Methods of Classifying and Identifying Microorganisms



Phenotypic Analysis

Microorganisms can be classified on the basis of cell structure, cellular metabolism, or on differences in cell components.

Learning Objectives

Distinguish between phenotypic characteristics for Bacteria, Archaea and Eukaryotes

Key Takeaways

Key Points

  • The relationship between the three domains ( Bacteria, Archaea, and Eukaryota) is of central importance for understanding the origin of life. Most of the metabolic pathways are common between Archaea and Bacteria, while most genes involved in genome expression are common between Archaea and Eukarya.
  • Microorganisms are very diverse. They include bacteria, fungi, algae, and protozoa; microscopic plants, and animals. Single-celled microorganisms were the first forms of life to develop on earth, approximately 3 billion–4 billion years ago.
  • The Gram stain characterizes bacteria based on the structural characteristics of their cell walls. By combining morphology and Gram-staining, most bacteria can be classified as belonging to one of 4 groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, and Gram-negative bacilli).
  • There are some basic differences between Bacteria, Archaea, and Eukaryotes in cell morphology and structure which aid in phenotypic classification and identification.

Key Terms

  • Gram stain: A method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative).
  • microorganism: An organism that is too small to be seen by the unaided eye, especially a single-celled organism, such as a bacterium.
  • domain: In the three-domain system, one of three taxa at that rank: Bacteria, Archaea, or Eukaryota.

Microorganisms are very diverse. They include bacteria, fungi, algae, and protozoa; microscopic plants (green algae); and animals such as rotifers and planarians. Most microorganisms are unicellular (single-celled), but this is not universal.

Single-celled microorganisms were the first forms of life to develop on earth, approximately 3 billion–4 billion years ago. Further evolution was slow, and for about 3 billion years in the Precambrian eon, all organisms were microscopic. So, for most of the history of life on earth the only forms of life were microorganisms. Bacteria, algae, and fungi have been identified in amber that is 220 million years old, which shows that the morphology of microorganisms has changed little since the Triassic period. When at the end of the 19th century information began to accumulate about the diversity within the bacterial world, scientists started to include the bacteria in phylogenetic schemes to explain how life on earth may have developed. Some of the early phylogenetic trees of the prokaryote world were morphology-based. Others were based on the then-current ideas on the presumed conditions on our planet at the time that life first developed.

Microorganisms tend to have a relatively rapid evolution. Most microorganisms can reproduce rapidly, and microbes such as bacteria can also freely exchange genes through conjugation, transformation, and transduction, even between widely-divergent species. This horizontal gene transfer, coupled with a high mutation rate and many other means of genetic variation, allows microorganisms to swiftly evolve (via natural selection) to survive in new environments and respond to environmental stresses.

The relationship between the three domains (Bacteria, Archaea, and Eukaryota) is of central importance for understanding the origin of life. Most of the metabolic pathways, which comprise the majority of an organism’s genes, are common between Archaea and Bacteria, while most genes involved in genome expression are common between Archaea and Eukarya. Within prokaryotes, archaeal cell structure is most similar to that of Gram-positive bacteria.

Phenotypic Methods of Classifying and Identifying Microorganisms

Classification seeks to describe the diversity of bacterial species by naming and grouping organisms based on similarities. Microorganisms can be classified on the basis of cell structure, cellular metabolism, or on differences in cell components such as DNA, fatty acids, pigments, antigens, and quinones.

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Bacterial Morphology: Basic morphological differences between bacteria. The most often found forms and their associations.

There are some basic differences between Bacteria, Archaea, and Eukaryotes in cell morphology and structure which aid in phenotypic classification and identification:

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The relative sizes of prokaryotic cells: Relative scales of eukaryotes, prokaryotes, viruses, proteins and atoms (logarithmic scale).

  • Bacteria: lack membrane -bound organelles and can function and reproduce as individual cells, but often aggregate in multicellular colonies. Their genome is usually a single loop of DNA, although they can also harbor small pieces of DNA called plasmids. These plasmids can be transferred between cells through bacterial conjugation. Bacteria are surrounded by a cell wall, which provides strength and rigidity to their cells.
  • Archaea: In the past, the differences between bacteria and archaea were not recognized and archaea were classified with bacteria as part of the kingdom Monera. Archaea are also single-celled organisms that lack nuclei. Archaea in fact differ from bacteria in both their genetics and biochemistry. While bacterial cell membranes are made from phosphoglycerides with ester bonds, archaean membranes are made of ether lipids.
  • Eukaryotes: Unlike bacteria and archaea, eukaryotes contain organelles such as the cell nucleus, the Golgi apparatus, and mitochondria in their cells. Like bacteria, plant cells have cell walls and contain organelles such as chloroplasts in addition to the organelles in other eukaryotes.

The Gram stain, developed in 1884 by Hans Christian Gram, characterizes bacteria based on the structural characteristics of their cell walls. The thick layers of peptidoglycan in the “Gram-positive” cell wall stain purple, while the thin “Gram-negative” cell wall appears pink. By combining morphology and Gram-staining, most bacteria can be classified as belonging to one of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, and Gram-negative bacilli). Some organisms are best identified by stains other than the Gram stain, particularly mycobacteria or Nocardia, which show acid-fastness on Ziehl–Neelsen or similar stains. Other organisms may need to be identified by their growth in special media, or by other techniques, such as serology.

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Gram-positive bacteria: Streptococcus mutans visualized with a Gram stain.

While these schemes allowed the identification and classification of bacterial strains, it was unclear whether these differences represented variation between distinct species or between strains of the same species. This uncertainty was due to the lack of distinct structures in most bacteria, as well as lateral gene transfer between unrelated species. Due to lateral gene transfer, some closely related bacteria can have very different morphologies and metabolisms. To overcome this uncertainty, modern bacterial classification emphasizes molecular systematics, using genetic techniques such as guanine cytosine ratio determination, genome-genome hybridization, as well as sequencing genes that have not undergone extensive lateral gene transfer, such as the rRNA gene.

Classification of Prokaryotes

Prokaryotic organisms were the first living things on earth and still inhabit every environment, no matter how extreme.

Learning Objectives

Discuss the origins of prokaryotic organisms in terms of the geologic timeline

Key Takeaways

Key Points

  • All living things can be classified into three main groups called domains; these include the Archaea, the Bacteria, and the Eukarya.
  • Prokaryotes arose during the Precambrian Period 3.5 to 3.8 billion years ago.
  • Prokaryotic organisms can live in every type of environment on Earth, from very hot, to very cold, to super haline, to very acidic.
  • The domains Bacteria and Archaea are the ones containing prokaryotic organisms.
  • The Archaea are prokaryotes that inhabit extreme environments, such as inside of volcanoes, while Bacteria are more common organisms, such as E. coli.

Key Terms

  • prokaryote: an organism whose cell (or cells) are characterized by the absence of a nucleus or any other membrane-bound organelles
  • domain: in the three-domain system, the highest rank in the classification of organisms, above kingdom: Bacteria, Archaea, and Eukarya
  • archaea: a taxonomic domain of single-celled organisms lacking nuclei, formerly called archaebacteria, but now known to differ fundamentally from bacteria

Evolution of Prokaryotes

In the recent past, scientists grouped living things into five kingdoms (animals, plants, fungi, protists, and prokaryotes) based on several criteria such as: the absence or presence of a nucleus and other membrane-bound organelles, the absence or presence of cell walls, multicellularity, etc. In the late 20th century, the pioneering work of Carl Woese and others compared sequences of small-subunit ribosomal RNA (SSU rRNA) which resulted in a more fundamental way to group organisms on earth. Based on differences in the structure of cell membranes and in rRNA, Woese and his colleagues proposed that all life on earth evolved along three lineages, called domains. The domain Bacteria comprises all organisms in the kingdom Bacteria, the domain Archaea comprises the rest of the prokaryotes, and the domain Eukarya comprises all eukaryotes, including organisms in the kingdoms Animalia, Plantae, Fungi, and Protista.

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Prokaryotes in extreme environments: Certain prokaryotes can live in extreme environments such as the Morning Glory pool, a hot spring in Yellowstone National Park. The spring’s vivid blue color is from the prokaryotes that thrive in its very hot waters.

The current model of the evolution of the first, living organisms is that these were some form of prokaryotes, which may have evolved out of protobionts. In general, the eukaryotes are thought to have evolved later in the history of life. However, some authors have questioned this conclusion, arguing that the current set of prokaryotic species may have evolved from more complex eukaryotic ancestors through a process of simplification. Others have argued that the three domains of life arose simultaneously, from a set of varied cells that formed a single gene pool.

Two of the three domains, Bacteria and Archaea, are prokaryotic. Based on fossil evidence, prokaryotes were the first inhabitants on Earth, appearing 3.5 to 3.8 billion years ago during the Precambrian Period. These organisms are abundant and ubiquitous; that is, they are present everywhere. In addition to inhabiting moderate environments, they are found in extreme conditions: from boiling springs to permanently frozen environments in Antarctica; from salty environments like the Dead Sea to environments under tremendous pressure, such as the depths of the ocean; and from areas without oxygen, such as a waste management plant, to radioactively-contaminated regions, such as Chernobyl. Prokaryotes reside in the human digestive system and on the skin, are responsible for certain illnesses, and serve an important role in the preparation of many foods.

Phylogenetic Analysis

The molecular approach to microbial phylogenetic analysis revolutionized our thinking about evolution in the microbial world.

Learning Objectives

Outline the approaches to perform phylogenetic analysis

Key Takeaways

Key Points

  • The purpose of phylogenetic analysis is to understand the past evolutionary path of organisms. Due to technological innovation in modern molecular biology and the rapid advancement in computational science, accurate inference of the phylogeny of a gene or organism seems possible in the near future.
  • The developing technology of nucleic acid sequencing, together with the recognition that sequences of building blocks in informational macromolecules can be used as ‘molecular clocks’ that contain historical information, led to the development of the three- domain model ( Archaea – Bacteria -Eucaryota).
  • As more genome sequences become available, scientists have found that determining these relationships is complicated by the prevalence of lateral gene transfer among archaea and bacteria.
  • Even using improved DNA-based identification methods, the total number of bacterial species is not known and cannot even be estimated with any certainty. Currently, there are a little less than 9,300 known species of prokaryotes.

Key Terms

  • Lateral gene transfer: Horizontal gene transfer (HGT), also lateral gene transfer (LGT) or transposition refers to the transfer of genetic material between organisms other than vertical gene transfer. Vertical transfer occurs when there is gene exchange from the parental generation to the offspring. LGT is then a mechanism of gene exchange that happens independently of reproduction.
  • microbial phylogenetics: The study of the evolutionary relatedness among various groups of microorganisms.

Microbial phylogenetics is the study of the evolutionary relatedness among various groups of microorganisms. The molecular approach to microbial phylogenetic analysis revolutionized our thinking about evolution in the microbial world. The purpose of phylogenetic analysis is to understand the past evolutionary path of organisms. Even though we will never know for certain the true phylogeny of any organism, phylogenetic analysis provides best assumptions, thereby providing a framework for various disciplines in microbiology. Due to the technological innovation of modern molecular biology and the rapid advancement in computational science, accurate inference of the phylogeny of a gene or organism seems possible in the near future.

Gene sequences can be used to reconstruct the bacterial phylogeny. These studies indicate that bacteria diverged first from the archaeal/eukaryotic lineage. The term “bacteria” was traditionally applied to all microscopic, single-cell prokaryotes. However, molecular systematics showed prokaryotic life to consist of two separate domains, originally called Eubacteria and Archaebacteria, but now called Bacteria and Archaea that evolved independently from an ancient common ancestor. The archaea and eukaryotes are more closely related to each other than to the bacteria. Due to the relatively recent introduction of molecular systematics and a rapid increase in the number of genome sequences that are available, bacterial classification remains a changing and expanding field. For example, a few biologists argue that the Archaea and Eukaryotes evolved from Gram-positive bacteria.

While morphological or metabolic differences allowed the identification and classification of bacterial strains, it was unclear whether these differences represented variation between distinct species or between strains of the same species. This uncertainty was due to the lack of distinctive structures in most bacteria, as well as lateral gene transfer between unrelated species. The developing technology of nucleic acid sequencing, together with the recognition that sequences of building blocks in informational macromolecules can be used as ‘molecular clocks’ that contain historical information, led to the development of the three-domain model (Archaea – Bacteria – Eucaryota) in the late 1970’s, primarily based on small subunit ribosomal RNA sequence comparisons pioneered by Carl Woese and George Fox.

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Evolutionary tree showing the common ancestry of all three domains of life: A highly resolved Tree Of Life, based on completely sequenced genomes. Bacteria are colored blue, eukaryotes red, and archaea green. Relative positions of some phyla are shown around the tree.

As more genome sequences become available, scientists have found that determining these relationships is complicated by the prevalence of lateral gene transfer (LGT) among archaea and bacteria. Due to lateral gene transfer, some closely related bacteria can have very different morphologies and metabolisms. To overcome this uncertainty, modern bacterial classification emphasizes molecular systematics, using genetic techniques such as guanine cytosine ratio determination, genome-genome hybridization, as well as sequencing genes that have not undergone extensive lateral gene transfer, such as the rRNA gene.

As with bacterial classification, identification of microorganisms is increasingly using molecular methods. Diagnostics using such DNA-based tools, such as polymerase chain reaction, are increasingly popular due to their specificity and speed, compared to culture-based methods. However, even using these improved methods, the total number of bacterial species is not known and cannot even be estimated with any certainty. Following present classification, there are a little less than 9,300 known species of prokaryotes, which includes bacteria and archaea. but attempts to estimate the true level of bacterial diversity have ranged from 107 to 109 total species – and even these diverse estimates may be off by many orders of magnitude.

There are four steps in general phylogenetic analysis of molecular sequences: (i) selection of a suitable molecule or molecules (phylogenetic marker), (ii) acquisition of molecular sequences, (iii) multiple sequence alignment (MSA), and (iv) phylogenetic treeing and evaluation.

Multilocus sequence analysis (MLSA) represents the novel standard in microbial molecular systematics. In this context, MLSA is implemented in a relatively straightforward way, consisting essentially in the concatenation of several sequence partitions for the same set of organisms, resulting in a “supermatrix” which is used to infer a phylogeny by means of distance-matrix or optimality criterion-based methods. This approach is expected to have an increased resolving power due to the large number of characters analyzed and a lower sensitivity to the impact of conflicting signals (i.e. phylogenetic incongruence) that result from eventual horizontal gene transfer events. The strategies used to deal with multiple partitions can be grouped in three broad categories: the total evidence, separate analysis, and combination approaches. The concatenation approach that dominates MLSAs in the microbial molecular systematics literature is known to systematists working with plants and animals as the “total molecular evidence” approach. It has been used to solve difficult phylogenetic questions such as the relationships among the major groups of cetaceans, that of microsporidia and fungi, or the phylogeny of major plant lineages. The total molecular evidence approach has been criticized because by directly concatenating all available sequence alignments. The evidence of conflicting phylogenetic signals in the different data partitions is lost along with the possibility to uncover the evolutionary processes that gave rise to such contradictory signals.

Nongenetic Categories for Medicine and Ecology

In medicine, microorganisms are identified by morphology, physiology, and other attributes; in ecology by habitat, energy, and carbon source.

Learning Objectives

Outline the traits used to classify: bacteria, viruses and microrganisms in ecology

Key Takeaways

Key Points

  • A pathogen causes disease in its host. In medicine, there are several broad types of pathogens: viruses, bacteria, fungi, eukaryotic parasites, and prions.
  • When identifying bacteria in the laboratory, the following characteristics are used: Gram staining, shape, presence of a capsule, bonding tendency, motility, respiration, growth medium, and whether it is intra- or extracellular.
  • Viruses are mainly classified by phenotypic characteristics, such as morphology, nucleic acid type, mode of replication, host organisms, and the type of disease they cause.
  • In ecology, microorganisms are classified by the type of habitat they require, or trophic level, energy source and carbon source.
  • Biologists have found that microbial life has an amazing flexibility for surviving in extreme environments that would be completely inhospitable to complex organisms; these are called extremophiles and many kinds exist.
  • Different species of microorganisms use a mix of different sources of energy and carbon. These may be alternations between photo- and chemotrophy, between litho- and organotrophy, between auto- and heterotrophy or a combination of them.

Key Terms

  • obligate: Able to exist or survive only in a particular environment or by assuming a particular role: an obligate parasite; an obligate anaerobe.
  • pathogen: Any organism or substance, especially a microorganism, capable of causing disease, such as bacteria, viruses, protozoa, or fungi. Microorganisms are not considered to be pathogenic until they have reached a population size that is large enough to cause disease.
  • extremophile: An organism that lives under extreme conditions of temperature, salinity, and so on. They are commercially important as a source of enzymes that operate under similar conditions.

Classifying microorganisms in medicine

A pathogen (colloquially known as a germ) is an infectious agent that causes disease in its host. In medicine, there are several broad types of pathogens: viruses, bacteria, fungi, eukaryotic parasites, and prions.

BACTERIA

Although most bacteria are harmless, even beneficial, quite a few are pathogenic. Each pathogenic species has a characteristic spectrum of interactions with its human hosts.

Conditionally, pathogenic bacteria are only pathogenic under certain conditions; such as a wound that allows for entry into the blood, or a decrease in immune function. Bacterial infections can also be classified by location in the body, for example, the vagina, lungs, skin, spinal cord and brain, and urinary tract.

When identifying bacteria in the laboratory, the following chatacteristics are used: Gram staining, shape, presence of a capsule, bonding tendency (singly or in pairs), motility, respiration, growth medium, and whether it is intra- or extracellular.

Culture techniques are designed to grow and identify particular bacteria, while restricting the growth of the others in the sample. Often these techniques are designed for specific specimens: for example, a sputum sample will be treated to identify organisms that cause pneumonia. Once a pathogenic organism has been isolated, it can be further characterised by its morphology, growth patterns (aerobic or anaerobic), patterns of hemolysis, and staining.

VIRUSES

Similar to the classification systems used for cellular organisms, virus classification is the subject of ongoing debate due to their pseudo-living nature. Essentially, they are non-living particles with some chemical characteristics similar to those of life; thus, they do not fit neatly into an established biological classification system.

Viruses are mainly classified by phenotypic characteristics,such as:

  • morphology
  • nucleic acid type
  • mode of replication
  • host organisms
  • type of disease they cause

Currently there are two main schemes used for the classification of viruses: (1) the International Committee on Taxonomy of Viruses (ICTV) system; and (2) the Baltimore classification system, which places viruses into one of seven groups. To date, six orders have been established by the ICTV:

  • Caudovirales
  • Herpesvirales
  • Mononegavirales
  • Nidovirales
  • Picornavirales
  • Tymovirales

These orders span viruses with varying host ranges, only some of which infect human hosts.

Baltimore classification is a system that places viruses into one of seven groups depending on a combination of:

  • their nucleic acid (DNA or RNA)
  • strandedness (single or double)
  • sense
  • method of replication

Other classifications are determined by the disease caused by the virus or its morphology, neither of which is satisfactory as different viruses can either cause the same disease or look very similar. In addition, viral structures are often difficult to determine under the microscope. Classifying viruses according to their genome means that those in a given category will all behave in a similar fashion, offering some indication of how to proceed with further research.

Other organisms invariably cause disease in humans, such as obligate intracellular parasites that are able to grow and reproduce only within the cells of other organisms.

CATEGORIES OF MICROORGANISMS IN ECOLOGY

In ecology, microorganisms are classified by the type of habitat they require, or trophic level, energy source and carbon source.

Habitat Type

Biologists have found that microbial life has an amazing flexibility for surviving in extreme environments that would be completely inhospitable to complex organisms. Some even concluded that life may have begun on Earth in hydrothermal vents far under the ocean’s surface.

An extremophile is an organism that thrives in physically or geochemically extreme conditions, detrimental to most life on Earth. Most known extremophiles are microbes. The domain Archaea contains renowned examples, but extremophiles are present in numerous and diverse genetic lineages of both bacteria and archaeans. In contrast, organisms that live in more moderate environments may be termed mesophiles or neutrophiles.

There are many different classes of extremophiles, each corresponding to the way its environmental niche differs from mesophilic conditions. Many extremophiles fall under multiple categories and are termed polyextremophiles. Some examples of types of extremophiles:

  • Acidophile: an organism with optimal growth at levels of pH 3 or below
  • Xerophile: an organism that can grow in extremely dry, desiccating conditions; exemplified by the soil microbes of the Atacama Desert
  • Halophile: an organism requiring at least 0.2M concentrations of salt (NaCl) for growth
  • Thermophile: an organism that can thrive at temperatures between 45–122 °C

Trophic level, energy source and carbon source

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The nutritional modes of an organism: A flowchart to determine if a species is autotroph, heterotroph, or a subtype.

  • Phototrophs: carry out photon capture to acquire energy. They use the energy from light to carry out various cellular metabolic processes. They are not obligatorily photosynthetic. Most of the well-recognized phototrophs are autotrophs, also known as photoautotrophs, and can fix carbon.
  • Photoheterotrophs: produce ATP through photophosphorylation but use environmentally-obtained organic compounds to build structures and other bio- molecules.
  • Photolithoautotroph: an autotrophic organism that uses light energy, and an inorganic electron donor (e.g., H2O, H2, H2S), and CO2 as its carbon source.
  • Chemotrophs: obtain their energy by the oxidation of electron donors in their environments.
  • Chemoorganotrophs: organisms which oxidize the chemical bonds in organic compounds as their energy source and attain the carbon molecules they need for cellular function. These oxidized organic compounds include sugars, fats and proteins.
  • Chemoorganoheterotrophs (or organotrophs) exploit reduced-carbon compounds as energy sources, such as carbohydrates, fats, and proteins from plants and animals. Chemolithoheterotrophs (or lithotrophic heterotrophs) utilize inorganic substances to produce ATP, including hydrogen sulfide and elemental sulfur.
  • Lithoautotroph: derives energy from reduced compounds of mineral origin. May also be referred to as chemolithoautotrophs, reflecting their autotrophic metabolic pathways. Lithoautotrophs are exclusively microbes and most are bacteria. For lithoautotrophic bacteria, only inorganic molecules can be used as energy sources.
  • Mixotroph: Can use a mix of different sources of energy and carbon. These may be alternations between photo- and chemotrophy, between litho- and organotrophy, between auto- and heterotrophy or a combination of them. Can be either eukaryotic or prokaryotic.
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Differing morphology in different Herpes viruses: Various viruses from the Herpesviridae family seen using an electron micrograph. Amongst these members is varicella-zoster (Chickenpox), and herpes simplex type 1 and 2 (HSV-1, HSV-2).