Bakteri hipolitik rare-actinomycetes merupakan kelompok bakteri yang mampu hidup di habitat dengan kadar nutrien rendah. Kelompok bakteri tersebut sulit untuk diisolasi. Penelitian ini bertujuan untuk mengisolasi, mengkarakterisasi, dan mengidentifikasi isolat-isolat hypolithic rare-actinomycetes dari sampel tanah di bawah batuan kuarsit (S3.3) di kawasan geotermal Cisolok, Jawa Barat. Sampel tanah diisolasi menggunakan dua metode; 1. metode enrichment dengan medium R2A cair 10% (cycloheximide 100 ppm + sodium azide 60 ppm) pada suhu 30°C selama 5 hari, kemudian disebar di atas permukaan R2A 10% agar dan diinkubasi pada suhu 30°C dan 45°C. 2. Metode tanpe enrichment, sampel tanah langsung ditebar pada medium R2A 10%, ISP 1 10%, dan NA 10% (cycloheximide 100 ppm + sodium azide 60 ppm), kemudian diinkubasi pada suhu 30°C dan 45°C. Koloni yang tumbuh dipurifikasi sebanyak tiga kali. Karakterisasi fenotipik melalui pengamatan makroskopis, pengecatan Gram, uji katalase, uji pertumbuhan pada variasi suhu (25–55°C), dan uji pertumbuhan pada variasi medium agar (International Streptomyces Project 1, 2, 3, Reasoner’s 2A, modified Bennet’s, dan Nutrient Agar). Karakterisasi genotipik dilakukan berdasarkan data sequence gen 16S rRNA menggunakan metode Neighbor Joining (NJ), Minimum Evolution (ME), dan Maximum Likelihood (ML). Lima isolat dipilih dari total 25 isolat dengan morfologi makroskopis menyerupai actinomycetes. Hasil analisis homologi sequence menunjukkan bahwa; Isolat S3.3.1 memiliki homologi tertinggi dengan Micromonospora solifontis PPF5-17T (99,20%), S3.3.2 dengan Streptomyces thermocarboxydovorans DSM 44296 T (99,10%), S3.3.3 dengan Kribbella aluminosa HKI 0478T (98,65%), S3.3.4 dengan Actinomadura nitritigenes DSM 44137T (99,65%), dan S3.3.5 dengan Streptomyces spiralis NBRC 14215T (99,79%). Hasil karakterisasi fenotipik menunjukkan kelima isolat merupakan bakteri Gram-positif, berfilamen, dengan warna koloni tangerine, white, hingga ivory, tumbuh pada seluruh variasi medium, menghasilkan soluble pigment pada medium MBA, dan memiliki aktivitas katalase. Rentang suhu pertumbuhan beragam untuk setiap isolat. Dua dari lima isolat tersebut (S3.3.2 dan S3.3.3) perlu dilakukan karakterisasi lebih lanjut karena berpotensi sebagai spesies baru.

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The hypolithic rare-actinomycetes bacteria are a group of bacteria capable of surviving in habitats with low nutrient availability. These bacteria are notoriously difficult to isolate using conventional methods due to their slow growth and sensitivity to environmental conditions. This study was conducted with the aim of isolating, characterizing, and identifying hypolithic rare-actinomycetes from soil samples collected beneath quartzite rocks (S3.3) in the geothermal area of Cisolok, West Java. Soil samples were isolated using two methods: 1. the enrichment method with 10% (w/v) liquid R2A medium (cycloheximide 100 ppm + sodium azide 60 ppm) at 30°C for 5 days, then spread onto the surface of 10% R2A agar and incubated at 30°C and 45°C. 2. The non-enrichment method, where soil samples were directly spread on 10% R2A, 10% ISP 1, and 10% NA media (cycloheximide 100 ppm + sodium azide 60 ppm), then incubated at 30°C and 45°C. Colonies that grew were purified three times. Phenotypic characterization was conducted through macroscopic observation, Gram staining, catalase test, growth test at temperature variations (25–55°C), and growth test on various agar media (International Streptomyces Project 1, 2, 3, Reasoner’s 2A, modified Bennet’s, and Nutrient Agar). Genotypic characterization was carried out based on 16S rRNA gene sequence data using the Neighbor Joining (NJ), Minimum Evolution (ME), and Maximum Likelihood (ML) methods. Five isolates were selected from a total of 25 isolates with macroscopic morphology resembling actinomycetes. The results of sequence homology analysis showed that: isolate S3.3.1 had the highest homology with Micromonospora solifontis PPF5-17T (99.20%), S3.3.2 with Streptomyces thermocarboxydovorans DSM 44296T (99.10%), S3.3.3 with Kribbella aluminosa HKI 0478T (98.65%), S3.3.4 with Actinomadura nitritigenes DSM 44137T (99.65%), and S3.3.5 with Streptomyces spiralis NBRC 14215T (99.79%). The results of phenotypic characterization showed that all five isolates were Gram-positive, filamentous bacteria, with colony colors ranging from tangerine, white, to ivory. They were able to grow on all tested media, produced soluble pigment on MBA medium, and exhibited catalase activity. The temperature growth range varied for each isolate. Two of the five isolates (S3.3.2 and S3.3.3) require further characterization as they have the potential to represent new species.