Lab Log Book

October 2016

12/10/2016 - Culturing bacteria in liquid medium

The Pigment bacteria set ordered from Carolina finally arrived. Can't wait to start culture it!

Fig 1. Pigmented bacteria set from Carolina Biological Supply.

The set includes 4 strains.

The set includes:

To culture the bacteria, I prepared 40mL LB broth and use a inoculation loop to transfer bacteria from tubes to LB broth. And then incubate it overnight at 30C.

Fig 2. Inoculation performed under aseptic technique.

Here's how I prepared my LB broth:

I weighted 4g ready-made LB broth powder (from Affymetrix) and added 120mL distilled water. Mix it well and then heat it up in microwave until it starts boil. Then wait for it to cool down and pour it into flasks.

13/10/2016

Let's see how our pigmented babies grow!

Fig 3. R. rhodochrous (left) and S. marcescens D1 (right)

after 24 hours incubation at 30C.

So far we observed the red pigmented Serratia marcescens D1 grow quite well, but the other 4 bacteria species did not show obvious bacterial growth.

14/10/2016

Fig 4. R. rhodochrous (left) and S. marcescens D1 (right)

after 48 hours incubation at 30C.

After 48 hours of incubation, the pink pigmented Rhodococcus rhodochrous (left) becomes cloudy, which indicates there is bacterial growth. However, it did not show pink color. There are several possible reasons:

• The volume too large, so it dilutes the pigment
• Another possible reasons is color change of pink is not obverse while the nutrient broth is yellow in color
• The third possible reason is contamination. Other bacteria outcompeted the pink pigmented babies
• The incubation temperature (30C) is not optimal for pigment formation. So I will try incubate it at 25C.

15/10/2016 - Centrifuge pigmented bacteria & Culture bacteria in agar plates

We transferred the Serratia marcescens D1 culture into 1.5mL centrifuge tube and centrifuge it at 4000 rpm (we use centrifuge brought from taobao) for 20 min. At the end we get a purple pellet. We repeated the process to get a larger pellet.

Fig 5. Purple pellet formed after centrifuging S. marcescens D1 at 4000rpm for 20 min.

Fig 6. All S. marcescens D1 culture centrifuged and LB both is removed. Large pellets is yielded after centrifuge for 4 times.

We also observed Serratia marcescens D1 culture under microscope. We observed rod shape bacteria at 400X magnification.

Fig 7. A slide of S. marcescens D1 culture is prepared and observed

under compound microscope (400X).

After reading the pigment extraction method from Charkoudian LK, Fitzgerald JT, Khosla C, Champlin A (2010) and after reflecting on the not satisfactory result from culturing pink, yellow and orange-yellow pigmented bacteria in liquid medium, I decided to inoculate the bacteria on agar plates.

Fig 8. Streaked agar plates for all four pigmented bacteria strains

and incubate overnight at 30C.

Accidental finding: We added 2 drops of blench to the confluent S. marcescens D1 culture, then we observed a color change from red to greenish-yellow. We wondered the NaOH in blench change the pH and thus change the chemical property of Prodigiosins

17/10/2016 - Observation of petri dishes

After 24 hours:

Fig 9. Streaked agar plates for all four pigmented bacteria strains

after incubated for 24 hours at 30C.

Fig 10. Spread agar plates for all four pigmented bacteria strains after

incubated for 24 hours at 30C.

The red babies grow crazily fast. It covers the whole plates after incubated for one night. The orange and yellow one grow slower. From Fig. 10, we can observe there are colonies cover S.A. (orange pigment) and M.L. (yellow pigment). However, I couldn't find and presence of colony of R.R. (pink pigment). Note that there are contamination of S.M. in the S.A. plate. This may be because we didn't practise aseptic technique properly... need to practise more! >.<

After 48 hours:

(a) (b)

Fig 11. Streak agar plates (a) and spread plate (b) of Sarcina aurantiaca

after incubated for 48 hours at 30C.

In Fig.1a, S. aurantiaca (orange) takes 2 days to grow. Fig 1b shows the comparison of R. rhodochrous (pink) and S. aurantiaca. Though R. rhodochrous plate is contaminated by the aggressive red babies, the orange babies still covers the whole plate as indicated by the orange color on S.A. plate. Sadly pink babies are still missing...