Transcript Microbiology Techniques

Microbiology
Techniques
2008
Media Types
How to hold an Inoculating Loop
Flaming the Loop
Streak Plate
http://www.sumanasinc.com/webcontent/anisamples/microbiology/streakpl
ate.html
Triple streak
Streaking and flaming
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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.
Transfer to tubes
Flaming tubes
Streaking a slant
Transfer 2
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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
3. Pass the mouth of the culture tube across the flame
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
6. Pick up the slant in your non dominant hand
7. Twist off the red cap
8. Flame the mouth of the slant tube
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.
11. Flame the mouth of the tube and replace the cap.
12. Flame your inoculating needle and replace in your rack.
Transfer 3
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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
Colony Morphology
Colony Morphology
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Colony morphology
Color
 Shape
 Margin
 Elevation
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The Smear
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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
Add a drop of water in the middle
Mix again
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
Let the slide cool
Place in the metal tray or in the rack
Gram Stain
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All staining work is to be done at the sink
Care should be taken to work directly over the sink
Place 1 drop of crystal violet stain on the smear ( 1 minute)
Rock or roll the slide to cover the area
Use the water bottle to drip water down the slide
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 saffranin on the slide for 1 minute
Rinse with water from the bottle
Let the slide air dry
Review of Bacterial Cell
Morphology
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http://science.nhmc
cd.edu/biol/wellmey
er/bacteria/bacmor
ph.htm
Streptococcus
Staphylococcus aureus
Gram negative bacilli