Biochemical Tests and gyrB qPCR

    This week we looked at the biochemical tests we inoculated last week. The lactose, sucrose, and citrate plates were all color changes while we had to add iodine to the starch plates and perform a procedure to see the MRVP results. 

    Deinococcus sonorensis was positive for lactose fermentation, inconclusive for sucrose fermentation, negative for the MR test and inconclusive for the VP test, positive for the starch test and inconclusive for the citrate test. The sucrose, VP and citrate tests all showed no difference between the negative control, positive control and test and will need to be redone. All off the TGY plates grew, with the 1/2 TGY plate having a smaller amount of growth and the double TGY plate thriving. However the only R2A plate that had growth was the regular recipe. 


Methyl red test 

1. Transfer 2.5 ml of culture into a new sterile culture tube.

2. Add 5 drops of the methyl red reagent. 

3. Compare the test organism to the control cultures to immediately interpret the result


Voges-Proskauer test

 1. Use the remaining 2.5 ml of culture grown in MR-VP broth.

 2. Add 0.6 ml (or 12 drops) of Barritt’s reagent A.

 3. Add 0.2 ml (or 4 drops) of Barritt’s reagent B.

 4.Carefully shake the tube for 30 seconds to 1 minute to expose the medium to atmospheric oxygen (necessary for oxidation of acetoin to obtain a color reaction).

 5. Allow the tube to stand for at least 30 minutes.

 6. Within 1 hour, compare the test result to control cultures to determine if the culture is VP positive 

Actively working on the MR and VP procedures. 
MR in the white tray, negative control, positive control than sono. 
VP in the pink tray, negative control, positive control than sono

Lactose test- sono, positive control, negative control

Sucrose test- sono, positive control, negative control

Starch test- sono, negative control, positive control

    We tried to make casein media so we could pour plates and inoculate this week however it is a tricky media that we didn't get right. The way we normally autoclave media ended up cooking the milk in the media, and i need to research another procedure before tying again. However we did manage to inoculate for the sporulation tests. These need to incubate for a few days before being left out to starve in order to get them to produce spores if they are able to do so.  Some of the group members also gave a presentation to the lab about different biochemical tests and what they each look for. This covered the Sucrose, Lactose, MRVP, Starch, Citrate, Oxidase, Catalase, SIM and Motility tests. 




    We also performed qPCR again, this time using the gene gyrB. The calculations were the same as the last time even though the gene was different. 

Tube 1 (T1)
- 16μl of T1 sample
- 13.6 water

Tube 2 (C1)
-8μl of C1 sample
-21.6μl water

Tube 3 (master mix)
-80μl reaction mix
-8μl enzyme mis
-6.4μl gyrB Forward
-6.4μl gyrB Reverse

Tube 4 (control)
-36.8μl water
-1.6μl gyrB Forward
-1.6μl gyrB Reverse


Wells A1 A2 and A3 each got 7.4μl of tube 1 and 12.6μl of tube 3
Wells B1 B2 and B3 each got 7.4μl of tube 2 and 12.6μl of tube 3
Well H1 got 20μl of tube 4




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