Identifying an unknown bacterial strain

Why identify a bacteria ?

Bacteria are everywhere, they are part of our environment and even of us. In fact, we are more bacteria than human ! Indeed, we have approximatively 1013 human cells and 1014 bacterials cells in us. Therefore, we encounter bacteria everywhere and it is sometimes necessary to identify them. Whether it is to determine the cause of a disease, to test if a certain food is safe to eat or simply to know what is present in a certain ecosystem, we have developed many techniques to identify bacteria.

Bacteria may seem like very simple organisms and you might think that most of them share many characteristics. In fact, every species is unique and has particular characteristics. This makes it possible to identify an unknown species.

In this article, I'm going to go over some of the simple tests you would perform on your unknown in order to identify it.


First some basics

Before going over the tests to identify an unknown bacterial species, we should remember some bases of manipulating bacteria.

It is important to always keep in mind that your unknown species is a potential pathogen. This means that it could be harmful to you. Therefore, when working with bacteria you must wear a lab coat, safety glasses and gloves. If you suspect that your bacteria might be an airborne pathogen (depending on where it comes from: if you took it from a sick patient, it has great chances of being harmful), it is recommended to work in a biohazard safety cabinet.

Moreover, you must use the proper aseptic techniques to keep out all unwanted organisms from your culture. If you use a loop or a needle to transfer bacteria from a medium to another, you must flame the loop or needle in the flame of a Bunsen burner for a few seconds and then wait for the wire to cool down to avoid killing your bacteria. You must always work in the area around our flame since microorganisms are present in the air. The area around the burner can be considered sterile. If you transfer your bacterium to or from a tube, you should flame the neck of the tube for a few seconds before and after. It creates a convection current and kills the cells that might have fallen in it during the manipulation.

Bacteria are cultured in either liquid or solid medium. Both contain agar, which is composed of complex polysaccharides, NaCl and yeast extract or peptone. It melts at 100°C and solidifies at around 40-45°C. In normal media, the concentration of agar is 1,5%.

Now that the basics are covered we can move on to start testing on our bacteria to determine which species it might belong to !

Example of a particular culture morphology


Culture morphology

When find an unknown bacterium, you first make a pure culture of it on an agar plate. A pure culture arrises from a single cell and thus contains only one type of microorganism. A colony is a visible mass of cells. Different bacterial species creates different culture morphologies. You can focus on the form, elevation, margin, surface, optical characteristics and pigmentation of your culture to describe it. Some species make very particular colonies. For example, Serratia marcescens forms bright red colonies and can be easily identified thanks to this pigmentation.

Unfortunately, a lot of bacteria have very common colonies (round, flat and white or creamy white) and this test is not enough to identify with certainty a species. But it's still a very useful first step and helps progress in the identification of the bacteria.

It's mostly a technique to rule out some options and to make sure we are dealing with a bacteria and not for example a mold.

Cell morphology

The second step to your identification is to put your unknown on a microscope slide and observe the morphology of your cell.

The most common shapes are :

  • Coccus (round)
  • Bacillus (rod-shaped)
  • Vibrio (comma-shaped)
  • Spirochete (spirale)

But some bacteria have very unique shapes and are therefore highly identifiable like that. For example some bacteria are square or star shaped.

Bacteria also grow in characteristic arrangements. They can grow by pairs and we add the prefix di-, in chains which is called strepto-, by four, in which case it's a tetrad or in clusters, to which we add the prefix staphylo-. For example, species from the Staphylococcus phylum are round bacteria that grow in clusters.

Common bacterial shapes



We talked about cell morphology earlier but it is true that bacterial cells are often colorless and therefore you wouldn't be able to see anything under the microscope. Therefore, different methods of staining exist to be able not only to see but also to differentiate bacteria.

A simple stain is the application of a single staining solution like methylene blue, carbon fushin or crystal violet to be able to see the morphological characters of your cell. The dying solution can be either basic or acidic. A basic dye, for example methylene blue, has a positively charged chromophore whereas an acidic dye like eosin has a negatively charged chromophore. Considering that the surface of bacteria is negatively charged, basic dyes go in the cell whereas acidic dyes are repelled and surround the cell.

A differential stain is the application of a series of reagents to show species or structural entities. There are many different stains to reveal different characteristic. We will go over them quickly.

The negative stain uses nigrosin which is an acidic stain. It therefore surrounds the cells which appear under the microscope. It's a gentle stain that doesn't require heat-fixing and thus doesn't distort bacteria. It is mostly used for observing bacteria that are difficult to stain.

The Gram stain is used to differenciate Gram-positive from Gram-negative bacteria. Gram-positive bacteria have a thicker peptidoglycan layer and therefore retain the primary stain (crystal violet) whereas Gram-negative cells lose it when treated with a decolourizer (absolute alcohol). They then take in the secondary stain (iodine). Gram-positive cells, like Staphylococcus aureus, are purple under the microscope and Gram-negative cells, for example Escherichia coli or Neisseria subflava, turn out red.

The acid fast stain differentiates bacterial cells with lipoidal cell call. Cells are treated first with carbol fushin which is heat-fixed, then with acid alcohol which decoulorize all cells except acid fast bacteria and finally with a counterstain (methylene blue). Under the microscope, acid fast cells are red and the others are blue. An example of an acid fast bacterial species is Mycobaterium smegmatis.

The cell wall stain stains, as its name suggests, the cell wall of bacteria. The cell wall is composed of lipopolysaccharides, lipoproteins, phospholipids and peptidoglycan. It surrounds the bacteria and gives it its shape. To perform a cell wall stain, you make the negatively charged cell wall positive with a cationic surface agent like cetylpyridinium, you then stain it with Congo red and finally counterstain with methylene blue. The cells will appear blue and the cell wall red. This is used to see whether or not the bacteria have a cell wall as some, like Mycoplasm species, lack a cell wall.

The spore stain is used to detect if the bacterial species produces spores. Spores are highly resistant cells formed by some species of bacteria to escape and germinate when it reaches more favorable conditions.The primary stain is malachite green which is heat-fixed followed by a counter stain with safranin. The spores stain green and the cells red. Bacillus subtilis creates a subterminal spore and Clostridium tetanomorphum has a terminal spore.

The capsule stain detects if your unknown bacterium has a capsule which is a secondary structure made of polysaccharides surrounding the bacteria to confer it additional resistance, nutrient storage, adhesion and waste dumping. An example of a species with a cell wall is Flavobacterium capsulatum. To perform a capsule stain, you need to smear you bacteria with nigrosin, then fix it with absolute alcohol and staining with crystal violet.

Finally, the flagella stain detects whether or not the bacteria possesses one or multiple flagella. Flagella are hair-like structure used by bacteria to move around. To do a flagella stain you need to use young cultures because they possess well formed, intact and less brittle flagella and you need to increase the thickness of the flagella with mordants like tannic acid and K+alum in order to be able to see it under the microscope. Pseudomonas fluorescens has one flagellum (it is called montrichous) and Proteus vulgaris has several flagella (peritrichous).

All those stains give you additional data on your unknown cell and brings you closer to knowing which species it belongs to. However, it is not enough information to be certain about its species. You might be starting to guess a phylum but you need to perform additional tests to know more about your cell.

Anaerobic jar



The next step to determining which bacteria you have is to know if it's aerobic or anaerobic. In other words, does it need oxygen to grow or can it use fermentation or anaerobic respiration. There are also bacteria that are facultative anaerobes, meaning that in presence of oxygen, they will use it but if they find themselves in anaerobic conditions, they'll be able to grow using fermentation pathways or anaerobic respiration. Another group is called microaerophiles and those grow best when the concentration in oxygen is inferior to 21%.

In order to know what group your bacteria falls into, you have several methods. You can either inoculate an agar plate and put it in an anaerobic jar or inoculate your bacteria directly into thioglycolate broth or cooked meat medium.

The anaerobic jar contains 5% of CO2, 10% of H2 and 85% of N2. It has a carbon dioxide generator that converts oxygen into hydrogen and carbon dioxide and a palladium pellet catalyst that takes hydrogen and oxygen to form water. It also contains an indicator that is blue when the jar contains oxygen and colourless when it's in anaerobic conditions. If your bacteria grows it is either an anaerobe or a facultative anaerobe. If it doesn't grow, it is aerobe.

The thioglycolate broth contains sulfhydryl groups which remove the oxygen from the medium. Anaerobic bacteria will grow everywhere in the medium, facultative anaerobes will grow everywhere with a preference for the top of the medium and aerobic bacteria will grow only at the top of the medium where there is still oxygen present.

Cooked meat medium contains heart tissues, meat containing cysteine residues. Those residues are rich in SH groups that can donate H to reduce the oxygen, forming water. Like in the thioglycolate broth, aerobes grow on top, facultative anaerobes grow everywhere but mostly on top and anaerobes grow everywhere. Moreover they produce H2S.

Biochemical properties (continued)

Another test is whether or not your unknown has an hemolytic reaction. Most bacteria are gamma-hemolytic, which means that they do not have an hemolytic reaction. This test is mostly used on streptococci species: it differentiates non pathogenic streptococci from pathogenic streptococci. This is tested on a blood agar plate: a beta-hemolysis creates a white discoloration around the colony whereas an alpha-hemolysis has a brownish green zone around the colony. Streptococcus pyogenes is not a pathogen and therefore is beta-hemolytic whereas Streptococcus pneumoniae or Streptococcus salivarius are alpha-hemolytic.

Another biochemical property is the production of H2S from the oxidation of sulfur containing compounds like cysteine or the reduction of inorganic compounds like thiosulfates, sulfates or sulfites. The media used is peptone-iron agar. The peptone has sulfur containing amino acids which are used by the bacteria to produce H2S and the iron detects the H2S by forming a black residue along the stab line. Proteus vulgaris for example produces H2S.

The following test is the coagulase test which shows if bacteria are capable of coagulating oxolated plasma. It is an indication of pathogenicity since if a bacteria can coagulate the blood, it can wall off from the immune system. Staphylococcus aureus can coagulate oxolated plasma and therefore blood. It is also capable of secreting gelatinase which is the enzyme that hydrolyzes gelatine into polypeptides and amino acids.

The following series of tests is called IMVIC which stands for Indole, Methyl red, Voges-Proskauer and Citrate.

  • The indole production test shows if the bacterial strain is capable of breaking down tryptophan by tryptophanophase into indole, ammonia and pyruvate. We can detect this reaction by using Kovac's reagent which is contained in amyl alcohol (not miscible in water). Kovac's reagent reacts with indole to form Rosindol dye, forming a red color that will rise to the top of the broth culture. This test is positive for Escherichia coli and Proteus vulgaris but negative for Enterobacter aerogenes for example.
  • The methyl red test tests for glucose fermentors. It turns red when the pH is inferior to 4,3. It is positive for E. coli but negative for E. aerogenes.
  • The Voge-Proskauer tests shows the production of acetoin. The reagent used is potassium hydroxide, a creatine solution. The medium turns red if the test is positive for E. aerogenes for example. It is negative for E. coli.
  • Finally, the citrate test is used to differentiate enterics. It tests if the bacterium has the permease required to take up the citrate and use it as the sole carbon source. The indicator used is bromothymol blue: the black medium turns blue if the citrate is used. E. aerogenes has the permease however E. coli doesn't.

Biochemical properties

The final step to determining your bacterial species is a series of tests to know its biochemical properties.

You can test if your bacterium can perform protein, starch or lipid hydrolysis. The method is simple: you streak your cells on a milk agar plate, a starch agar plate and a tributyrin agar plate. If a clear zone forms around your colony on the milk agar plate, it means that it has protease, the enzyme that breaks down proteins (in this case the protein is casein). Bacillus cereus for example is capable or protein hydrolysis. If a bluish brown color appears on your starch plate when you flood it with iodine, it means that your species possesses amylase, the enzyme that turns starch into dextrans, maltose, glucose. An example of a bacterial strain with this enzyme is also Bacillus cereus. Finally, your unknown has the enzyme that hydrolyses lipids into glycerol and fatty acids (lipase), if a clear zone appears around the colony. It might be Pseudomonas fluorescens.

You can then test for nitrate reduction (denitrification). You place your bacterial strain in a medium containing nitrate and an indicator. If the result is negative, it might mean that the bacteria do not reduce nitrate but it might also mean that the nitrate was reduced to nitrite and then further reduced to ammonia. In this case, you add some zinc powder to your tube: the zinc reacts with nitrate thus creating a color change. If the bacteria have further reduced the nitrogen, there will be no color change. Pseudomonas aeruginosa and Serratia marcescens reduce nitrate while Bacillus subtilis doesn't.

The next test consists in placing your bacteria in fermentation tubes with glucose, lactose or sucrose and an indicator (phenol red). The indicator is red at a neutral pH and turns yellow in an acidic pH. Here are some example of bacteria and what they ferment: Staphylococcus aureus ferments glucose, lactose and sucrose and doesn't produce gas, Bacillus subtilis only ferments glucose with no gas production, Proteus vulgaris ferments glucose and sucrose and creates gas, Pseudomonas aerugenosa doesn't ferment anything and Escherichia coli ferments glucose and lactose with gas formation.

You can also test for inulin fermentation. Inulin is fructose containing oligosaccharides. You test this in a cystine trypticase agar tube with phenol red as an indicator. It's a way to differenciate Streptococcus pneumoniae from other alpha-hemolytic streptococci. Another way of distinguishing S. pneumoniae for the others is through a bile solubility test using sodium deoxycholate solution as a reagent.

Identifying your unknown

You now have a lot of informations about your species. Putting all of it together, you should be able to have a good guess as to which species it belongs to or at least which phylum.

All those tests are done in laboratories, in hospitals, etc. in order to know what they are dealing with. Unfortunately they can't be used on any bacterium as some of them are uncultivable or do not belong to any known group. More precise techniques are used in some cases but some bacteria remain a mystery.

Diversity of bacteria

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