This update is written by the Ustav Molekularnej Biologie, Slovakia, one of the SAGA Project partner, who describe their preparation for their trip to the Historical Archives of the EU in Florence Italy where they are tasked with analysing archive materials for bacteria, fungus and dna.
When analysing any object/environment, the first key step is the sampling. This is preceded by thorough preparation of complete material, mainly if sampling is not carried out directly in the laboratory, but in an external environment. It is necessary to decide what types of samples we want to analyse, whether objects or the surrounding environment. Because on the “site” we cannot just run back to the laboratory for something we have forgotten. So, logistics are extremely important. This is how we proceeded before our trip to the first archive within the SAGA project, to the Historical Archives of the European Union in Florence (IT).
Since we knew that we would analyse archive items and also the air environment, we planned the necessary material accordingly, especially focusing on bacteria and moulds. For sampling from fragile, delicate material as documents, negatives, pictures etc. we commonly use sterile disposable cotton swabs and nitrocellulose membranes, which we gently pass over the object, then the cotton swab/membrane are carefully stored and subjected to further analysis in the laboratory. If the given object allows, a special dermatological adhesive tape is used, which is gently applied onto the object/place and is then immediately placed on sterile glass slides and kept in a box until arrival to laboratory.
For air sampling, we can proceed in two ways:
1) sampling into a sterile solution or
2) direct suction of air on specific culture media.
The first option requires a biosampler (impinger), which captures airborne microorganisms in 20 ml of phosphate buffer. The suspension is then transferred to the laboratory for further analysis. The second air sampling approach utilises an air-sampler, coupled to specific microbiological media prepared in advance. In this case, agar media specific for this purpose are selected and prepared: LB (Luria Bertani) and R2A for bacteria; DRBC (Dichloran Rose Bengal Chloramphenicol) and PDA (Potato Dextrose agar) for fungi.
The required weight from the given powdered culture media is mixed with distilled water and sterilised, to avoid any possible microbial contamination.
Sterilisation is ensured by autoclaving, where sterilised media are exposed to 120 ˚C for 20 minutes. To be sure that the media are sterile, we use the sterilisation tape to mark them, the colour changes of the tape attests the successful sterilisation process.
After the sterilisation process it is necessary to cool media to about 50°C before the next manipulation. For ensuring the specific growth of moulds and bacteria, the media for moulds are supplemented with an antibiotic, while in the media for bacteria an antimycotic is added. If these antimicrobial compounds are added to a hot medium, the temperature would destroy them and therefore they would not fulfil the desired function.
When the agar media have cooled down and adequate amounts of prepared antibiotic/ antimycotic is added (according to the desired concentration), the medium must be well mixed and poured in an amount of 20 ml for each Petri dishes. All of this is done in a sterile laminar box. Such prepared plates are cooled down and solidify due to the agar present in the medium.
As you notice, the word “sterile” is mentioned everywhere. All the used material must be sterilised, prepared in a sterile environment, to be sure that what we isolate is really isolated from the analysed object/environment. Therefore, even when sampling in terrain, we use laboratory gloves, sterile swabs, sterile microscope slides, sterile scissors, sterile tweezers, sterile solutions, 96% ethanol, sterile falcons, sterile equipment and sterile media themselves. This entire preparation takes almost 5 days and took place under the hands of our skilled laboratory assistant Katarína.
The collected samples will be further processed in our laboratory, either by culture-dependent approach, but also by modern culture-independent methods (DNA-based approaches)
After preparing all the necessary materials, another question came up. How to transport everything to Florence? Our skilful PhD. students Dominika and Nikola got involved here and, together with a lot of bubble wrap and 2 suitcases, they safely packed what was needed. Now we just have to hope that everything will arrive to destination in good conditions.