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Microbiology Techniques 2008. Media Types How to hold an Inoculating Loop.

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Presentation on theme: "Microbiology Techniques 2008. Media Types How to hold an Inoculating Loop."— Presentation transcript:

1 Microbiology Techniques 2008

2 Media Types

3 How to hold an Inoculating Loop

4 Flaming the Loop

5 Streak Plate ate.html ate.html ate.html

6 Triple streak

7 Streaking and flaming Flame the loop to sterilize it and let cool. 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). 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. 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. 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 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. 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 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. Repeat as necessary for the third and fourth phases. After streaking the plate, flame sterilize the loop before setting it down.

8 Transfer to tubes

9 Flaming tubes

10 Streaking a slant

11 Transfer 2 1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer 1. 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 counter 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 counter 3. Pass the mouth of the culture tube across the flame 3. Pass the mouth of the culture tube across the flame 4. Direct the inoculating needle into the broth. 4. Direct the inoculating needle into the broth. 5. Flame the mouth of your broth culture tube and replace the cap. Place it in your rack 5. Flame the mouth of your broth culture tube and replace the cap. Place it in your rack 6. Pick up the slant in your non dominant hand 6. Pick up the slant in your non dominant hand 7. Twist off the red cap 7. Twist off the red cap 8. Flame the mouth of the slant tube 8. Flame the mouth of the slant tube 9. Direct the inoculating needle into the tube and stab the agar in the base( butt) 9. 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. 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. 11. Flame the mouth of the tube and replace the cap. 12. Flame your inoculating needle and replace in your rack. 12. Flame your inoculating needle and replace in your rack.

12 Transfer 3 Steps for Transfer of Broth to Broth Steps for Transfer of Broth to Broth Hold loop or needle with dominant hand( right ) Flame the loop Hold culture tube in left hand Remove red cap with pinkie of right hand Flame mouth of culture tube Place loop into broth( water) Flame mouth of culture tube and close Open culture tube with broth( should be labeled) Dip loop into new broth and mix Flame mouth of tube and close Flame loop Place to the side of your rack

13 Colony Morphology

14 Colony morphology Colony morphology Color Color Shape Shape Margin Margin Elevation Elevation

15 The Smear Using 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. Using 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 slide Make a circular motion in the middle of the circle to spread the cells equally in this region of the slide Add a drop of water in the middle Add a drop of water in the middle Mix again Mix again Let Air dry Let Air dry Run the slide through the flame until the slide is warm ( The frosted side should be down) This fixes the bacteria to the slide Run the slide through the flame until the slide is warm ( The frosted side should be down) This fixes the bacteria to the slide Let the slide cool Let the slide cool Place in the metal tray or in the rack Place in the metal tray or in the rack

16 Gram Stain All staining work is to be done at the sink All staining work is to be done at the sink Care should be taken to work directly over the sink Care should be taken to work directly over the sink Place 1 drop of crystal violet stain on the smear ( 1 minute) Place 1 drop of crystal violet stain on the smear ( 1 minute) Rock or roll the slide to cover the area Rock or roll the slide to cover the area Use the water bottle to drip water down the slide Use the water bottle to drip water down the slide Place 1 drop of iodine on the slide ( 1 minute) Place 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 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 minute Place 1 drop of saffranin on the slide for 1 minute Rinse with water from the bottle Rinse with water from the bottle Let the slide air dry Let the slide air dry

17 Review of Bacterial Cell Morphology cd.edu/biol/wellmey er/bacteria/bacmor ph.htm cd.edu/biol/wellmey er/bacteria/bacmor ph.htm cd.edu/biol/wellmey er/bacteria/bacmor ph.htm cd.edu/biol/wellmey er/bacteria/bacmor ph.htm

18

19 Streptococcus

20 Staphylococcus aureus

21 Gram negative bacilli


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