LAB 5 : DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACT

Procedure :

Part 1 : Determination of bacteriocin activity via agar diffusion test

Step 1 : Draw a line  at bottom of all petri dishes and label which lactid acid bacteria (LAB) and
             pathogen to insert.

Step 2 : Three type of lactic acid bacteria is poured into three centrifuge tube and take to
             centrifuge at 3500rpm for 15 minutes.

Step 3 : Pour adequate amount of trypticase soy-yeast extract agar (TSAYE) into the labeled petri
             dish and wait for it solidify.

Step 4 : 10 mL of  three types of pathogen is added to the three 50mL Brain heart infusion solution
              in scott bottle and mix well.

 ( S.aureus, K.pneumonia and P.aeruginosa)

 (Brain heart infusion solution)

Step 5 : Pour the BHI solution on the solidified TSAYE layer in the same petri dish and wait for
             solidify. Then the sterile filter paper disk is dipped into the supernant of three LAB and
             placed on the solidified BHI agar according to the label.

(LAB after centrifuged is separated into pellet and supernant.)

(Sterile filter paper disk)

Step 6: Inverse the petri dish and inocubate for 24-48hr at 37ºC.

Part 2 : Determination of bacteriocin activity via optical density.

Step 1 : 20mL of L. plantarum culture (lactic acid bacteria) is poured into centrifuge tube and
            take to centrifuge at 3500rpm for 15 minutes.Then it is used as extracellular extract.

Step 2 : Prepare a serial dilution of the extracellular extracts. (dilute 0x, 2x, 10x, 50x,100x)

(The extracellular extract is transfer to 50 mL of
   distilledwater)

Step 3 : Add all 5mL double-strength MRS with 1 ml cultures containing spoilage/pathogenic
             bacteria and shake the mixture to mix well.

 Step 4 : Add 5ml of each dilution of extracellular extract (2x, 10x, 50x, 100x) into each universal

              bottles according to the label

Step 5 : Incubate the mixtures for 12-15 hr at 37ºC.

Step 6 : Prepare a positive-control using 5 mL of double-strength MRS, 1mL of the cultures
              containing spoilage bacteria, and 5 mL of sterile peptone. Incubate the mixture
              for 12-15hours at 37ºC.

Step 7 : Preparea negative-control for "auto-zero" via the spectrophotometer. Add 5 mL of double-
             strength MRS with 2 mL of distilled water. (Need not incubate)

Enjoy reading our laboratory report 5...^^