Purpose: Aseptic Transfer of Sterile Nutrient into different commonly used vessels. ​



(Instructor's Notes: Dispose of used pipettes in Biohazard receptacle.)



​Materials: 

Bunsen Burner

Striker

10mL Sterile Pipettes

Sterile Cell Culture Dish

Pipette Aid

100mL Sterile LB (Luria Broth) (Fisher Scientific Lot # 082482, BP 1426-2)

Disinfectant (CiDecon CAT#8504, Lot #0110, Exp 01/2012)

Sterile Empty Glass Bottle

​

Test Tubes: 2 x 50mL Conical tubes

Biohazard Disposal container

1 LB Agar sterile plate

Cotton tipped applicators (Fisher Brand 23-400-124)

Lab labeling tape

Lab Gloves



Method: 



1. Connected burner to gas line after inspecting hose for cracks or leaks and used the striker to ignite.
2. Adjusted the flame for optimal inner blue cone approximately 3 cm. 
3. Loosened caps on conical tubes
4. Flamed 100mL bottle of LB. (cap on)
5. Flamed 100mL bottle of LB. (cap off)
6. Aseptically pipetted 10mL from 100mL bottle of LB and transferred into first conical tube while holding tube at an angle to prevent air contaminants from falling straight into tube. Tightened vented tube cap.
7. Aseptically pipetted 10mL from 100mL bottle of LB and transferred into the second conical tube while holding tube at an angle to prevent air contaminants from falling straight into tube.. Tightened vented tube cap. Flamed 100mL bottle's rim and tightened cap again.
8. Aseptically transferred another 10mL of LB from original 100mL container to 125mL bottle, flaming after cap removed and before cap was replaced. Then I extinguished the burner.
9. Used lab tape to label tubes and placed them in the bacterial incubator. 
10. Used sterile cotton swabs to swab bench area. Then streaked agar plate using the lid to shield plate. Labeled plate and disposed of swab in the biohazard receptacle. 
11. Relit burner and adjusted again for optimal flame. 
12. Flamed original source bottle of LB and pipetted 5mL into Cell Culture dish using lid to shield. Then flamed and closed source bottle of LB and extinguished burner.
13. Verified all remaining containers were labeled correctly and put them into the bacterial incubator also. Incubator set at 37 degrees C.



Data and Results: 

10/01/2012 Will be checking on transfers of broth the rest of the week for evidence of contamination with bacterial or mold growth.



10/02/2012 and after: 



Vessel:                           24 Hours                    48 Hours                 72 Hours

Glass bottle of LB           Not turbid                 Unchanged            Zero contamination!

                                    Broth is yellow.

                                   No evidence of 

                                   change or of 

                                   contamination. 





LB Agar Plate           No evidence of                One colony.         One colony formed. 

from bench             colonies present                                          Additional growth is minimal.

swab.

​

Cell Culture Plate      Lots of condensation      Unchanged          Zero contamination!

                               Not turbid. Broth

                                appears unchanged/

                                light yellow (less 

                                concentrated in 5mL)



Tube 1                      No turbidity/no pellet.      Unchanged         Zero contamination!

Tube 2                      Same as Tube 1.               Unchanged         Zero contamination!









Discussion: 

When I executed this exercise I was ill/recovering from being ill over the weekend. I was especially vigilant NOT to touch gloves to face and to cough directly into inner elbow.



When transferring I was careful to hold containers at an angle away from my face and did not speak while working to minimize potential for contamination.