2. Remove the tube cultivation from its rack or jar with the left hand and ignite the cotton-wool plug by holding it to the flame of the Bunsen burner. Extinguish the flame by blowing on the plug, whilst rotating the tube on its long axis, its mouth directed vertically upward, between the thumb and fingers. (This operation is termed "flaming the plug," and is intended to destroy any micro-organisms that may have become entangled in the loose fibres of the cotton-wool, and which, if not thus destroyed, might fall into the tube when the plug is removed and so accidentally contaminate the cultivation.) 3. Hold the tube at or near its centre between the ends of the thumb and first two fingers of the left hand, and allow the sealed end to rest upon the back of the hand between the thumb and forefinger, the plug pointing to the right. Keep the tube as nearly in the horizontal position as is consistent with safety, to diminish the risk of the accidental entry of organisms (Fig. 68). 4. Take the handle of the loop between the thumb and forefinger of the right hand, holding the instrument in a position similar to that occupied by a pen or a paint-brush, and sterilise the platinum portion by holding it in the flame of a Bunsen burner until it is red hot. Sterilise the adjacent portion of the aluminium handle by passing it rapidly twice or thrice through the flame. After sterilising it, the loop must not be allowed to leave the hand or to touch against anything but the material it is intended to examine, until it is finished with and has been again sterilised. 5. Grasp the cotton-wool plug of the test-tube between the little finger and the palm of the right hand (whilst still holding the loop as directed in step 4), and remove it from the mouth of the tube by a "screwing" motion of the right hand. 6. Introduce the platinum loop into the tube and hold it in this position until satisfied that it is quite cool. (The cooling may be hastened by touching the loop on one of the drops of moisture which are usually to be found condensed on the interior of the glass tube, or by dipping it into the condensation water at the bottom; at the same time care must be taken in the case of cultures on solid media to avoid touching either the medium or the growth.) 7. Remove a small portion of the growth by taking up a drop of liquid, in the case of a fluid culture, in the loop; or by touching the loop on the surface of the growth when the culture is on solid medium; and withdraw the loop from the tube without again touching the medium or the glass sides of the tube. 8. Replace the cotton-wool plug in the mouth of the tube. 9. Replace the tube cultivation in its rack or jar.