In performing a lumbar puncture, first the patient is usually placed in a left (or right) lateral position with their neck bent in full flexion and knees bent in full flexion up to their chest, approximating a fetal position as much as possible. It is also possible to have the patient sit on a stool and bend their head and shoulders forward. The area around the lower back is prepared using aseptic technique. Once the appropriate location is palpated, local anaesthetic is infiltrated under the skin and then injected along the intended path of the spinal needle. A spinal needle is inserted between the lumbar vertebrae L3/L4, L4/L5 or L5/S1 and pushed in until there is a "give" that indicates the needle is past the ligamentum flavum. The needle is again pushed until there is a second 'give' that indicates the needle is now past the dura mater. Since the arachnoid membrane and the dura mater exist in flush contact with one another in the living person's spine (due to fluid pressure from CSF in the subarachnoid space pushing the arachnoid membrane out towards the dura), once the needle has pierced the dura mater it has also traversed the thinner arachnoid membrane and is now in the subarachnoid space. The stylet from the spinal needle is then withdrawn and drops of cerebrospinal fluid are collected. The opening pressure of the cerebrospinal fluid may be taken during this collection by using a simple column manometer. The procedure is ended by withdrawing the needle while placing pressure on the puncture site. The spinal level is so selected to avoid spinal injuries.In the past, the patient would often be asked to lie on their back for at least six hours and be monitored for signs of neurological problems, though there is no scientific evidence that this provides any benefit. The technique described is almost identical to that used in spinal anesthesia, except that spinal anesthesia is more often done with the patient in a seated position.

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