During the first few weeks of our internship, we got the opportunity to experiment with Halobacterium salinarum.
By Kedus Getaneh
Objective
The goal of the experiment was to test if Halobacteria were able to grow in newly-made Complete Media that we prepared. Complete Media is a growth medium for Halobacteria that enhances cell growth by providing the cells with rich nutrients, such as amino acids and salts. This creates a stable environment where the cells can grow and achieve maximum growth. However, to see if our Complete Media provided a stable environment for the cells to grow, we had to compare our results with a standard Complete Media. This standard Complete Media had already been tested in previous experiments to ensure that Halobacteria can grow in it. For this reason, we know that Halobacteria successfully grows in the Standard Media. By comparing our data with the data from the standard media, we can conclude if our media is sufficient for optimal Halobacteria growth.
Procedure
- Preparation of Complete Media
- Hypothesis through Signature Curve
- Prepping experiment tubes for incubation
- Taking baseline Optical Density (OD) Readings
- Recording Data
- Graphing Data
- Conclusion
Preparation of Complete Media
This media can be prepared in larger quantities, but this procedure is for making 1 liter of Complete Media.
Materials: 
- 250 g NaCl
- 20 g MgSO4
- 2 g KCl
- 3 g NaH2C6H5O7 (Sodium Citrate)
- 10 g Oxoid Peptone
- Stir Plate & Bar (or a Stir Stick if not available)
- 1 L Beaker
- 1 L Distilled Water
- 1 L Graduated Cylinder
- 1 L Bottle
- 0.01 g – 300 g capable Scale
- Weigh boats and spatulas
- Autoclave
- Optional: Autoclave tape and Pen
Instructions:
1. Measure out all substances except peptone using weigh boats into beaker. Place beaker onto stir plate and add distilled water until solution volume is 800 ml.
2. Place a stir stick into beaker and center beaker on stir plate. Turn stir plate on low setting (may have to turn it on high setting momentarily to allow stir stick to spin through salt).
3. Let contents mix until fully dissolved and integrated.
4. While solution is stirring, measure out oxoid peptone into a weigh boat (it is acceptable to use one weigh boat through entire procedure, but do not contaminate stock supplies with stir sticks or scoops). When beaker mixture is fully stirred, gradually add peptone to beaker with stir plate off.
5. Restart stir plate on low setting to incorporate peptone. When peptone is dissolved, turn off stir plate and add distilled water to the beaker from a graduated cylinder until beaker volume measures 1000 ml (1 L).
6. Transfer solution to bottle. Place lid on bottle loosely to allow for expanding air and water vapor to escape bottle in autoclave. Wrap a small piece of foil around bottle opening and place autoclave indicator tape on foil.
7. Label bottle with date, contents, initials and station (ex. ‘Halo’).
8. Place bottle in autoclave station and clean workstation.
Hypothesis through Signature Curve
To make our hypothesis/ prediction for this experiment, we needed to look at a signature curve of Halobacteria growing in standard Complete Media:
Halobacteria are Archaeans that are know to divide once every six hours. With this information, we can infer that at the beginning of their growth cycle, their population grows slowly, then increases exponentially, and should stabilize around 1.2 Optical Density units.
Preparation of Samples for Incubation
To test our hypothesis, we had to prepare ten samples to compare Halobacteria growth in the new ‘Student’ media and in the control ‘Standard’ media.
Out of the ten samples, 8 samples with the following solutions were incubated:
- 1 tube - Student CM
- 1 tube - Standard CM
- 3 tubes - Student CM + Halo
- 3 tubes - Standard CM + Halo
The following 2 samples were not incubated:
- 1 tube - Student CM (stock)
- 1 tube - Standard CM (stock)
Before incubation, we collected the starting data using a spectrophotometer to measure the Optical Density (OD) of the solutions. A spectrophotometer is a device that uses a range of light waves to measure the OD of a solution. When in use, it emits a ray of light that either passes through the sample, is absorbed, or is refracted by the particles. The OD number given by the spectrophotometer indicates the concentration of particles in a solution. A numerically higher OD value indicates a higher concentration of particles, while a numerically lower OD value indicates a lower concentration of particles. In our case, we use the spectrophotometer to measure the growth of Halobacteria in our samples. The two samples that were not incubated and the two samples of only CM served as controls in our experiment. We measured the growth of Halobacteria using the spectrophotometer over a 3 day period. From this data, we created a graph that represented all ten samples.
Results
The results we obtained after three days were similar to our hypothesis made previously. At the beginning of their growth cycle (6 hours), the Halobacteria population grew slowly until they started to divide exponentially and hit their mid-log phase at around 43 hours. After this point, the growth rate decreased and the population stabilized around 1.2 OD. This phase phase is known as the stationary phase in which the growth and death rate reach equilibrium. Overall, our Halobacteria grew successfully.
Conclusion
After approximately three days of growth and measurement, it is clear that Halobacterium salinarum grow successfully in the newly-prepared “Student” CM. The student cultures actually grew faster compared to the cultures in the standard CM. However, during the stationary phase, both cultures in the Student CM and the cultures in the standard CM ended up at the same level of cell growth. With the information provided by the two graphs, we concluded that the Student Complete Media can be used to successfully grow Halobacterium salinarum.