Part 1: The purification and growth of bacteria host system.
Bacteria, in particular Escherichia coli, are marvelous organism (i.e. when they are not causing disease.) that are used widely in microbiology, biotechnology and molecular biology. This is due to several factors:
- Relative ease of isolation.
- Ease of handling.
- Easy propagation in prepared standard media.
- Viability in solid or liquid media
- Quick generation time (30 minutes/ binary fission)
- Ability to survive long term storage by freezing or freeze-drying and reconstitution.
- Potentially competent to receive foreign DNA.
- Ability to express foreign DNA under the right conditions.
- Easy detection of phenotypic change via colony characteristics.
- Silent regardless of manipulation
- Do not bleed, bite or no violent behavior.
As with any specific microorganism, sterile technique is important. Firstly, do not contaminate the organism and your experiment. Secondly, do not contaminate yourself or others. The bacterial growth curve is illustrated as follows
The optimal growth conditions are at the log or exponential phase. At this time, the cells will be actively growing and it is at this stage the cells should be harvested for DNA preparations.
After this stage, with the gradual depletion of nutrients and the accumulation of metabolites and waster products, the culture reaches a stationary phase, after which the cells start to die unless transferred to fresh media. Once the cells rupture and die, there will be a release of various enzymes that will quickly degrade the nucleic acids. DNA isolation at this point is not recommended.
1. A vial of liquid culture of E. coli grown in nutrient broth is given.
2. The inoculation loop was heated up till I inch, in the blue flame until red-hot. Cooled on the side of the agar.
3. Cooled sterile loop was carefully dipped into culture. Streaked onto plates using dilution method to obtain single pure colonies.
4. Loop was flamed in between directional streaking. Eg. 1-flame-2flame-etc.
5. Dish was covered and incubated upside down in a 37°c incubator overnight.
Part 2: Growing an overnight culture.
1. Plate was removed from the incubator and inspected for presence of single, well separated colonies.
2. Colour, clarity and morphology of colony were observed and recorded.
3. Contamination by other organisms in plates and control was checked.
4. Using a sterile flamed loop, a well defined single colony was picked. (A colony is considered pure because it is derived from a single cell.)
5. The mouth of the universal containing the media was flamed. The culture was inoculated with
6. The bottle was capped, not too tightly and placed in a 37°c incubator, shaked at 150-200 rpm overnight.
1. The bacteria strain used as host in this experiment is Escherichia coli DH5a and Escherichia coli with ampicilin resistance plasmid (pUc19). Both strains are provided in the Falcon tube with LB broth. In this practical, we have to transfer the Escherichia coli DH5a from the LB broth in Falcon tube and streak it onto plates using dilution method to obtain single pure colonies. Bacterial loop is used in this process. Besides, we also transfer 100μl of E. coli with pUc19 using micropipettor onto LB plate with ampicilin 50μg/ml and spread it with a hockey stick.
2. Ampicilin is a type of antibiotics that kills bacteria except those with ability to confer resistance to that antibiotic. E. coli with pUc19 consists of plasmid with ampicilin resistance gene which enables it to grow in LB agar with ampicilin.
3. A sterile flamed loop is used to pick a well defined single colony from the plate and dip it into LB broth in Falcon tube, this is to minimize the contamination of Falcon tube during transfer of the single colony.
4. The broth must be checked to see whether it is clear or turbid. Turbidity means there is already presence of bacteria and the broth cannot be used anymore.
5. In the bacterial grow curve, the lag phase lasts about 10-15 minutes where the bacteria lie dormant. During log or exponential phase, there is growth by binary fission. At this stage, the media should look between clear and slightly cloudy with swirling flower formation. DNA is still intact and viable for DNA preparations. Then, the culture reached stationary phase of plateau after gradual depletion of nutrients and accumulation of metabolites and waste products. The bacteria are lysed and dead.
6. One should check for bacteria after culture within 16-19 hour as the bacteria will die due to depletion of nutrients. Once the cells rupture and die, there will be release of various enzymes that degrade the nuclei acids rapidly. Isolation of DNA at this point is not recommended.
In bacterial purification, bacteria dilution streaking is used to produce single colonies. Turbidity and cloudiness of the broth indicate that there is bacteria growth in the broth.