Specimen Collection CSF - Lumbar Puncture Clean the puncture site with antiseptic solution and alcohol before needle insertion. Insert a needle with stylet at the L3-L4 or L4-L5 or L5-S1 interspaced. When the subarachnoid space is reached, remove the stylet and spinal fluid will appear in the needle hub. Slowly drain the CSF into the sterile leak-proof tubes. Three separate tubes are generally required for Microbiology, Hematology, and Chemistry testing. The second tube will generally go to Microbiology and the third to Hematology. Specimens for viral culture are sent on ice.