LAB 4: ASEPTIC TECHNIQUEAND

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 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

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

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 - 9000B. 10,000 – 50,000C. >10,000

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