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 have 19 The 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%CO2 Can promote growth, the optimal growth temperature is 35℃, slow growth,3 Only visible after the day 1~2mm Small circular bacterial colonies.
according to GB/T 40392-2021 The detection of Legionella in circulating cooling water requires inoculation of Legionella samples GVPC After agar, in 2.5%CO2, 36℃±1℃, relative humidity ≥95%Cultivate in the environment 10 Heaven, and in the 3 Starting 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 inoculated BCYE and BCYE-Cys Validate 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 on CO The dependence on gas environment suggests that the sample preparation process should also be considered 2.5%Conducted in a CO2 environment to avoid low air conditions CO The compensatory changes in stress caused by Legionella in the environment.
–Control gas workstation–
use Punmicro of Control gas workstation It 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 cavity CO 2 is stable 2.5%±0.1%The level; ▲ Internal cavity directional airway forced convection can ensure internal cavity CO The uniformity of the concentration of 2 can also ensure the uniform and stable temperature inside the cavity 36℃±0.5℃； ▲ The automatic transfer gate can pre prepare the gas environment during the sample transfer process, avoiding low external pressure CO The destruction of the internal gas environment by air ensures that the sample culture is in a continuous and stable state; ▲ Equipped with built-in HEPA Filter, intake end HEPA The 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 red 1~2mm Identifying 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 necessary 1~2mm The 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–
use Punmicro of ICON Automatic colony counter It 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; ▲AI Artificial intelligence technology can accurately distinguish and judge complex bacterial colony morphology, and 10 Complete 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.