LAB 4: ASEPTIC TECHNIQUE AND ISOLATION OF BACTERIA

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LAB 4 ASEPTIC TECHNIQUEANDISOLATION OF BACTERIA
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Microorganisms to be used this semester

• Many of the microorganisms we will use this
• semester will be Biosafety Level 1 (not shown to
• cause disease in humans) but several will be
• Biosafety Level 2 (can cause disease in humans).
• Because of this potential risk we ask that you
  treat ALL bacterial cultures as if they cause
  infection!

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ASEPTIC TECHNIQUE TERMS

• Aseptic Technique
• Procedure to prevent contamination of medium or
  bench surface.
• Pure culture
• Contains only 1 type of microorganism
• Mixed culture
• Contains 2 or more types of microorganisms
  living/growing together

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ASEPTIC TECHNIQUE TERMS

• Inoculation
• Act of placing bacteria (and other
  microorganisms) onto culture medium.
• Contaminant
• Unwanted microbes present in culture medium or
  lab bench surface.
• Sterile Media
• Media prepared and then sterilized prior to use.
• Always inspect media to ensure no visible
  contaminants are present prior to use.
• Media is sterilized by autoclaving or filtration
  during preparation

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DEVICES FOR PERFORMING ASEPTIC TECHNIQUE

• Inoculating Loop (a)/Needle (b)
• Metal wire used to transfer organisms.
• Incinerator
• Heat source that is used to remove any unwanted
  microorganisms on the inoculating loop/needle.

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TYPES OF MEDIA

• ENRICHED selects for certain microorganisms by
  including a nutrient that the desired
  microorganism or group can use and its
  competitors can not
• SELECTIVE selects for growth of certain
  microorganisms in a mixed population by using an
  ingredient that inhibits the growth of other
  microorganisms, but not the desired species or
  group
• DIFFERENTIAL does not select for any particular
  group by inhibiting or enhancing their growth
  over competitors, but it does show a visible
  difference between or among groups of
  microorganisms
• NOTE MEDIA CAN BE 1, 2, OR ALL OF THE
• ABOVE

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MEDIA TYPES AND USES

• BROTH a liquid medium. Advantage tube is easy
  to store and transport. Disadvantage can not see
  colony morphology.
• SLANT tube of solid medium at an angle.
  Advantage tube is easy to store and transport,
  can see colony morphology. Disadvantage small
  surface area.
• AGAR DEEP tube of solid or semi-solid medium.
  Good for organisms that prefer reduced O2 and to
  evaluate motility.

Broth Slant Agar deep
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MEDIA TYPES AND USES

• PETRI DISH/PLATE SOLID MEDIUM ON A FLAT SURFACE.
  
• This is the MOST COMMON METHOD TO OBSERVE COLONY
  MORPHOLOGY AND TO WORK WITH INDIVIDUAL COLONIES
  FOR DIAGNOSTIC METHODS.

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Removing inoculum from broth
Removing inoculum from a solid medium
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INOCULATING BACTERIA ON AN AGAR SLANT

• DO NOT gouge the agar with the inoculating loop,
  instead gently graze the surface.

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INOCULATING BACTERIA INTO A DEEP AGAR

• Stab the needle containing bacteria directly into
  and straight out of the deep agar.

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INOCULATING A PLATETHE STREAK PLATE TECHNIQUE
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URINE PLATE TECHNIQUE
CALIBRATED LOOP 0.001 uL vs. 0.01
uL Inoculation dip calibrated loop in
urine, streak down middle of agar plate, then
with the same loop go back and streak across the
center inoculum to dilute
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URINE TYPE LOOP COLONY COUNT (cfu/mL)
Non-invasive urine examples Clean-voided Foley catheter Ileal loop Green LOOP 0.001 mL 1/1000th of a mL 1 colony 1,000 cfu/mL
Invasive urine examples Straight catheter Cystoscopic Kidney Blue LOOP 0.01 mL 1/100th of a mL 1 colony 100 cfu/ml
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RESULTS
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THE STREAK PLATE TECHNIQUE
THE PURPOSE IS TO DILUTE OUT AND SEPARATE THE
BACTERIA PRESENT TO GET ISOLATED COLONIES.
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WHY IS THE STREAK PLATE ISOLATION METHOD IMPORTANT

• SAMPLES FROM PATIENTS OR THE ENVIRONMENT ARE NOT
  PURE, I.E. ONE TYPE OF MICROORGANISM PRESEND.
  SAMPLES USUALLY CONTAIN MIXTURES OF MULTIPLE
  TYPES OF BACTERIA.
• LABORATORY IDENTIFICATION AND SUSCEPTIBILITY
  TECHNIQUES REQUIRE A PURE CULTURE OF A SINGLE
  MICROORGANISM.
• THE STREAK PLATE ISOLATION METHOD ALLOWS ONE TO
  SEPARATE OUT INDIVIDUAL BACTERIAL COLONIES.

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IMPROPER STREAK PLATE TECHNIQUE
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PATTERNS OF GROWTH IN BROTH
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PATTERNS OF GROWTH ON A SLANT
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PATTERNS OF GROWTH IN AGAR DEEP
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PATTERNS OF GROWTH ON AN AGAR PLATE
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The 0.001 calibrated loop was used. Given the
selections, what is the number of cfu/mL in the
original sample?

1. 1000 - 9000
2. 10,000 50,000
3. gt10,000

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The 0.01 calibrated loop was used. What is the
number of cfu/mL in the original sample?

1. 10
2. 100
3. 1,000
4. 10,000