5. Inoculation methods
Preparation of spore suspension
Inoculation methods for packaging material need to be carried out in a professional way in order to achieve results that are indisputable.
General consideration regarding spores
- Spores of bacteria's or moulds should be from a germ which is listed in an official type culture collection and commercially available.
- For spore suspensions, there should be different, independent suppliers available.
- The spores need to be prepared in a water suspension free from salts and organic material that could provide a residue during drying.
- Spore dilutions need to be prepared in pure sterile water. No ringer solution may be used.
- Regarding spores of moulds, they need to be free from organic material coming from cultivation. Normally the spore suspensions are washed 3-4 times and centrifuged in order to remove any kind of foreign matter.
There are three different methods for the inoculation of the packaging material:
Inoculation with a single dot:
The dot has a volume of 10µl and is placed with a micro pipette on the surface of the material that needs to be tested. When starting from a defined concentrated spore suspension (e.g. 108 spores/ml), dilutions will be prepared in order to have the final correct concentration level of spores. The inoculation area will be marked with a circle.Inoculation with multi dots:
The dots will have a total volume of 10ìl and are placed with a micro pipette on the surface of the material that needs to be tested. When starting from a defined concentrated spore suspension (e.g. 108 spores/ml), dilutions will be prepared in order to have the final correct concentration level of spores. The sum of the individual dots concentration will be equal to the concentration level that is required. If the final level is 10,000 spores and 10 dots are placed on the material, each dot will have an amount of 1,000 spores. The inoculation area will be marked with circles.Spray inoculation:
A sprayer that delivers a constant volume and a homogeneous fine film will be used to inoculate the inside of the packaging material. The difficulty is to carry out constantly the same distribution of inoculation. Training of the operators needs to be carried out.