Creating a Bacterial Culture With Proper Sterile Technique

Microbiology is a very important part of science utilized by every sector of biology, whether that is to study medicines or create special substances that we need, such as insulin (the title picture above is not mine, but this is a colony of GFP, green fluorescent protein, producing bacteria). The "instructions" for these cells to create such useful products is contained on plasmids, small circular strands of DNA separate from the bacteria's main genome. The plasmids can be created by biologist and then inserted into bacteria such as E. coli, where they will be maintained and copied into future generations of bacteria. These special bacteria can be held in a -80 C freezer until needed and then thawed out and put into a broth solution, facilitating their rapid growth. Overnight, a scientist can have millions of copies of a plasmid-containing bacteria ready to produce the desired product.

But it is very important to use proper technique when conducting such research since there are airborne pathogens that can easily contaminate the cell cultures. In this Instructable, I will be showing you how to use a Bunsen burner to maintain sterility while manipulating LB broth and bacteria.

Materials

• LB broth (we get ours from Fischer Scientific)
• BD Falcon 5 mL polystyrene round bottom tube
• Kanamycin (or other antibiotic such as ampicillin)
• bacteria carrying desired plasmid with necessary antibiotic resistance
• reverse osmosis filtered water (Nanopure Water)

Tools

• scale
• scoop and weigh boat
• tube rack
• 500 mL glass bottle
• micropipettes (p20, p200 and p1000) with tips
• Pipet Aid and pipets
• Bunsen burner
• fire starter
• stir bar
• stir plate
• autoclave machine
• standard PPE: nitrile gloves, lab coat, goggles

Weigh out the prescribed amount of powder broth mixture per the manufacturers instructions. The specific type of broth we use calls for 25 g in 1 L of water. Therefore, since we are making 500 mL (0.5 L), weigh out 12.5 g of broth onto the weigh boat and add it to the glass bottle. Then add 487.5 mL of nanopure water to top off the bottle (it is usually a safe assumption that the powder broth takes up 1 mL per gram). This doesn't need to be exact, but try to get as close as possible. Then add the stir bar and stir on the plate at a good speed until it is fully dissolved. Finally, to ensure sterility, autoclave the LB broth solution.

We add antibiotic to ensure that only our bacteria with our gene of interest (GoI) are present in the culture. This is done by using an antibiotic that our bacteria is resistant to since we have a protecting gene that is right next to our GoI. Therefore, we add some of our antibiotic, Kanamycin, to a 50 mL aliquot. The steps are laid out below and it is important to stay within the vicinity of the flame to maintain a sterile environment. The flame creates an upward airflow and heated environment to prevent bacteria from getting into out LB broth. Use the video for reference on completing the following steps:

1. light the Bunsen burner in an open space on your lab bench
2. pre-loosen the cap on the 50 mL conical tube
3. remove the cap from the LB 500 mL bottle; don't put it on the lab bench and keep it face down
4. "flame" the lip by lightly putting it near the flame
5. place the bottle down near the flame while still holding the cap
6. aspirate out broth using a Pipet Aid
7. replace the cap onto the LB bottle
8. pick up the conical tube and unscrew and remove the cap with one hand (the Pipet Aid should be in the other hand)
9. lightly flame this lip (careful not to get burned or melt anything)
10. add in the broth and replace the cap on the tube
11. repeat as many times as necessary to add 50 mL, making sure to stay near the flame
12. now add 5 microliters of Kanamycin (my Kanamycin is at 50 mg/mL to start with so that amount you add may change depending on your starting concentration) to the LB aliquot in a similar fashion as when aliquoting the broth (you don't need to flame the Kanamycin, though)
13. cap and mix or vortex the solution

Now we want to aliquot our Kanamycin-spiked LB broth to a small BD Falcon 5 mL polystyrene round bottom tube. This has a special lid that doesn't tighten fully, allowing for some gas to escape, which is why we use this to grow our bacteria since they produce gas as they grow and live. This means, though, that we must be careful not to tip it over or mix it through inversion.

So use the same technique as before to add 4 mL of our antibiotic LB broth to 5 mL Falcon tube, keeping everything near the flame. Then we need to add our bacteria. We store our bacteria glycerol in a -80 C freezer and this gives the solution a gel-like consistency. Therefore, we can just stick in the tip of a pipette into our mother colony and then dip it into the broth. This won't transfer very much bacteria, but enough to form a colony overnight.

To finish up, we leave it overnight on a shaker in an incubator set at 37 C. In the morning we can retrieve our colony which will have a hazy look to it. Congratulations, those are our bacteria with our GoI!