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Microbiology is the study of microscopic, usually unicellular organisms that can develop to macroscopically visible colonies at appropriate growth conditions on the finest culture media. The main morphological and physiological fundamentally different groups are viruses, bacteria, actinomycetes, yeasts and molds.
Furthermore different test methods lead to the desired result. One differentiates between three common methods that are practiced in microbiological laboratories.
In this method the agar is liquefied first. For this purpose the tube or the bottle of agar is positioned in a boiling water bath and maintained until the agar is completely dissolved. The dissolved agar is then cooled to about 45 degrees celsius.
Then 1 ml of the sample to be examined is pipetted into a sterile petri dish. After addition of about 7 ml (at a 60 mm petri dish) or 15-20 ml (at a 90 mm petri dish) of the cooled agar both components are thoroughly mixed.
After the agar has solidified the incubation is carried out with the lid facing downwards. The incubation conditions (temperature and time) are guided by the agar nutrient medium type and the growth requirements of the targeted organisms. Finally the number of colonies is determined by counting the visible with 6- to 8-fold loupe colonies and should be defined per ml.
Similar to the pour plate method, the first step in the membrane filtration method is the liquefaction of the agar culture medium in a boiling water bath followed by cooling to 45 degrees celsius.
To prepare the agar plates the cooled agar is poured into sterile petri dishes. In this case about 7 ml for a 90 mm Petri dish and about 15-20 ml for a 60 mm petri dish are used. Then you can let the plates rest until the agar has solidified. Filtration of the test sample through a suitable membrane filter is carried out according to the manufacturer of the filtration system.
After the filtration process gently take the membrane filter with a sterile forceps from the frit and set it onto the agar surface without creating air bubbles. Incubation takes place with the lid facing down. The incubation conditions (temperature and time) are guided by the agar nutrient medium type and the growth requirements of the targeted organisms.
Open a pouch with ten pieces inside and remove the petri dish with pickled nutrient pad (NCP). The nutrient pad in the petri dish is then moistened with 3 to 3.5 ml of sterile, distilled or demineralized water. An optimal humidification level can be recognized by a significant excess liquid in the petri dish between NCP and shell edge.
Then open the sealed package of the membrane filter, remove the membrane using sterile forceps, place it on the frit of the filter holder and then install the filter funnel. Filter the specimen, rinse with sterile water or peptone and carefully aspirate with vacuum completely.
Lastly the membrane filter is carefully removed from the frit with sterile forceps, placed on the prepared nutrient pad without creating air bubbles and the petri dish is incubated with the lid facing upwards. The incubation conditions are dependent on the NCP-variety.