Practical Secrets of the Bacterial World for the Uninitiated

As a protein biochemist where bacteria were mere workhorses, imagine my surprise when I began work in a bonafide micro lab! I discovered that bacteria could be much fussier than my good ol’ cloning and expression friends E. coli DH5ɑ or BL21. One broth would not do for all, some even required blood! No, no, not human blood, sheep’s blood, coming in nice rounded 500ml bottles. Learning about the complexity and adaptiveness of bacteria has been a marvelous and sometimes frustrating journey. Whether you are culturing your microbes to perform molecular techniques like PCR or sequencing or want to see them up close using microbiology microscopy you need to know how to take care of them. Here are some practical microbiology guidelines for anyone starting out in bacterial microbiology after coming from a different field.

1. Food

One of the amazing things about bacteria is their ability to adapt to different food niche markets (there are bacteria that eat nuclear waste!) and there is in turn a vast selection of growth media, each optimized to promote the growth of a particular researchers bug. For example, LB stands for Lysogeny broth and although it was originally optimized for E. coli phage lysogeny, it’s now been adopted as a very standard broth for growth of a variety of bugs.

LB is not always the best media to use as explained on the Small Things Considered blog. While E. coli are fairly unfussy in terms of what they can and will grow on, some buggy friends need a lot more care and consideration, especially if you want speedy growth. Helicobacter pylori for example is notoriously fussy, requiring blood containing growth media and special air requirements. The bottom line is that if you are starting work with a new bug that you just got after lengthy efforts from a distant colleague, you want to make sure it grows well; it is worth doing some literature searching or asking the group you got it from (!)  to investigate the right conditions before you start!

Talking about speedy growth, the time taken for one bacteria to become two, referred to as the doubling time, varies enormously between bacteria. For example, under optimal growth conditions, E. coli has a doubling time of 20 minutes where as that of Mycobacterium tuberculosis is 24 hr, which can have huge implications when planning experiments!

2. Air

As I mentioned, H. pylori is a fussy guy in terms of media but it also requires 10% CO₂. It does not tolerate being exposed to regular air for long periods. The naming system for buggy air requirements will clue you in on how to treat your bugs:

• Obligate aerobes require oxygen to respire, e.g., Mycobacterium tuberculosis also known as TB.
• Faculative anaerobes can grow both in the presence and absence of oxygen, e.g., E. coli
• Obligate anerobes cannot growth in the presence of oxygen. They use alternative electron acceptors such as nitrate or sulfates. For example, Clostridium tetani, the cause of tetanus, is one of these.
• Microaerophiles need oxygen, but not too much of it, and often also require CO₂ such as H. pylori. This makes sense because the preferred habitat of H. pylori is the mucus lining of your stomach where oxygen is low and CO₂ is higher because of respiring stomach cells.
• Aerotolerant bugs do not need or use oxygen to respire but are not killed by it either, e.g., the gastroenteritis inducing Campylobacter jejuni.

3. Temperature

Generally, in the medical microbiology field, organisms that grow at 37°C are the norm because this is the temperature of the human body and these are known as mesophiles. They operate in a temperature range of 30 to 37°C. Many will continue to grow slowly at lower temperatures and can be stored in the fridge on an agar plate.

4. Nomenclature

Everyone likes to be called by their proper name, right? Bacteria are referred to by their genus and species names which are written in italics, e.g., Escherichia coli (genus, species). This is often shortened to a capital letter for the genus name. For example, E. coli, still in italics with a period after the capital letter.

Once you become friends or even brief acquaintances, nicknames are also acceptable, and informally, microbiologists may use just part of the name when referring to strains they commonly use—though there do seem to be various unspoken rules that I suspect also vary between labs. For example, Enterococcus faecalis becomes faecalis, Staphylococcus aureus becomes Staph, and Staphylococcus epidermidis becomes Staph epi.

You’ll see also that there are different strains of bacteria such as E. coli DH5?. This refers to a strain with deliberately created genotypic differences. Subspecies are also genotypically different, but not engineered this way, and are part of the bacterial classification.

Salmonella is a treasure trove for this; for example, Salmonella enterica has 6 subspecies two of which are Salmonella enterica subsp. enterica and Salmonella enterica subsp. salamae. You will also see strains denoted to have immunological differences (serovars or serotypes) such as Salmonella ser. Typhi, where the serotype is not italicized and has a capital first letter. You can read more about Salmonella nomenclature here.

This can all get somewhat confusing, so stick to your bacteria to start and once you have your bug down, adding on others will be a breeze. However if classification is “your thing”, a good website for more in depth detail on bacterial classification can be found here.

5. Nomenclature for genes and proteins

As your research progresses, layers may be added but in the beginning here’s what you’ll need for understanding references to genes and proteins:

• Wild type gene names are given 3 letter abbreviations, written in italics in lower case (e.g., abc). If there are genes of the same protein but a different subunit, the subunit is given a letter (e.g., xyzA, xyzB)
• Deleted genes or mutants are written in the same way with a greek delta symbol (ΔxyzA) or a – sign following (xyzA-). When referring to a strain where the gene has been replaced by an antibiotic resistance marker such as kanamycin it is written as abc1::kan. To distinguish WT genes from the mutant genes if neither a greek delta symbol or a minus sign is used, then the WT gene is followed by a + and the mutant/deleted gene has no symbol. (e.g., xyzA+) for the WT and xyzA for the mutant.
• For proteins from the gene in question, lose the italics but retain the capital letter Abc1. Watch out though – not all proteins are called the same name as their gene! I work a good deal with E. coli, as a pathogen and as a commensal (a micro-organism that is part of the human flora and does good works for your body!) and two good resources are Ecogene and Ecocyc. Several micro-organisms have their own databases – your colleagues will be able to let you know about internet resources and failing that, there is everyone’s search and retrieve colleague – Google!

The Journal of Bacteriology has a more detailed description of accepted nomenclature guidelines here.

So, to be besties with your medical bug, find its optimal food, air and temperature requirements and don’t call it names! It could be the beginning of a beautiful friendship.

What are your tips for working with bugs? Any practical microbiology tips? Let us know in the comments below!