ProGurt Summer II: Fourth Week (7/29 ~ 8/4)

Fourth Week (7/29 ~ 8/4)

7/30/18 MON: Preparation for the Quantification and PCR.

1)   Procedure of making buffer solution

We needed 900 micro liters because we were doing colonies.

1.    Calculate how much solution we need.

            àFor example, if we do 4 colonies, we need 3600 micro liters (900 x 4)

2.    Then, calculate the solution

200 micro liters of EDTA 

3 micro liters of protein (small one)

+          1 micro liters of tween 20 (brown one)

            

= 204 micro liters of solution X N = M (in this case, M should be more than 3600 micro liters) 

     EDTA                                          Protein                                              Tween 20

2)   Making sample

1.    Put 900 micro liters of buffer solution in each small container with beads in them.

2.    Label containers 

3.    Took sample from Albanian Yogurt 10^-4and 10^-5, put it in the container with solution.

àTwo containers for each sample (10^-4and 10^-5). 

3)   Mixing samples

To mix samples well, we put the colonies in first Vortex (power 3 for 3 min) and second Centralfuge (power 13 for 1 min)

      Vortex                                                         Centralfuge

4)   Second dilution of Albanian yogurt on MRS plates

5)   First Dilution of Greek yogurt on MRS plates

7/31/18 TUE: 

1)   Results of Second Dilution of Albanian yogurt on MRS plates:

CFUs (10^-4): 1.4 x 10^7

For Serving Size (150ml): 2.1 x 10^9

   

2)   Quantification

1.    2 beads for one qubit and label them.

2.    [ reagent (1 x n micro liter) + cubit buffer (199 x n micro liter)] / 3 (because we can transfer 1000 micro liter at a time)

3.    10 micro liters of DNA solution to each cubit.

4.    put each cubit in the Qubit (device) and check the DNA quantity 

-      dsDNA

-      high sensitivity 

-      read standard 1 and 2

5.    Record all the quantities.

3)   PCR (polymerase chain reaction)

For each DNA samples: 

50 micro liter total (25 micro liter àQPCR)

-      46 micro liters of pure water

-      1 micro liter of 27 F

-      1 micro liter of 1525 R

-      2 micro liters of DNA

For Negative control:

50 micro liter total

-      46 micro liters of pure water

-      1 micro liter of 27F

-      1 micro liter of 1525R

-      2 micro liters of pure water

4)   Third Dilution of Albanian yogurt

5)   Second Dilution of Greek Yogurt

8/1/18 WED: Gram Stain And Last Dilution of Greek Yogurt

1)    Results of Third Dilution of Albanian yogurt:

CFUs (10^-4): 1.7 x 10^7

For Serving Size (150 ml): 2.6 x 10^9

2)    Gram Stain

1.    Get slides as many as you need.

2.    Using pipets, put one drop of water on each slide. Test tubes with sterile water has to be sterilized with Bunsen burner. 

3.    Transfer small amount of colony of bacteria on each water drop.

àMake sure you make it really thin.

4.    Dry slides.

5.    After you dry slides, fix them using Bunsen burner with PIN; move four times.

6.    Bring it to the sink and get ready to dye cells

àFollow the procedures in the pictures.

3)    Last Dilution of Greek yogurt

4)    Results of Greek Yogurt

8/2/18 THU:

Gram Stain results of Greek yogurt on MRS media(Diluted on 7/30/18):

Gram Stain Results of Albanian yogurt on MRS media(Diluted on ):

Gram Stain Results of Albanian yogurt on TSA media(Diluted on 7/23/18):

Gram Stain Results of Indian yogurt on TSA media(Diluted on 7/19/18):