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

 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 LOOP COLONY COUNT
(cfu/mL)

Non-invasive urine Green LOOP 1 colony =

examples: •0.001 mL 1,000 cfu/mL
• Clean-voided •1/1000th of a mL
• Foley catheter
• Ileal loop

Invasive urine Blue LOOP 1 colony =

examples: • 0.01 mL 100 cfu/ml
•Straight catheter • 1/100th of a mL
•Cystoscopic
• Kidney
 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