lab 4: aseptic technique and isolation of bacteria
TRANSCRIPT
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LAB 4: ASEPTIC TECHNIQUEAND
ISOLATION 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 PLATE:THE STREAK PLATE TECHNIQUE
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URINE PLATE TECHNIQUECALIBRATED 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 urineexamples:•Straight catheter•Cystoscopic• Kidney
Blue LOOP• 0.01 mL• 1/100th of a mL
1 colony =100 cfu/ml
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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?
A. 1000 - 9000B. 10,000 – 50,000C. >10,000
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The 0.01 calibrated loop was used. What is the number of cfu/mL in the original sample?
A. 10B. 100C. 1,000D. 10,000