7Streaking and flaming Flame the loop to sterilize it and let cool. Position the plate so that the spot of inoculum is nearest the hand not holding the loop (the opposite hand).Lift the plate lid with the opposite hand; just enough to get the loop inside and touch the loop to the inoculum spot. It is often helpful to treat the inoculating loop as if it were a pencil - steadying the loop by resting the heel of the hand against the lab bench.Move the loop back and forth across the spot and then gradually continue toward the center of the plate as you sweep back and forth. Use a very gentle and even pressure.When creating each phase, do not worry about keeping each pass across the plate separate from previous ones.When about 30% of the plate has been covered by the first streaking phase, remove the loop and flame sterilize it.Repeat the above procedure for the second phase, but this time pick up some inoculum by crossing into the first phase 2-3 times and then not passing into it again (Figure 1-5).Repeat as necessary for the third and fourth phases. After streaking the plate, flame sterilize the loop before setting it down.
11Transfer 21. Wrap fingers of non dominant hand around the culture tube containing broth for transfer 2. Using the pinkie finger of your dominant hand twist the red cap from the tube. Hold in your pinkie and do not place it on the counter3. Pass the mouth of the culture tube across the flame4. Direct the inoculating needle into the broth.5. Flame the mouth of your broth culture tube and replace the cap. Place it in your rack6. Pick up the slant in your non dominant hand7. Twist off the red cap8. Flame the mouth of the slant tube9. Direct the inoculating needle into the tube and “ stab” the agar in the base( butt)10. Withdraw on the entry line and when you reach the surface make a simple streak along the face.11. Flame the mouth of the tube and replace the cap.12. Flame your inoculating needle and replace in your rack.
12Transfer 3 Steps for Transfer of Broth to Broth Hold loop or needle with dominant hand( right )Flame the loopHold culture tube in left handRemove red cap with pinkie of right handFlame mouth of culture tube Place loop into broth( water)Flame mouth of culture tube and closeOpen culture tube with broth( should be labeled)Dip loop into new broth and mixFlame mouth of tube and closeFlame loopPlace to the side of your rack
15The SmearUsing aseptic technique remove a colony from a plate or cells from your slant. Be carefully to gently touch the surface of your culture with the inoculating loop.Make a circular motion in the middle of the circle to spread the cells equally in this region of the slideAdd a drop of water in the middleMix againLet Air dryRun the slide through the flame until the slide is warm ( The frosted side should be down) This fixes the bacteria to the slideLet the slide coolPlace in the metal tray or in the rack
16Gram Stain All staining work is to be done at the sink Care should be taken to work directly over the sinkPlace 1 drop of crystal violet stain on the smear ( 1 minute)Rock or roll the slide to cover the areaUse the water bottle to drip water down the slidePlace 1 drop of iodine on the slide ( 1 minute)Place 1 drop of alcohol on the slide 10 seconds ( KEY – do not leave on longer than 10 seconds or it will decolorize)Place 1 drop of saffranin on the slide for 1 minuteRinse with water from the bottleLet the slide air dry