Mastering basic bacterial culturing practices is a must if you are planning a career in microbiology!
Growing bacteria might be one of the easiest things to do as a scientist. Also, as you’ve probably discovered, it’s even easier to do when you’re trying to prevent bacteria from growing where it shouldn’t be!!
When we go about culturing bacteria, we want to create an environment to selectively grow desired bacterial species only. This article will take you through how you can culture a specific bacterial species in an efficient manner.
Ladies and gentlemen, this is the “welcome mat” to the world of microbiology.
Learning how to culture bacteria is the first step to many a project, so let’s start laying the foundation for your future Nobel Prize!
Why Do We Want to Culture a Single Bacterial Species?
Bacteria culturing is carried out for a plethora of reasons, including:
- to isolate a single species
- to isolate a single genetic clone of a particular species
- to isolate a mixed culture of many species
- to identify an unknown species
- to aid in the clinical diagnosis of pathogens (or their absence) in patient samples
The goal is to culture only the bacterial species that you want! This can be tricky, especially if you are working with environmental or clinical samples. Many different bacterial species live in the environment around us (and in the lab too!) and in clinical material, such as feces or sputum. To combat this problem, sterility and aseptic technique are some of your most critical allies.
Sterilize Your Bacterial Culture Tools Before Use!
You can easily sterilize utensils, liquids and agar media via a number of methods in the lab. While you work, you can dip glass or metal culturing tools (such as inoculating loops or cell spreaders) in alcohol and hold them in the flame of a Bunsen burner for about 30 seconds to ensure complete sterility before use. Remember to allow 20-30 seconds for cooling before proceeding to streak/inoculate your bacteria, so as not to set your precious clones ablaze!
Alternatively, you might choose to purchase many of the commonly used culturing tools in a pre-sterilized disposable form. You can also purchase a number of buffers and ready-made media pre-sterilized.
All of these techniques and approaches seek to eliminate stray bacteria that may hop on your agar plate and overtake your culture before they become a problem.
Keeping Your Bench Clean and Aseptic Technique
Another important thing to keep in mind while trying to achieve sterility is your work space. Areas where you culture bacteria should be treated in a manner similar to how you treat a PCR work space:
- clutter free
- wiped down with alcohol spray before use
In an effort to prevent bacteria from literally falling into cultures, it is wise to work with an open flame (Bunsen) nearby. This creates an “umbrella” of heat over your workspace and can kill any ubiquitous bacteria floating around. Just don’t set yourself on fire!
Alternatively, you can perform this work in a sterile hood, which eliminates the need for the open flame, but you may need to fight anyone doing tissue culture for that space! They don’t like bacteria around their media, for good reason because bacteria easily infect mammalian cell culture.
Check out an earlier article at Biosize Bio for more details on how to work aseptically in the lab.
Use Antibiotics to Prevent Unwanted Bacterial Growth
As well as practicing aseptic technique, another common way to avoid unwanted bacteria is to take advantage of an antibiotic selection marker provided to your clone via plasmid transformation. This will allow you to grow your species or clone of interest in the presence of one of a number of different antibiotics, such as ampicillin or kanamycin, while killing anything else that is unable to break down these compounds. This strategy is very common when molecular techniques involving mutagenesis or protein expression vectors are used.
Plates? Liquid media? Tubes? Oh my!
You can use a number of different types of media to grow bacteria, and all of them can be supplemented with these antibiotics as required by the plasmids or experimental goals.
To isolate a single clone or colony, or to identify an unknown species via morphology, assessing growth on agar plates is probably your best bet. This will allow for the separation of a single genetic clone that can be analyzed microscopically or isolated for further analysis.
To expand bacterial cultures for DNA (e.g., plasmid) or protein isolation, liquid media is typically used. Microliter-sized cultures can easily be up-scaled to larger cultures as required. Liquid media is easy to prepare in large amounts, and growth parameters such as the presence of antibiotics, aeration, and temperature can easily be controlled and adjusted as needed.
Finally, if you’re specifically interested in bacteria that grow under low-oxygen conditions, use a stab tube (a test tube filled with soft agar). The stab tube will allow your inoculation loop reach deep down into the media where free oxygen is at a minimum. Inoculating liquid media in a test tube and leaving it to stand (no shaking!) can also allow you to grow colonies under low-oxygen conditions in liquid media.
Bacterial Growth Conditions – Optimum Temperature
Conditions in which bacterial cultures are grown can really impact your overall success. Typically, bacteria are grown at 37°C, because this closely simulates the temperature of the human body. Not all bacterial species, however, grow optimally at 37°C, so identifying an optimal growth temperature is important. Guidelines for growth are always given when cultures are purchased from culture banks. The scientific literature is another great place to find out how to grow your bacteria.
Suboptimal incubation temperatures can cause growth rates to decrease or even stop completely and protein expression levels can change dramatically. Growth in liquid culture can easily be monitored via spectroscopy by measuring light absorbance at 600 nm. Graphing this as a function of time illustrates a growth curve.
Position Your Bacteria for Success!
Having these tools at your disposal can go a long way towards putting yourself in the best position for long term success as you go back to frozen cultures time and time again. Early failures can lead to headaches that last years, so be careful from the start!