. Since 2008, SPL has participated in Legionella proficiency programs:

        • The U.K. Public Health England (PHE)
          In 2007, no Legionella proficiency programs were available in the United States. That’s why SPL started participating in the international program. The program in Europe serves laboratories of the European Working Group on Legionella Infections, as well as other interested testing labs. Managed by the U.K. Public Health England (PHE), formerly Health Protection Agency (HPA), SPL is one of the few labs in the US that participates in this program.
          The PHE proficiency program is accredited by internationally recognized organizations and is used in more than 70 countries. The international organization of the European Working Group on Legionella Infections (EWGLI) recommends participation in the Legionella proficiency program provided by the PHE.
        • CDC-Elite Program
          The Centers for Disease Control began a Legionella proficiency program called the Environmental Legionella Isolation Techniques Evaluation (ELITE) program in April 2009.  The CDC had recognized the need for laboratories to be capable and competent in performing Legionella testing. Some laboratories have failed to detect Legionella and outbreaks have occurred because the institution was unaware of the presence of Legionella in their water system(s).

 

Accreditations and Proficiency
We are an accredited laboratory (A2LA and NELAP) and participate in national and international Legionella
testing proficiency programs, CDC-ELITE and PHE. SPL is also registered in Connecticut and New York for Legionella testing in potable and nonpotable water and also in New York for Heterotrophic Bacteria and Total Coliform and E.coli

 


GSA Contract Holder
SPL provides laboratory and consulting services to government organizations through the General Services Administration. Contact us for more information.

 


Measure of Uncertainty
A parameter associated with the result arising from random variation of measuring an analyte (organism) by some (specified) procedure is available for our LegionellaP. aeruginosa, and Heterotrophic Plate Count tests upon request.

Role of Environmental Surveillance in Determining the Risk of Hospital-Acquired Legionellosis: A National Surveillance Study With Clinical Correlations.  Stout et al. Infection Control Hospital Epidemiology 2007; 28:818-824.