Judul: Homogeneity and Stability of Freeze-Dried Cultures of Salmonella Typhimurium and Confirmation by real-time PCR
Abstrak: Biakan kering beku Salmonella Typhimurium sebagai objek uji profisiensi harus memiliki karakteristik homogen dan stabil. Viabilitas sel harus dipertahankan selama pendistribusian sehingga peserta uji profisiensi mendapatkan objek uji dengan performa yang sama. Penelitian ini bertujuan untuk menentukan konsentrasi kultur awal untuk mencapai limit deteksi (pada pencawanan) dalam pembuatan biakan kering beku S. Typhimurium, mengevaluasi tingkat homogenitas dan stabilitas biakan kering beku S. Typhimurium secara kualitatif dan kuantitatif di limit deteksi, dan mengonfirmasi biakan kering beku S. Typhimurium secara kualitatif dan kuantitatif menggunakan real-time PCR. Metode dalam penelitian ini adalah penentuan konsentrasi kultur awal yang menghasilkan limit deteksi (melalui pencawanan), uji homogenitas dan stabilitas, serta konfirmasi dengan real-time PCR. Krioprotektan yang bersifat stabil terhadap biakan kering beku S. Typhimurium yaitu susu skim 10% + trehalosa 4%. Setelah liofilisasi, viabilitas menurun dari konsentrasi awal 7 log CFU/mL menjadi 2,25 ± 0,04 log CFU/mL dan ditetapkan sebagai limit deteksi metode cawan. Hasil uji homogenitas dan stabilitas menunjukkan bahwa biakan kering beku homogen dan stabil pada standar deviasi uji profisiensi 25% dengan rata-rata sebesar 2,35 ± 0,17 log CFU/mL. Hasil pembacaan dengan real-time PCR menunjukkan konsentrasi DNA sampel sebesar 0,34 ng/μL (4,50 log copies) lebih besar daripada jumlah koloni hasil pencawanan (2,35 log CFU/mL). Hal tersebut terjadi akibat terdapat bakteri dalam kondisi viable but non-culturable (VBNC)., Freeze-dried cultures of Salmonella Typhimurium as proficiency test object must have homogeneous and stable characteristics. Cell viability must be maintained during distribution so that the proficiency test participants get the test object with the same performance. This research aimed to determine the concentration of the initial culture to reach the detection limits (on platting) in the production of freeze-dried S. Typhimurium, evaluate the level of homogeneity and stability of the freeze-dried S. Typhimurium qualitatively and quantitatively at the detection limits, and confirm the freeze-dried cultures of S. Typhimurium qualitatively and quantitatively using real-time PCR. The methods in this research were to determine the initial culture’s concentration that produces the detection limits (by platting), test the homogeneity and stability, and confirmation by real-time PCR. Stable cryoprotectant against freeze-dried culture of S. Typhimurium was 10% skim milk + 4% trehalose. After lyophilization, viability decreased from initial concentration of 7 log CFU/mL to 2.25 ± 0.04 log CFU/mL, which was defined as the detection limits of platting method. The homogeneity and stability test exhibited that freeze-dried culture was homogeneous and stable at a proficiency test’s standard deviation of 25% with an average of 2.35 ± 0.17 log CFU/mL. The real-time PCR exhibited that the sample’s DNA concentration was 0.34 ng/μL (4.50 log copies) greater than the number of colonies from platting results (2.35 log CFU/mL). This happens due to the presence of bacteria in a state of viable but non-culturable (VBNC).
Keyword: freeze-dried culture, homogeneity, real-time PCR, Salmonella Typhimurium, stability