Bacterial Taxonomy 1 – Classification Based on Morphology and the Gram Stain

Taxonomy, is literally the science of classification. Look at the picture above, and imagine that all those little divisions, like “firmicutes” are different phyla under the kingdom of bacteria. Then those phyla are further subdivided into different classes, then orders, then families, then genera, and then finally species! Take a look at how this works for one particular bacteria, called streptococcus mutans.

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Wow, there’s a lot to classify, probably why it’s taxonomy: it’s such a taxing job.

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Ahem, right, so. As you’ve probably noticed, Streptococcus mutans is named using its Genera and its Species name. Similarly, all organisms have a scientific name comprising of two parts: The genus, followed by the species. It is very important to classify organisms in this way because:

  1. It establishes criteria for identifying organisms.
  2. Allows arrangement of related organisms into groups.
  3. Provides important information on how organisms evolved.

Bacteria are classified, usually, according to their morphological, metabolic and biochemical differences, although genetic and immunologic factors are also now being considered.

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(Picture courtesy Principles and Explorations in Microbiology, 8th ed. – J. Black).

One of the earliest, and most fundamental methods of classifying bacteria depended on the use of the Gram Stain.

Gram Stain

Unlike large organisms like humans, parrots and dra-, erm, Komodo Dragons, which are easy to spot and have a distinct appearance to the eye, bacteria are colourless and invisible to light microscopy. Thus, gram staining had to be developed to give bacteria a colour, and visualize them. Since bacteria would either respond to the stain, or not, all bacteria were subsequently classified into gram-positive and gram-negative bacteria.

There are 4 steps to the Gram Stain Procedure.

  1. Pour crystal violet stain (a blue dye) and wait for 60 seconds.
  2. Wash off with water and flood with iodine solution. Wait for 60 seconds.
  3. Wash off with water and then “decolourize” with 95% alcohol solution.
  4. Counter-stain with safranin (a red dye). Wait 30 seconds and then wash off with water.

Basically, when viewed under the microscope, cells that absorb the crystal violet dye and hold on to it become blue: These are gram-positive. Alternatively, if the crystal violet is washed off by the 95% alcohol, the cells absorb the safranin and appear red. These are gram-negative.

Gram Positive = Blue 

Imagine yourself sitting by the beach, opposite crystal blue waters, or kayaking across deep waters, or even river tubing across the bluest of rivers.. won’t you say yes to that? So, Blue = Positive. Note that Gram-Positive bacteria may also appear purple if the red safranin is not effectively washed off. This is because blue + red is purple.

Gram Negative = Red

Now you’re sitting in sweltering red heat, sweat pouring down your body, the sun red in the sky. You don’t want that, do you? Or for comic book fans, you can picture Superman’s face when he sees a Red Sun in the sky. NOPE, thinks Superman. So Red = Negative.

This difference occurs due to a difference in morphology of the bacteria.

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Gram Positive vs Gram Negative Bacteria 

Unlike eukaryotic animal cells which contain only one cell membrane composed of phospholipid separating the nucleus from the ECF, both gram-positive and gram negative bacteria contain more than 1 layer: the layer outside the bacterial cytoplasmic membrane is the peptidoglycan layer.

The peptidoglycan layer is composed of repeating disaccharide units that contain 4 amino acid groups attached to an O-C(CH3) unit.

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These amino acids then form side linkages with other disaccharide units in neighboring chains. This allows the formation of cross linkages. The enzyme that allows the formation of these amino acid-amino acid peptide bonds across chains is transpeptidase, located in the inner cytoplasmic membrane. As a side note, this is the enzyme targeted by penicillin, vancomycin and cephalosporins.

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The major difference between the gram-positive and gram-negative is the thickness of the peptidoglycan layer. The gram-positive cell wall is very thick due to a very high degree of cross-linking and high transpeptidase activity. In contrast, the gram-negative cell wall is very thin due with a simple and not complex cross-linking pattern. To visualize this easily, imagine two lines, representing the cell walls, with a space in between to specify the thickness of the cell wall. Now try squashing a +, as in Gram positive, in between the two lines. The two lines must have a high space in between to support the + shape. In contrast, a – sign is easily squashed by the two lines. This way, you can remember that:

“+” is thick peptidoglycan layer is gram positive.

“-” is thin peptidoglycan layer is gram-negative.

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There is also a difference in the structure of the outer layer between gram-positive and gram-negative bacteria.

Gram-positive bacteria possess a cell wall composed of a thick peptidoglycan layer, as described previously, alongside teichoic acid, polysaccharides and other proteins. Teichoic acid is composed of polymers of either ribitol phosphate or glycerol phosphate.very important as an antigenic marker, making it important in serologic identification of gram-positive species. It should be noted that teichoic acid that remains bound to the peptide linkages keeps the name, however, teichoic acid bound to lipids from the inner cell membrane, is then called lipoteichoic acid. Furthermore, the inner surface of the cell wall touches the cytoplasmic membrane. The cytoplasmic membrane, in turn, contains proteins that span the lipid bilayer. Thus, the outer cell wall and inner cytoplasmic membrane comprise the cell envelope of the gram-positive bacteria.

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In contrast, Gram-Negative bacteria possess a cell envelope made of three layers. It still possesses a peptidoglycan cell wall and an inner cytoplasmic membrane; however, a Gram-negative bacteria also possesses an outermost cell membrane.

The inner cell membrane, as in Gram-positive bacteria, still contains a phospholipid bilayer with embedded proteins. However, unlike in gram-positive bacteria, where the inner surface of the cell wall touches the cytoplasmic membrane, there exists a definite periplasmic space between the thin peptidoglycan layer and the inner cytoplasmic membrane. This space is filled with a gel-like matrix, the periplasm, that contains proteins and enzymes. In some bacteria, the periplasm contains Beta- lactamases that break down penicillin, making them more resistant to penicillin like drugs.

The thin peptidoglycan layer also does not contain any teichoic acid, instead containing a small, helical lipoprotein known as murein lipoprotein. This lipoprotein originates from the peptidoglycan layer and extends outwards to attach to the outer cell membrane.

This outer cell membrane is a very typical cell membrane, with a phospholipid bilayer structure and hydrophobic tails in the centre. It is unique, however, due to the presence of LPS or lipopolysaccharide on the outermost portion of the bilayer.

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The LPS, or lipopolysaccharide is an endotoxin. It is responsible for many of the features of disease during infection by these organisms, such as fever and shock. It is different from an exotoxin since it is a vital part of the structure of the bacteria, and thus cannot be removed from the body as an exotoxin would be. An LPS is composed of 3 covalently linked components:

  1. Outer carbohydrate chains of 1-50 oligosaccharide units that extend into the surrounding media. These differ from one organism to another and are antigenic determinants. This is known as the O-specific side chain, or O-Antigen. Since it is outermost, and is an antigenic determinant, O-antigen should come to mean “Outer-Antigen.”
  2. The middle is composed of a water soluble core polysaccharide.
  3. Innermost, lies lipid A, a disaccharide with multiple fatty acid tails reaching into the membrane. Lipid A is the toxic component of the LPS, and is the endotoxin of the gram-negative bacteria. The endotoxin of the gram-positive bacteria is in fact, the less powerful teichoic acid. In any case, when bacterial cells are lysed by our own immune system during infection, fragments of membrane containing lipid A are released into circulation, causing fever, diarrhea and possible fatal endotoxic shock (septic shock).
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(Pictures Courtesy Clinical Microbiology Made Ridiculously Simple 6th ed. – M, Gladwin)

Also embedded in the outer membrane of the gram-negative bacteria, lies proteins known as porins. These porins allow passage of nutrients into the bacteria, and are only necessary in gram-negative bacteria.

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 To remember ALL the differences between the Gram-positive and Gram-negative bacteria, take a look at this pretty nifty way of remembering the differences!

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A LONG PPT, as in a boring, long powerpoint, is at least of some help now, right?

This difference between gram-positive and gram-negative bacteria yields different medical consequences and treatment paths.

Gram-positive bacteria: The thickly meshed peptidoglycan layer, while providing a diffusion barrier, does not block diffusion of low molecular weight compounds. This means that substances that damage the cytoplasmic membrane can pass through, such as antibiotics, detergents and dyes. This is the reason that the 95% alcohol does not wash off the crystal violet dye, and the gram-positive bacteria appear blue.

Gram-negative bacteria: The negative outer LPS-containing membrane blocks the passage of substances that can damage the cytoplasmic membrane and fragile peptidoglycan layer. Thus, antibiotics, penicillin and lysozyme are unable to pass through.

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Taxonomy Based on Bacterial Morphology

Bacteria can have 4 major shapes:

1) Cocci: spherical

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2) Bacilli: rods. Especially short bacilli are called coccobacilli. (Imagine compressing a rod length wise – it gets more and more spherical, hence adding the “cocco” in the name.

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3) Spiral Forms (Spirilla): Comma-shaped, S-shaped or spiral shaped.

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4) Pleomorphic: Lacking a distinct shape.

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These creatures all organize together to form more complex arrangements and patterns.

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For example, 2 cocci pairing together forms diplococci.

cluster of cocci form staphylococci, like a staff picture of your teachers plotting to destroy you with hard exams.

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^Pictured above, staphylococci (think staffylococcus). 

Alternatively, they can be arranged in long strips, in which case they are streptococci (think striptococci).

If the cocci are arranged in a cuboidal pattern, it implies that the bacteria belong to the genus of sarcina.

FInally, 4 cocci arranged together form a tetrad.

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Notice that the bacilli follow a similar concept as the cocci. Noteworthy however, is the absence of the staph variant of bacilli. Thus, there is only streptobacilli, there is no staphylobacilli. Instead, clusters of bacilli fall under palisades.

Taxonomy Based On Staining

The only bacteria you need to remember are the gram-positive ones. This is because there are only 12 gram-positive bacteria; all the rest are gram-negative (with a few exceptions, which will be mentioned).

Gram Positive Bacteria 

Of the 12 gram-positive bacteria, are cocci and 6 are bacilli.

The gram-positive cocci are:

  1. Streptococcus
  2. Staphylococcus
  3. Enterococcus 
    1. Enterococci were once considered streptococci, but were put into a separate category because they can appear either as diplococci or short chains.
  4. Micrococcus
    1. Appears in tetrads.
  5. Peptostreptococcus
    1. Occur in short chains, as diplococci, or even individually.
  6. Peptococcus

(So yes, ALL streptococci and staphylococci are gram-positive!)

The 6 Gram-positive bacilli are:

  1. Bacillus
    1. This may sound confusing. But it’s really just poor naming. You see, a bacillus is a rod-shaped bacteria, as discussed earlier. However, Bacillus, capitalized and italicized, refers to a specific genus of bacteria that is rod shaped (sigh).
    2. Noteworthy because it produces spores, which are spheres that protect a dormant bacterium from the harsh environment).
  2. Clostridium
    1. Also produces spores.
  3. Corynebacterium

    1. Appears in palisades
  4. Listeria
    1. Appears in palisades
  5. Erysipelothrix
    1. Appears in palisades
  6. Propionobacterium
    1. Appears in palisades

There are also filamentous bacilli that are gram-negative and show extensive branching, and these are the Actinomyces, Nocardia, Arachnia and Streptomyces. 

Gram-Negative Bacteria 

There are only 3 gram-negative cocci. Since all the streptococci and the staphylococci are gram-positive, it follows that the gram-negative bacteria are all diplococci. They are:

  1. Neisseria
  2. Moraxella
  3. Veillonella 

Similarly, there is only 1 group of spiral shaped bacteria, or spirilla. This group is:

  1. Spirochetes.
    1. The spirochetes include several bacteria, and of these, the disease causing bacteria are:
      1. Treponema pallidum – causes syphillis
      2. Leptospira species – causes leptospirosis
      3. Borrelia burgdorferi, B. garinii, B. afzellii – cause Lyme Disease
      4. Borrelia recurrentis – Relapsing fever (think recurrentis as in recurring, hence relapsing)
      5. Brachyspira pilosicoli and Brachyspira aalborgi – causes intestinal spirochaetosis

ALL OTHERS gram-negative bacteria are either bacilli, coccobacilli or pleomorphic.

The pleomorphic bacteria include rickettsiae and chlamydiae, that are very small bacteria and cannot be seen under the light microscope effectively.

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Bacteria that Stain Under Special Circumstances

As mentioned previously, some bacteria do not neatly fall into the categories of gram-positive or gram-negative. These are:

Mycobacteria: Mycobacteria are only weakly gram-positive. This is because there is a very high amount of lipid in the cell wall, and thus, the dye does not easily penetrate. Instead, you have to use a dye known as the acid-fast stain. To remember this, simply think of the mycobacterium as an obese bacteria, trying to fast to get thinner.

So mycobacteria = high lipid content in cell wall —> acid fast stain required.

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 (Courtesy midlandstech)

Spirochetes: Don’t be mistaken. Spirochetes are still gram-negative. The reason they are in this list however, is because even though they will stain with a gram stain, they are too small to be seen under a light microscope. Thus, a special type of microscope, the darkfield microscope, must be used for these bacteria.

Spirochetes are also unique morphologically. While, like other gram-negative bacteria, they contain an inner cytoplasmic membrane, thin peptidoglycan cell wall, and outer cytoplasmic membrane fitted with LPS, they also contain an additional fourth outer membrane, with very few embedded proteins. This is believed to help spirochetes avoid antigenic identification, making spirochetes a “stealth organism.” A ninja, really. To remember this, think of how cheaters need to be stealthy to get away with what they’re doing. Well, so do spirochetes. Furthermore, spirochetes possess an axial flagella, that emerges from the spirochete cell wall, but runs sideways along the spirochete under the outer membrane sheath. Thus, the flagella is referred to as a periplasmic flagella. Rotation of this periplasmic flagella generates thrust, that propels the spirochete.

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Mycoplasma: Mycoplasma actually have no cell wall at all! They only have a cell membrane, and thus they are neither gram-positive, nor gram-negative.

Rickettsiae and Chlamydiae: These pleomorphic bacteria are extremely small, and intracellular, and cannot be seen under the light microscope despite being gram-negative.

Legionella Pneumophila: Has poor uptake of red safranin stain, and thus, a more intense application of safranin is required.

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(Courtesy Review of Medical Microbiology and Immunology, 12th ed)

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(Courtesy Clinical Microbiology Made Ridiculously Simple, 6th ed.)


That’s all guys! Hope this was useful for you all. You should now have an idea for names of all the bacterial genera, and be able to classify them as gram negative or gram positive. Additionally, you should be able to identify the differences between a gram-positive and gram-negative bacteria.

Here are some other resources for you guys!

VIDEOS:

Classification of Bacteria 

QUESTIONS:

1) A patient comes into your office complaining of small, painless sores and ulcers around her genital area. She claims she is sexually active. What bacteria is most likely to cause her condition?

A. Treponema pallidum

B. Rickettsiae 

C. Borrelia recurrentis

D. Staphlyococcus Aureus 

2) Which of the following is not representative of gram-positive bacteria:

A. Staphylococcus

B. Peptococcus

C. Spirochetes

D. Corynebacterium

ANSWERS:

A, C

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