Download Microbiology (BIO

LAB 4: ASEPTIC TECHNIQUE
AND
ISOLATION OF BACTERIA
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!
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
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
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.
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
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
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.
Removing inoculum
from broth:
Removing inoculum from a solid
medium:
INOCULATING BACTERIA ON AN
AGAR SLANT
DO NOT gouge the agar with the inoculating
loop, instead gently graze the surface.
INOCULATING BACTERIA INTO A
DEEP AGAR
Stab the needle containing bacteria directly
into and straight out of the deep agar.
INOCULATING A PLATE:
THE STREAK PLATE TECHNIQUE
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
URINE TYPE
Non-invasive urine
examples:
• Clean-voided
• Foley catheter
• Ileal loop
Invasive urine
examples:
•Straight catheter
•Cystoscopic
• Kidney
LOOP
COLONY COUNT
(cfu/mL)
Green LOOP
•0.001 mL
•1/1000th of a mL
1 colony =
1,000 cfu/mL
Blue LOOP
• 0.01 mL
• 1/100th of a mL
1 colony =
100 cfu/ml
THE STREAK PLATE TECHNIQUE
THE PURPOSE IS TO DILUTE OUT AND SEPARATE THE BACTERIA PRESENT TO
GET ISOLATED COLONIES.
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.
IMPROPER STREAK PLATE
TECHNIQUE
PATTERNS OF GROWTH IN BROTH
PATTERNS OF GROWTH ON A
SLANT
PATTERNS OF GROWTH IN AGAR
DEEP
PATTERNS OF GROWTH ON AN
AGAR PLATE
The 0.001 calibrated loop was used.
Given the selections, what is the number of cfu/mL in the original
sample?
A. 1000 - 9000
B. 10,000 – 50,000
C. >10,000
The 0.01 calibrated loop was used.
What is the number of cfu/mL in the original sample?
A. 10
B. 100
C. 1,000
D. 10,000