Introduction to Legionella Detection Methods

Legionella is an environmentally infectious pathogen that causes acute respiratory infections, namely legionellosis. Legionella is widely distributed, and various water sources such as water supply systems for hospitals and hotels, and cooling systems for air conditioners are easy to breed and contaminate Legionella, and cause the risk of Legionella infection. Legionella may be present in water-related areas.

Legionella transmission and infection have the following characteristics:

1. Infected by air and water, the spread is seasonal, and more than 50% of cases occur in the summer.

2, breeding in the hospital, hotel water supply system, through the faucet, bathroom spread.

3, through the air conditioning cooling system, spread by the vents and infected objects.

Legionella Gram-negative bacilli, obligate aerobic, no spores, no capsules, Legionella requires cysteine ​​and iron, and without cysteine, Legionella cannot grow. Biochemical characteristics include no fermentation or oxidation of sugar, no reduction of nitrate, oxidase test negative or weak positive, urease negative, the optimum pH for Legionella growth is 6.9-7.0, 2%-5% CO2 can promote part Growth of the strain. Legionella grows slowly and is easily covered by other colonies. The colonies of Legionella are diverse in color, usually white, gray, blue or purple, and can also be dark brown, grayish green, and deep red. The colonies are neat, the surface is smooth, and there is fluorescence under the UV lamp. Legionella is currently divided into 64 serotypes and 42 species.

Second, the detection method of Legionella---ISO11731:1998

1 Scope

This standard describes a method for culturing Legionella for the isolation and estimation of the number of Legionella in environmental samples. This method is applicable to all environmental samples, including drinking water, industrial water and natural water, as well as sediment, sediment and clay/lime related samples.

2. Medium and reagents

2.1: CP0020 BCYE plate (legionella growth plate)

2.2: P0030 BCYE-CYS plate (BCYE without L-cysteine ​​plate)

2.3: CP0040 GVPC selective plate (legionella selective plate)

2.4: Acid treatment buffer

3. sampling

3.1 sampling container

The water sample can be made of glass, polyethylene or the like. Containers that have been used before should be cleaned, dehydrated, and autoclaved at 121 °C for 15 min. If the container cannot withstand autoclaving, it should be treated in flowing hot water or flowing steam above 70 °C for at least 5 min.

3.2 Samples containing biopreservatives

If the water sample contains or is considered to contain an oxidizing biopreservative, it should be neutralized by adding an inactive reagent at the time of sampling or prior to sampling. If the water sample contains a disinfectant, the neutralizing agent should be added before or after sampling. The chlorine or oxidizing disinfectant can be used as a neutralizing agent with sodium thiosulfate or potassium thiosulfate. In principle, the laboratory should perform microbiological analysis as soon as possible after receiving the water sample, preferably on the day of sampling, especially for samples known to contain biological preservatives. Therefore, it is recommended that the interval from sample collection to preparation of the concentrated sample is preferably 2d, no more than 5d, and no longer than 14d.

3.3 Sample transportation

Samples should be transported at 6-18 ° C. Heat and light should be avoided. It is best to send the samples to the designated laboratory within 1 day and no more than 2 days.

4. sample

4.1 General

If the amount of Legionella in the liquid sample is estimated to exceed 105 CFU/L, direct plating can be used, that is, directly applied to the GVPC plate. Concentration techniques are required to ensure detection of Legionella in samples below this amount. In order to concentrate the water sample, membrane filtration (4.2) or centrifugation (4.3) may be employed.

4.2 membrane filtration method

If the sample is cloudy, opaque or colored, centrifugation should be used. Membrane filtration was carried out using an MFS membrane filtration apparatus using a sterile white matrix black membrane having a diameter of 47 mm, a pore diameter of 0.22 μm and a 0.45 μm. The filtered membrane should be placed in a sterile container with a lid, cut with sterile scissors, and added with 5 ml to 25 ml of sterile diluent or sterile deionized water for at least 2 min. The container can also be placed in the ultrasound for 2-10 minutes.

4.3. Centrifugation

Take 200ml sample in a centrifuge bottle of 300-500ml volume, centrifuge at 6000g for 10min or 3000g for 30min, keep the temperature at 15-25 °C, discard the supernatant, suspend the sediment in 2-20ml sterile diluent or sterile Deionized water.

to cultivate

5.1. The prepared sample (concentrated or unconcentrated) is divided into 3 parts, 1 part is not treated; 1 part is heat-treated; 1 part is subjected to acid treatment.

5.2 Heat treatment: Add 1±0.5ml sample in a sterile container and treat in a water bath at 50±1°C for 30±2min.

5.3. Acid treatment: Add 1-10ml sample in a screwed container, centrifuge at 6000g for 10min or 3000g for 30min, use a sterile tip to remove half of the supernatant, resuspend the residue by vortex and treat with acid. The buffer fills the original volume. The mixture was allowed to stand for 5 ± 0.5 min.

5.4. Gram staining: The colony grows slowly, and the colonies growing the next day can be discarded directly. Legionella colonies are diverse in color, usually white, gray, blue or purple, and can also be dark brown, grayish green, and deep red. The colonies are neat, the surface is smooth, and there is fluorescence under the UV lamp. Legionella is a Gram-negative bacillus.

5.4. Colony verification: select several suspicious colonies from each plate, inoculate BCYE, BCYE-Cys plates and blood plates for subculture. Any bacteria that grow on BCYE plates but not grow on BCYE-Cys plates are likely It is Legionella.

5.5. Legionella count: The plates with the highest number of confirmed bacteria were selected from the three GVPC plates, and divided by the concentration multiple, as an estimate of the number of Legionella.