How to deal with Legionella testing | Punmicro brings you the ultimate experience from cultivation to counting

Legionella bacteria（Legionella）Gram negative bacteria have been found to have19The species can cause human pneumonia, with Legionella pneumophila being the most susceptible to disease. Legionella has special nutritional requirements, requiring oxygen,2.5~5.0%CO2Can promote growth, the optimal growth temperature is35℃, slow growth,3Only visible after the day1~2mmSmall circular bacterial colonies.

according toGB/T 40392-2021The detection of Legionella in circulating cooling water requires inoculation of Legionella samplesGVPCAfter agar, in2.5%CO2，36℃±1℃, relative humidity ≥95%Cultivate in the environment10Heaven, and in the3Starting from the day, check the culture dish and at the end of the cultivation period, record the number of suspected Legionella colonies according to the typical morphology of Legionella colonies. Typical Legionella colonies are white, gray, blue, or purple in color, with a smooth surface morphology, or colonies that produce fluorescence under ultraviolet light irradiation. Suspected colonies are inoculatedBCYEandBCYE-CysValidate on the culture medium.

Question one/cultivate

In the entire inspection process, the formation of typical colonies of Legionella is a crucial step for the inspection results. However, although Legionella has strong resistance in natural environments, it is prone to the phenomenon of not forming typical colony morphology when separated from the optimal cultivation environment.

Considering the impact of Legionella onCOThe dependence on gas environment suggests that the sample preparation process should also be considered2.5%Conducted in a CO2 environment to avoid low air conditionsCOThe compensatory changes in stress caused by Legionella in the environment.

-Control gas workstation-

usePunmicroofControl gas workstationIt can achieve strict and stable operation and cultivation environment in the optimal environment for Legionella bacteria.

The gas control system with automatic feedback regulation can ensure the internal cavityCO2 is stable2.5%±0.1%The level; ▲ Internal cavity directional airway forced convection can ensure internal cavityCOThe uniformity of the concentration of 2 can also ensure the uniform and stable temperature inside the cavity36℃±0.5℃； ▲ The automatic transfer gate can pre prepare the gas environment during the sample transfer process, avoiding low external pressureCOThe destruction of the internal gas environment by air ensures that the sample culture is in a continuous and stable state; ▲ Equipped with built-inHEPAFilter, intake endHEPAThe filter and built-in ultraviolet lamp can ensure the cleanliness of the internal cavity and increase the biological safety protection for operators.

Problem Two/Counting bacteria

Throughout the entire inspection process, identifying typical Legionella colonies is a challenging task. Under visible light, the color of Legionella colonies exhibits variability, including white, gray, blue, purple, and even brown, gray green, and dark red1~2mmIdentifying small bacterial colonies poses a challenge for inspectors and is also an important source of result bias.

GB/T 40392-2021 also provides a method for identifying fluorescent colonies through UV light irradiation, but UV light has lethal effects on live bacteria and only allows a few seconds of irradiation time. However, in complex colonies, naked eye recognition is necessary1~2mmThe fluorescence of small bacterial colonies and accurate recording of the number of fluorescent colonies are difficult to complete in just a few seconds.

-Automatic colony counting-

usePunmicroofICONAutomatic colony counterIt can automatically and quickly identify and count fluorescent colonies.

Equipped with an automatic sample chamber door and using a completely black environment for detection, it can accurately obtain weak fluorescence signals of small bacterial colonies; Low power ultraviolet lamps serve as fluorescence excitation sources, significantly reducing the killing effect of ultraviolet light on live bacteria and effectively protecting bacterial activity; ▲AIArtificial intelligence technology can accurately distinguish and judge complex bacterial colony morphology, and10Complete full dish analysis within seconds without cutting the edges of the culture dish to avoid forced negative deviation; ▲ Comprehensive data generation function for images and reports, customizable testing reports in specialized formats for testing units.