1. LAB 4: ASEPTIC TECHNIQUE AND ISOLATION OF BACTERIA

2. 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!

3. ASEPTIC TECHNIQUE TERMS
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

4. 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

5. 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.

6. TYPES OF MEDIA NOTE: MEDIA CAN BE 1, 2, OR ALL OF THE ABOVE
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

7. 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

8. 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.

9. Removing inoculum from broth:
Removing inoculum from a solid medium:

10. INOCULATING BACTERIA ON AN AGAR SLANT
DO NOT gouge the agar with the inoculating loop, instead gently graze the surface.

11. INOCULATING BACTERIA INTO A DEEP AGAR
Stab the needle containing bacteria directly into and straight out of the deep agar.

12. INOCULATING A PLATE: THE STREAK PLATE TECHNIQUE

13. CALIBRATED LOOP: 0.001 uL vs. 0.01 uL
URINE PLATE TECHNIQUE
CALIBRATED LOOP: uL vs 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

14. 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
100 cfu/ml

15. RESULTS

16. THE STREAK PLATE TECHNIQUE
THE PURPOSE IS TO DILUTE OUT AND SEPARATE THE BACTERIA PRESENT TO GET ISOLATED COLONIES.

17. 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.

18. IMPROPER STREAK PLATE TECHNIQUE

19. PATTERNS OF GROWTH IN BROTH

20. PATTERNS OF GROWTH ON A SLANT

21. PATTERNS OF GROWTH IN AGAR DEEP

22. PATTERNS OF GROWTH ON AN AGAR PLATE

23. The 0.001 calibrated loop was used.
Given the selections, what is the number of cfu/mL in the original sample?
10,000 – 50,000
>10,000

24. 10 100 1,000 10,000 The 0.01 calibrated loop was used.
What is the number of cfu/mL in the original sample? 10
100
1,000
10,000