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Legionella pneumophila – Guidelines for Laboratory Interpretation

Legionella pneumophila is a Gram negative, aerobic bacteria that is characterized as an opportunistic pathogen. It is the cause of Legionnaires’ Disease, a severe form of pneumonia and, it is the cause of Pontiac fever, a non-pneumonic form of L. pneumophila infection. Legionella spp.’s mode of transmission is through aerosols or aspiration of contaminated water.

The Public Health Agency of Canada has posted an informative pathogen safety data sheet on Legionella pneumophila which can be viewed at:

http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/legionella-eng.php

Legionella spp. can be isolated from numerous places. Samples should be transported to the lab immediately and kept cool in sterile containers. Chlorinated water should be treated with sodium thiosulfate to neutralize the chlorine. Water samples should be brought to the lab ideally within a few hours of sampling and should be stored at room temperature (20 + 5°C) in the dark.

Examples of sampling areas:

A. Potable water (1 L in sterile container):

– Municipal water (hot water tanks), hoses, well water, faucets, water fountains, sinks, respiratory therapy equipment, showers, eye washers, bottled water or cooler water systems.

Note: Collect 2 water samples from shower heads, tap water faucets, respiratory therapy equipment, water fountains, etc. First a pre-flush or first draw sample by draining the first 1 L of water from the faucets or flush drains into a bottle. Allow the water to run for approximately 1 minute and collect the second draw of 1 L of water.

B. Non-potable water (250 mL in sterile container):

– Cooling towers, humidifiers, decorative fountains, HVAC systems, dire sprinkler systems, storage tanks, basin water, hot tubs, surface water from reservoirs, misters.

C. Swabs:

– Faucet aerators, condensate pan, shower heads, condenser coils.

D. Bulk samples:

– Sludge from the bottom of a condensate pan scraped off a cooling coil.

E. Legionella Air Sampling requires the following which can be provided by MBL Inc.:

Latex gloves
70% Isopropyl alcohol to sterilize sampler after every other sample
SKC QuickTake 30 pump with Andersen N-6 single stage Impactor
BCYE (Buffered Charcoal Yeast Extract) agar

Interpretation of Legionella results:

Table 1. The following chart can be used as a guideline for interpretation of Legionella spp. results received from the laboratory.

Action (see below)

Cooling tower

100

1,000

Domestic water

100

Humidifier

Action 1: Prompt cleaning and/or biocide treatment of the system.
Action 2: Immediate cleaning and/or biocide treatment. Take prompt steps to prevent employee exposure.

(Source: From OSHA Technical Manual (Section III: Chapter 7, Legionnaires’ Disease), which was adapted from George K. Morris, PhD, and Brian G. Shelton, Pathcon Technical Bulletin 1.3, Legionella in Environmental Samples: Hazard Analysis and Suggested Remedial Actions, June 1991)

For more information on Legionella testing or to find out about our other bacteria and mold testing services, please contact Mold & Bacteria Consulting Laboratories, call our Ontario, Mississauga Office at 905-290-9101 or the British Columbia, Burnaby Office at 604-435-6555.

Mycotoxins in Indoor Environment

Lyn Gandham

Molds or fungi can be found in at least small amounts almost anywhere due to their release of tiny spores. Spores can be hazardous to human health and potentially cause allergic reactions, fungal infection (mycosis) or release mycotoxins. Mycotoxins are secondary metabolites (toxic chemical products) that can be present with the spores, the mycelium of fungi and even the surface where the fungi grow.

Not all fungi however produce mycotoxins and, specific environmental conditions are required for these potent compounds to be produced. Increased mycotoxin biosynthesis within a fungal spore is affected by the following environmental conditions: temperature, water activity and pH. Other factors that affect susceptibility to mycotoxins vary by individual. Such factors include the length and frequency of exposure, the concentration of the mycotoxins, the age, health and sex of the exposed individual, etc.

At a high enough exposure, certain mycotoxins can be harmful or even lethal to both humans and animals. They can enter the body through ingestion, skin exposure and inhalation. Many mycotoxins are allergenic, immunosuppressive, carcinogenic, or can cause kidney toxicity. However, extreme exposure to mycotoxins is rare even in residences with serious mold problems. Further, some mycotoxins can be beneficial to humans; such is the case of penicillin which is used to treat many bacterial infections. The major groups of mycotoxins are: Aflatoxins, Citrinin, Ergot Alkaloids, Fumonisins, Ochratoxin, Patulin, Trichothecenes, and Zearalenone.

Exposure to mycotoxins has a greater likelihood of occurring in areas of the world that do not practice proper food handling and storage of food and, do not have proper government screening and regulation programs. They can appear in the food chain through crop fungal infections or, crops eaten by livestock as feed or directly by humans. Many mycotoxins are also highly resistant to decomposition, digestion, and even heat treatment which allow them to persist in meat and dairy products. Most control methods involve high-performance liquid chromatography (HPLC) techniques to access mycotoxin levels in food. Examples for food products that are currently tested include peanuts and peanut products, tree nuts, corn and corn products, cottonseed, and milk.

Preventative measures such as good agricultural practices and adequate drying of crops post-harvest can be taken to control exposure. Ongoing biotechnology research is also being performed into using bio-control agents, developing crops that have enhanced antifungal genes, and targeting regulatory mycotoxin genes.

Mycotoxins in the indoor air environment:

Mycotoxin detection in indoor air is not well documented and, it is therefore difficult to identify their presence. Current research suggests the concentrations present in a normal home, office or school are too low to trigger an immune response. Examples of molds that are known to have the potential to produce myxotoxins include Alternaria, Aspergillus (many forms), Penicillium, and Stachybotrys.

Of course, the best preventative measure in preventing myxotoxin production in the indoor air is to prevent mould growth. For more information and to find out about our mold testing services, please contact Mold & Bacteria Consulting Laboratories, call our Ontario, Mississauga Office at 905-290-9101 or the British Columbia, Burnaby Office at 604-435-6555.

References:

Bennett JW, M Klich. 2003. Mycotoxins. Clin Microbiol Rev 16(3):497-516.

Jarvis BB, Miller JD. 2005. Mycotoxins as harmful indoor air contaminants. Appl Microbiol Biotechnol 66(4):367-372.

Keller NP, Turner G, Bennett JW. 2005. Fungal secondary metabolism-from biochemistry to genomics. Nat Rev Microbiol 3(12):937-947.

Richard JL. 2007. Some major mycotoxins and their mycotoxicoses-an overview. Int J Food Microbiol 119(1-2):3-10.

Robbins CA, Swenson LJ, Nealley ML, Gots RE, Kelman BJ. 2000. Health effects of mycotoxins in indoor air: a critical review. Appl Occup Environ Hyg 15(10):773-784.

Shephard GS. 2008. Determination of mycotoxins in human foods. Chem Soc Rev 37(11):2468-2477.

Turner NW, Subrahmanyam S, Piletsky SA. 2009. Analytical methods for determination of mycotoxins: a review. Anal Chim Acta 632(2): 168-180.

The Indoor Mold Is An Early Warning Device

Jackson Kung'u

The mold (or mould, in the Queen’s English) has been the victim of a bad press. Mention the word and it conjures up an image of something rotting or in the process of decomposition. And yet, were it not for the mold that inspired the discovery of penicillin millions of lives would have been lost. If you love cheese, wines such as Auslese, Sauternes and Tokaji or the protein rich meat-like product called quorn, you can thank the mold for that, too. The mold is a network of thread-like filaments. Some molds are good, some are bad. One of the more undesirable types is the indoor mold. Apart from being a potential health hazard, indoor mold is unsightly. You usually find them in damp sections of the building such as the attic or basement, or in the kitchen and bathroom. The mold is also likely to raise its unsightly presence in parts of the home that have been flooded or have poor ventilation.

Indoor mold is a health hazard

The indoor mold can cause health problems, especially with those suffering from asthma. A greenish-black, slimy variety known as Stachybotrys chartarum has earned the name “toxic mold” and grows on wet organic material rich in cellulose such as wood or paper (but not on concrete or tile). It may cause severe problems for humans as well as pets, and is one of the causes of “sick building syndrome”. This indoor mold can produce allergic reactions like watery eyes, headaches, frequent sneezing and coughing.

Indoor mold is an early warning device

The upside is that an indoor mold can serve as an early warning device. It tells you that an area of the home is not properly ventilated, has excess moisture and that the air quality is inferior. You can then take steps to reduce moisture in the area and remove materials that cause mold growth.

Cleaning up indoor mold

The mold is everywhere and can survive under all sorts of conditions, even in places like the refrigerator. Some molds are resistant to anti-fungal soap or highly-acidic solvents. Therefore, when cleaning up a mold-infested area, you should always wear proper protection like a respirator mask, rubber gloves and the right clothing.

The key to cleaning up an area with indoor mold is to solve the moisture problem. Dry up the excess moisture and remove all the materials that have been infested by the mold. Get rid of items like carpets and wallpaper that cannot be cleaned and dried. First spray the area with water to keep down the dust and debris while you are removing the items. The materials should then be sealed in a double-bag and the bag wiped clean to prevent the mold from spreading to other areas.

If the area with indoor mold cannot be removed, it should be cleaned with soap or detergent. After that, it helps to disinfect the area with a diluted bleach solution (care should be taken when using bleach. It is a hazard too). Do not sand or scrape the surfaces and avoid using a hammer to remove drywall that is mold-infested.

If the affected area is larger than 10 square feet, you may need to turn the job over to a professional mold remediator. You can find companies that specialize in restoring homes damaged by fire and water whose services include indoor mold remediation.

If you would like to receive more information regarding our laboratory services or our prices, please call us at 905-290-9101 (Ontario) or 604-435-6555 (British Columbia). You can also complete the form below and we shall get back to you within 2 business days.

Bacteria School Science Projects

Iveta Kukurova

Hi kids, do we look familiar to you? You could use us in your school science projects.

We are bacteria and since we are very small and can only be seen under very powerful microscopes, people call us microorganisms. The cartoons you see above show how we could look like in your imagination. one reason why you should consider us in your school science projects is that we grow very fast and hence in a short time, you’ll be done with your project.

As you use us in your science projects, remember some bacteria are good and can be beneficial to your body; some are bad and can make you very sick…

Did you know that you can find bacteria everywhere? You can find us in the air, in the soil, on your hands, clothes, shoes, on your cell phone, or computer, on the door knobs, on money, on the handles on the bus; on the animal fur… there are so many places you can find bacteria on.

When we are grown on special media in a laboratory we may look like the pictures below :

If you would be interested in growing us for your school project MBL Laboratories can help you. Explore the amazing world of bacteria growth by doing your science project at MBL.

If you are looking for an elementary, middle or high school project ideas in the life science –environmental microbiology – MBL Laboratories are ready to help you!

MBL Laboratories can help you with bacteria testing in different matrices:

If you have any other ideas on the testing for your original science project simply give us a call at 905 290 9101 (1-866-813-0648) or email us at info@moldbacteria.com. We will be more than happy to assist you.

Hey, the cartoons for the bugs above were drawn by Daniel Gaczol. We greatly appreciate his help.

Heterotrophic Plate Count: What is HPC and when is the right time to use it?

Lyn Gandham

Heterotrophs are a group of microorganisms (bacteria, moulds and yeasts) that use organic carbon sources to grow and can be found in all types of water. In fact, the majority of bacteria found in drinking water systems are considered heterotrophs. Heterotrophic plate count (HPC) is a method that measures colony formation on culture media of heterotrophic bacteria in drinking water. Thus the HPC test (also known as Standard Plate Count) can be used to measure the overall bacteriological quality of drinking water in public, semi-public and private water systems.

The Limitations of HPC:

As stated by the Health Canada guidelines on HPC testing, “HPC results are not an indicator of water safety and, as such, should not be used as an indicator of potential adverse human health effects.” The World Health Organization (WHO) states that methods such as coliform testing are better indicators than HPC to test the sanitary conditions of water.

The HPC method does not indicate the specific heterotrophic bacteria present or their sources. Instead, HPC testing indicates the culturable organisms present, which could be as low as 1% of the total bacteria present. There are several factors that affect the genera of bacteria and their level of presence recovered by HPC. These factors include the type of medium used to grow the bacteria, what temperature is used for incubation, how long the plates are incubated, where the water sample was collected, the time of year and the age of the sample. It is also important to note that the concentrations and types of bacteria that are recovered at the same sampling location can vary over time.

Typically high levels of HPC bacteria in a distribution or plumbing system result from bacterial regrowth where bacteria that resisted treatment grow or those that were injured during treatment recover.

How do we interpret HPC results?

For HPC, regulations set out by Health Canada are followed under the Canadian Drinking Water Quality Guidelines: “No maximum acceptable concentration (MAC) is specified for heterotrophic plate count (HPC) bacteria in water supplied by public, semi-public, or private drinking water systems. Instead, increases in HPC concentrations above baseline levels are considered undesirable.”

In other words, sudden increases in HPC above levels typically seen may indicate a change in the quality of raw water or, that bacterial regrowth has occurred in the distribution system or plumbing. When steady increases of HPC are seen over time, this indicates a gradual decline in raw water quality.

As stated by the National Primary Drinking Water Regulations established by the U.S. EPA a “lower concentration of heterotrophic bacteria in the drinking water is linked to a better maintenance of the treatment and distribution systems.” According to these regulations, treatment techniques should aim to control HPC concentrations in surface waters and groundwaters influenced by surface waters to less than 500 CFU/mL (using standard methods). Note: “This is not a health-based standard, but reflects the concern that at concentrations above 500 CFU/mL, heterotrophic bacteria can interfere with some total coliform and E. coli recovery methods.”

Importance of HPC counts:

High HPC counts indicate ideal conditions for bacterial regrowth and should be corrected. Bacterial regrowth can lead to pipe corrosion, encourage slime growth, increase the need for disinfectants, cause foul-tasting water, and harbour secondary respiratory pathogens (ex. Legionella). Thus, HPC can be used as a marker for the underlying causes of some aesthetic problems (WHO, 2002).

Does having a positive HPC results mean the overall water quality is poor?

No, not necessarily.

Unlike other indicators, such as Escherichia coli or total coliforms, low concentrations of HPC organisms will still be present after drinking water treatment. In general, water utilities can achieve heterotrophic bacteria concentrations of 10 colony-forming units (CFU) per millilitre or less in finished water.

What are the health effects associated with HPC levels?

At an international meeting of experts in Geneva, Switzerland, it was concluded that heterotrophic bacteria in drinking water is not a health concern to the general public. However, some bacteria present in a heterotrophic population are opportunistic pathogens that could infect individuals with weakened immune systems.

“Heterotrophic bacteria belonging to the following genera have been associated with opportunistic infections: Acinetobacter, Aeromonas, Chryseobacterium (Flavobacterium), Klebsiella, Legionella, Moraxella, Mycobacterium, Serratia, Pseudomonas, and Xanthomonas. These organisms have been mainly associated with nosocomial (hospital acquired) infections, including wound infections, urinary tract infections, post-operative infections, respiratory infections, and infections in burn patients.”

What methods can be used to measure HPC?

There are 3 methods used for routine testing of heterotrophic bacteria:

Pour plate method (least desirable method because embedded colonies are slower growing and microaerobic environment is not ideal for growth and, bacteria could undergo heat shock during plating).
Spread plate method (heat shock is eliminated by using solidified agar but only a small volume (0.1-0.5 mL) can be tested).
Membrane filtration method (most flexible method because volumes upto 10L can be tested, heat shock is eliminated by membrane filter, and it is ideal for HPC less than 10 CFU/mL but multiple dilutions may be required to count colonies on filter).

Here at MBL, we use membrane filtration to measure heterotrophic plate count. If you need to determine the level of heterotrophic bacteria, fecal coliforms, E.coli, total coliforms, Legionella or other type of bacteria in water call our Mississauga office at 905-290-9101 or our Burnaby office at 604-435-6555 or, our toll free number at 1-866-813-0648.

References:

Health Canada (1996) Guidelines for Canadian Drinking Water Quality, 6th edn. Minister of Public Works and Government Services Canada, Ottawa, Ontario

Heterotrophic plate counts and drinking-water safety: The significance of HPCs for water quality and the human health. Edited by J. Bartram, J. Cotruvo, M.Exner, C. Fricker, A. Glasmacher. Published on behalf of WHO by IWA Publishing © WHO 2003: http://www.who.int/water_sanitation_health/dwq/HPCFull.pdf

Guidelines for Canadian Drinking Water Quality: Guideline Technical Document: Heterotrophic Plate Count (Prepared by the Federal-Provincial-Territorial Committee on Drinking Water of the Federal-Provincial-Territorial Committee on Health and the Environment (February 2006). http://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/heterotrophic-heterotrophes/index-eng.php

For access to the BC Drinking Water Protection Act and the BC Drinking Water Protection Regulation, please refer to the following website: http://www.health.gov.bc.ca/protect/dw_index.html

Guidance on the Use of Heterotrophic Plate Counts in Canadian Drinking Water Supplies (Document for Public Comment Prepared by the Federal-Provincial-Territorial Committee on Drinking Water – Consultation period ended May 27, 2011): http://www.hc-sc.gc.ca/ewh-semt/consult/_2011/Heterotrophic-heterotrophes/Heterotrophic-heterotrophes-eng.php

US EPA (1989) 40 CFR Parts 141 and 142 Drinking Water; National Primary Drinking Water Rules and Regulations; filtration, disinfection; turbidity, Giardia lamblia, viruses, Legionella, and heterotrophic bacteria; final rule. US Environmental Protection Agency. Fed. Regist. 54(124), 27486–27541.

US EPA (2001) National Primary Drinking Water Standards. EPA 816-F-01-007, March, US Environmental Protection Agency, Washington, DC (www.epa.gov/safewater).

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