Starting bacterial cultures

The Bio 6B lab explores bacterial plasmids and operons through a set of connected experiments over multiple lab days. The concepts behind these labs are presented in a set of related pages on this site:

In addition, there are multiple pages for the experimental methods, which you'll find in the menus.

Background

This page is about the methods for culturing bacteria. You'll use these techniques multiple times throughout the quarter. For the first day of lab, you should read the Bacterial strains and plasmids page before you start with the techniques described on this page.

Bacterial culture techniques and media

A culture medium is the mix of ingredients used to grow bacteria in the lab. We will be using a recipe called LB, which contains all the nutrients needed by E. coli for rapid growth. A growth medium such as LB can be produced in liquid form or mixed with agar to make a solid medium which is poured into Petri dishes to make plates.

  • Liquid media, in which cells grow suspended in a broth inside cultures tube or flasks, are good for rapidly producing large numbers of cells. The liquid medium with added bacterial cells is incubated in a shaker incubator, which shakes the culture so the cells will get better access to nutrients and oxygen.
  • Plate media, on which colonies of bacteria grow on top of agar, are used for analyzing cells (for example, to see if they grow in the presence of antibiotics) and for isolating single colonies. If you spread a small number of cells onto a plate, each cell can grow into a colony. The colony is the original cell and all the new cells that came from it; all these cells are normally genetically identical, so the colony is a clone.

When you add cells to a culture, you are inoculating the culture. You'll do that on the first day of lab, and several times thereafter.

"Media" is the plural of "medium."

Selective media

When you add antibiotics to a liquid or plate medium, you are creating a selective medium: a culture medium that allows one strain to grow while another cannot. The selective medium selects for a particular strain, such as the strain containing the plasmid. For example, if the growth medium contains ampicillin, then only cells that are ampicillin-resistant will be able to grow. If you are working with a plasmid that contains an ampicillin resistance gene, then ampicillin plates (or liquids) will select for the cells that contain the plasmid. (For background on antibiotics and resistance mechanisms, see Bacterial cultures and plasmids.

Differential media

A differential medium will allow more than one strain to grow, but different strains will look different. You'll use LB with arabinose as a differential medium in the pGLO lab later this quarter. The arabinose in the medium will induce cells containing the pGLO plasmid to produce a fluorescent protein (Green Fluorescent Protein, or GFP). Cells that don't contain the pGLO plasmid can still grow on arabinose plates, but they won't produce the fluorescent protein.

You will also use LB plates containing both ampicillin and arabinose to make a medium that is both selective and differential.

Where your media come from

Lidia makes them in the back room. We buy LB broth or agar in powdered form, and hard-working lab techs like Lidia mix up the powders in water, steam sterilize the solutions in an autoclave, and pour the melted media into sterile petri dishes for plate media or culture tubes for liquids. The Bio department goes through hundreds of plates per quarter, so it's a lot of work.

The main ingredients in LB are yeast extracts. LB provides all the essential amino acids, salts, and other nutrients needed by E. coli, but with a low concentration of sugar. This turns out to be beneficial for our experiments.

Calculating the amount of antibiotics

You'll need to add a small amount of a concentrated stock solution of ampicillin or streptomycin to a liquid culture to get the appropriate final concentration of the antibiotic for your selective medium. (You won't normally need to do this with plates, because the antibiotics are usually added to the LB agar before it's poured into plates.) This is a dilution, so to figure out how much antibiotic to add, you can use the equation C1V1=C2V2. For an explanation of how to use it, look at the Calculations page (especially the "Dilutions" part and the "Adding ampicillin" part.

Using micropipettes

For everything you do in the molecular biology lab, you'll need to handle small amounts of liquid. The fundamental tool for that is a micropipette such as a Rainin Pipetman, which we have in lab. I will demonstrate how to use them in lab. You'll use them every day; in today's lab you'll need to pipet the correct amount of antibiotic solution into your liquid cultures.

Here's an excellent video on how to use them: Using a Micropipette from the University of Leicester.

Methods

For today, each lab group will need to start two plate cultures and two liquid cultures from starter plates that have already been prepared.

Materials

Before you get your other materials, put on gloves and clean your workspace (probably half your table) with disinfectant. Get all your personal stuff out of your working space. Now obtain the following:

  • Starter plate with colonies of E. coli HB101
  • Starter plate with colonies of E. coli S17/pARO180
  • 1 LB/amp plate
  • 1 LB/Strep plate
  • 2 LB liquids (culture tubes containing 4 ml sterile LB broth)
  • 1 tube of ampicillin stock solution
  • 1 tube ot streptomycin stock solution
  • Inoculating loop (metal)

Start liquid cultures

We usually use plates with selective or differential media to isolate bacterial colonies and learn something about them (for example, do the cells contain a particular plasmid?). Once we have isolated the particular cells we want, we use liquid cultures to produce large numbers of cells so we can analyze the DNA or protein from the cells. When you start a liquid culture, it's best to start from a single colony.

Using a bacti-cinerator to sterilize a loop.

  1. Start your bacti-cinerator first; it will take a long time to warm up. It's not ready to use until it's hot enough to glow red inside. If you use it before it's fully heated, you will probably contaminate your cultures. Danger! It's hot!
  2. Label your two liquid culture tubes by putting tape on the tube and writing on the tape. Label both tubes with your initials and the date, and label one tube "HB101" and the other "pARO." Don't write directly on the tubes or lids, because we will reuse them. Don't put your tape labels on the lids, because you might mix them up.
  3. Prepare your liquid media by adding the appropriate antibiotics. Put ampicillin in the "pARO" tube, and streptomycin in the "HB101" tube. Use the amounts you calculated earlier.
  4. Sterilize the loop: Wait until your bacti-cinerator is completely hot; it should be glowing orange inside. Sterilize your loop by holding it in the bacti-cinerator until the wire glows orange. (Don’t leave the loop in the bacti-cinerator when you’re not holding it; it’s likely to overheat and burn someone, or melt the loop, or burn the lab table when it falls out.) When you take the loop out, it’s hot; let it cool for 10 seconds or so (don’t set it down or wave it around; it’s likely to get contaminated.)
  5. Get some bacteria from your HB101 starter plate: The loop might still be hot. First touch a region of the agar that doesn’t have any colonies. This will cool down the loop. Then gently touch the loop to a colony on your culture plate. You don’t need to scrape up the whole colony; if you can see any bacteria on your loop, that will be plenty to inoculate a new culture. 
  6. Transfer the HB101 colony to the HB101 liquid culture, which already has streptomycin. Dip the loop into the LB liquid and spin it briefly. You'll probably see a tiny blob of bacterial colony fall into the liquid, but even an invisible amount of bacterial cells is enough to inoculate a culture.
  7. Sterilize the loop and repeat the process, this time transferring S17/pARO180 bacteria into the "pARO" tube.
  8. Sterilize the loop again when you're done.
  9. Put your two inoculated liquid cultures into the shaker incubator. We use the shaker with liquid cultures to keep the bacteria stirred up so they get better access to nutrients and oxygen. The instructor will show you where it is.

Start plate cultures

Spreading bacteria onto a plate is called "streaking;" it requires more technique than starting liquid cultures, because it's important to get isolated colonies.

  1. Your bacti-cinerator should already be hot.
  2. Label your plates by writing directly on the bottom of the plate. Put your initials and the date on each. Label the LB/amp plate "pARO180" and the strep plate "HB101." Divide your plates into 4 sections by drawing lines or making marks.
  3. Sterilize the loop, as described above.
  4. Get some bacteria from your starting culture plate: The loop might still be hot. First touch a region of the agar that doesn’t have any colonies. It will cool down instantly. Then gently touch the loop to a colony on your culture plate. You don’t need to scrape up the whole colony; if you can see any bacteria on your loop, that will be plenty to start colonies on the new plate. (In a future lab, you will start plates from liquid cultures. For that, just dip the loop into the liquid. The wire loop will hold a few microliters.)
  5. Streak the bacteria onto section 1 of your new plate. Make 4 or 5 zigzags across the section. Don't try to fill in the whole section. This section is likely to produce dense growth, with no isolated colonies.
  6. Sterilize the loop again. Spread the bacteria from section 1 into section 2 by making 4 or 5 zigzags across both sections. Don’t put more bacteria on the plate at this point; just spread. Section 2 should give you thin streaks and maybe some isolated colonies.
  7. Sterilize the loop again and spread bacteria from section 2 onto section 3. This section should give isolated colonies.
  8. Sterilize the loop again and spread bacteria from section 3 onto section 4. This section will give colonies only if your initial culture was very dense.
  9. Sterilize the loop one more time, and you’re done.
  10. Incubate your plate upside down in the plate incubator.
Plate streaked with quarter method

What your plates should look like:

The great thing about this streaking technique is that it will work to produce isolated colonies regardless of the density of your original culture. When you get isolated colonies on an antibiotic plate, you can be sure that they are actually antibiotic resistant and not satellites.

This photo shows a typical streaking result. Section 1 has a dense smear of bacterial growth. If you pick cells from this area, you can't be sure whether those are truly antibiotic-resistant cells or non-resistant satellite colonies. Section 2 is less dense, and section 3 is perfect for picking plenty of isolated colonies that are clearly antibiotic resistant.

If you start with a smaller number of cells, you might only see colonies in the first two sections. That's fine, as long as you get some isolated colonies.

Before you leave

You should have two liquid cultures in the shaker incubator:

  • S17/pARO180 in LB/amp
  • E. coli HB101 in LB/strep

Two plates in the plate incubator:

  • S17/pARO180 in LB/amp
  • HB101 on LB/strep

Now clean up your workspace and put things away:

  • Used pipet tips go in the biohazard waste container.
  • Beaker goes in the used glassware tub on the bottom shelf of the cart.
  • Everything else goes back where you found it.
  • Disinfect your workspace.
  • Throw your gloves in the regular trash and wash your hands.

That's all for today. You'll see your culture results next time.

Alternate protocol: pipet liquid onto the plate

You won't do this technique on the first day of lab, but you may do it later this quarter. If you're streaking a plate from a liquid culture, you can normally just dip the loop into the liquid and spread it on the plate as described above. The loop only transfers a small amount of liquid (2-8 μl) onto the plate. This normally works well, because the liquid culture will contain a lot of cells, and you only need a few. However, in some cases, the cells you want will be present only at low concentration, so you'll need to transfer more liquid to the plate. For example, in both the Conjugation and pGLO transformation labs, you will start with a large number of cells, but only a small percentage will take up the plasmid and become ampicillin resistant. To insure that you get enough colonies, start by pipetting some of the liquid onto one section of the plate, and then spread the bacteria, modifying the technique described above. Your instructor will advise you on how much liquid to start with.

Lab day 2: view your results

You'll need to check whether your cultures grew. For your liquid cultures, you should see cloudy liquid with a blob of bacterial cells on the bottom of the tube. For the plates, you should see clearly defined colonies. Ideally the plates will show one area of dense growth (section 1), and then other sections of the plate with more dispersed colonies.

If your cultures grew as expected, you're ready for your next experiments. If not, you'll have to come up with new cultures from another source. Your instructor will advise you on this.

Review

You should understand these terms, concepts, and techniques:

  • Antibiotics and antibiotic resistance
  • Calculations: know how to calculate the amounts of antibiotics used in today's lab
  • Colonies on plates; why a colony is usually a clone
  • Culture media
  • Differential media
  • Inoculating cultures (what this term means and how to do it)
  • LB
  • Micropipette use: know how to read the volume
  • Plasmids and their role in antibiotic resistance
  • Plate vs. liquid media, and why we use them
  • Selective media
  • Shaker incubator and plate incubator

References & further reading

Videos

How To Perform Streak Plate Technique and How To Use Aseptic Technique from Bio-Rad Explorer. These overlapping videos demonstrate methods for transferring bacteria without contaminating the cultures. The techniques in this video are different from the techniques you'll use in the 6B lab; we don't use bunsen burners.

Streaking technique

How to Streak for Single Colonies from Synthetic Biology One. This video starts by explaining the rationale for streaking plates. It shows the traditional method of streaking, which I think is a little harder to get right than the simplified version I described above. Also, the video shows the use of disposable plastic loops, but we use metal ones that can be sterilized.

Micropipette use

Using a Micropipette video from the University of Leicester.

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