Chapter 29 Sub-culturing of Bacteria, Fungi and Actinomycetes Microbial growth is defined as increase in number and/or biomass. All microorganisms require food, oxygen, moisture, and space for growth. Like any other living organisms, they age, and their growth is inhibited in the absence of food, space, oxygen, etc. In order to sustain any microbial culture in laboratory conditions, sub-culturing is required. Sub-culturing is a procedure of transferring of microor- ganism into fresh nutritive medium from its stock culture. It includes transfer of culture from slant to slant, slant to plate, plate to plate, plate to slant, solid medium to broth, and broth to solid media. Sub-culturing is done to maintain culture in its active form (prolong- ing life and/or increase the number of cells) for varied applications. Materials: Petri plates, filter paper, medium slant or plate contain- ing pure culture of microorganism, Bunsen burner, 70% ethanol, sterilized cotton, Sabouraud dextrose agar (SDA) or any other medium, inoculation loop (for culturing of bacteria and actinomy- cetes) and inoculation needle (for culturing of fungi), fresh medium plates/slants/broth, laminar airflow. Procedure: 1. Clean the surface of laminar airflow with 70% ethanol, close hood, and switch on the UV light. 2. Switch off the UV light after 10 min and open fluorescent light. 3. Switch on blower and open the hood and wipe the working surface with 70% ethanol. 4. Allow the surface to get dry for 2–3 min and ignite the Bunsen burner. 5. Dip the wire of inoculation loop (or needle) in 70% ethanol. Aakanchha Jain et al. Basic Techniques in Biochemistry, Microbiology and Molecular Biology: Principles and Techniques, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-4939-9861-6_29, © Springer Science+Business Media, LLC, part of Springer Nature 2020 101