Microbiology Techniques

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Microbiology Techniques

• 2008

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Media Types
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How to hold an Inoculating Loop
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Flaming the Loop
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Streak Platehttp//www.sumanasinc.com/webcontent/
anisamples/microbiology/streakplate.html
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Triple streak
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Streaking 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.

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Transfer to tubes
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Flaming tubes
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Streaking a slant
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Transfer 2

• 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.

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Transfer 3

• 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

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Colony Morphology
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Colony Morphology

• Colony morphology
• Color
• Shape
• Margin
• Elevation

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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.
• 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

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Gram Stain

• 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
•  

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Review of Bacterial Cell Morphology

• http//science.nhmccd.edu/biol/wellmeyer/bacteria/
  bacmorph.htm

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Streptococcus
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Staphylococcus aureus
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Gram negative bacilli