General Methods of Classification-Dr C R Meera

Ø    Goals of Classification

A classification system should have two qualities.

a.              Stability

b.             Predictability

Stability- Frequent changes in the classification system can cause confusion. Therefore, it is important to strive for the development of a classification system that requires only minor adjustments when new information becomes available. This will help minimize confusion and ensure stability in the classification system.

Predictability- By knowing the characteristics of one member of a taxonomic group, it should be possible to assume that the other members of the same group probably have similar characteristics.

Predictability is an important characteristic of a classification system. It refers to the ability to anticipate and understand how the system categorizes and organizes information. A predictable classification system follows consistent rules and criteria, allowing users to have a clear understanding of how entities are classified and how they relate to each other.

Ø    General Methods of Classification

1.             Intuitive method

2.             Phenetic or Phenotypic classification

3.             Phylogenetic or Phyletic classification

4.             Genotypic or Genetic classification

5.             Numerical taxonomy or Adansonian classification

 1.             Intuitive method

         In this method, a microbiologist who has been studying the properties of the organisms for several years may decide that the organisms under study represent one or more species or genera. The drawback of this system is that the characters considered important by one person may not be so important to others. So different taxonomists may arrive at very different groupings. This is a primitive method that is not in use now.  However, some schemes based on intuitive methods have proved to be useful.

2.             Phenotypic classification

      For a very long time, microbial taxonomists relied on this system. This classification system is based on the mutual similarity in the phenotypic or morphological characteristics of the organisms. This system succeeded in bringing order to biological diversity. To some extent, this system also clarified the function associated with morphological structures. For eg: Flagella and motility are associated in most organisms. So, it could be assumed that flagella are involved in at least some types of motility.

Phenetic studies can also reveal evolutionary relationships. However, this system is not dependent on phylogenetic analysis. The phenetic system is based purely on morphological characters. This system will compare as many traits as possible, giving equal weightage to all traits studied. The best phenetic classification is the one constructed by comparing as many attributes/ morphological characters as possible. Organisms sharing many characteristics (>70%) are grouped into a single taxon.

3.             Phylogenetic or Phyletic classification

      With the publication of Darwin’s “On the Origin of Species’ in 1859, biologists began to develop phylogenetic or phyletic classification system. This classification system is based on the evolutionary relationships of organisms. The term “phylogeny” means ‘evolutionary development of a species’. In Greek, ‘phylon’ means ‘tribe or race’ and ‘genesis’ means ‘origin or generation.’ However, for much of the 20^th century, microbiologists could not effectively employ phylogenetic classification systems mainly due to the lack of good fossil records. When Carl Woese and George E Fox proposed the use of rRNA nucleotide sequences to assess evolutionary relationships among microorganisms, the doors opened for phylogenetic classification systems. 16S ribosomal RNA (or 16S rRNA) is the RNA component of the 30S subunit of a prokaryotic ribosome (SSU rRNA). It binds to the Shine-Dalgarno sequence and provides most of the SSU structure. The genes coding for it are referred to as 16S rRNA gene and are used in reconstructing phylogenies, due to the slow rates of evolution of this region of the gene. The validity of this approach is widely accepted and over 2 lakh different 16S and 18S rRNA sequences are saved in the International databases-  GenBank and the Ribosomal Database Project (RDP-II).

4.             Genotypic or Genetic classification

     In the genotypic classification system, genetic similarity between the organisms is evaluated. In this method comparison of either the individual gene or whole genome (haploid set of chromosomes in a microorganism) is done. Since 1970, it is widely accepted that if the genome of prokaryotes shows similarity in more than 70%, they could belong to the same species. Microbial genomes could be compared in many ways. The simplest method is DNA Base Composition determination. Other ways include Nucleic acid hybridization, Nucleic acid sequencing, and Genomic fingerprinting (evaluation of genes that evolve more quickly than those that encode rRNA.

5.             Numerical taxonomy or Adansonian classification

     Numerical taxonomy, also known as numerical phenetics or phenetic taxonomy, is a method used in biological classification to group organisms based on their overall similarity with the help of computers. Unlike traditional taxonomy, which relies on morphological characteristics and evolutionary relationships, numerical taxonomy employs quantitative data from various attributes or characteristics of organisms. This approach aims to create classifications based on the overall similarity or dissimilarity of organisms rather than their shared ancestry.

The process of numerical taxonomy involves several steps:

1. Data Collection: Quantitative data is collected from the organisms under study. This data can include various traits such as morphological features, biochemical characteristics, physiological measurements, or genetic markers.

2. Data Standardization: The collected data is standardized to ensure compatibility and comparability across different organisms. This step may involve converting measurements to standardized units or transforming data to eliminate biases or variations.

3. Similarity or Dissimilarity Calculation: A similarity or dissimilarity matrix is constructed based on the standardized data. Different mathematical methods can be used to calculate the degree of similarity or dissimilarity between pairs of organisms. Common methods include the Jaccard coefficient or correlation coefficients.

4. Cluster Analysis: Cluster analysis is performed using the similarity or dissimilarity matrix to group organisms into clusters or taxa.

5. Taxonomic Hierarchy: The clusters obtained from the cluster analysis are organized into a taxonomic hierarchy. This hierarchy can include higher-level groupings, such as classes or orders, as well as lower-level groupings, such as genera or species.

Numerical taxonomy has been widely used in fields such as ecology, microbiology, botany, and zoology. It provides a systematic and quantitative approach to classify organisms based on overall similarities, without relying on subjective interpretations or expert judgments. However, it is important to note that numerical taxonomy does not consider evolutionary relationships explicitly, which can be a limitation in certain contexts where phylogenetic information is crucial for understanding the evolutionary history of organisms.

Pure culture techniques- streak, spread and pour plate methods-Dr C R Meera

 

In natural habitats microorganisms usually grow in complex, mixed populations containing several species. This presents a problem for the microbiologist because a single type of microorganism cannot be studied adequately in a mixed culture. One needs a “pure culture”- that's a population of cells arising from a single cell to characterize an individual species. The development of pure culture techniques by the German bacteriologist Robert Koch transformed microbiology. Within about 20 years after the development of pure culture techniques, most pathogens responsible for major human bacterial diseases had been isolated.

 A single bacterial cell on suitable culture media will give rise to a number of genetically identical daughter cells by binary fission. These are called “clones” of cells forming into a colony on culture media. A pure culture is usually made up of a succession of cultures and is often derived from a single colony. All strains of bacteria isolated and identified till now are maintained in American Type Culture Collection (ATCC) and Microbial Type Culture Collection (MTCC).  Each strain is designated by an identifying number and its history is recorded (the source from which the isolation was made, name of the person who made the isolation, date of isolation and the culture collection in which the strain is maintained and from which it can be obtained for study).

A variety of techniques have been developed whereby isolation into pure culture can be accomplished. Each technique has certain advantages and limitations, and there is no one method that can be used for all bacteria. Most commonly used pure culture techniques include streak plate method, spread plate method and pour plate method. Streak plate is considered only as a qualitative method. Unlike the streak plate technique, the pour plate and spread plate techniques may be performed in a quantitative manner to determine the number of bacteria present in a specimen.

 Methods of isolating pure cultures

 A)  The streak plate technique

The streak culture or surface plating method is routinely employed for the isolation of bacteria in pure culture from clinical specimens. A platinum loop is charged with the specimen to be cultured.  Owing to the high cost of platinum, loops for routine work are made of nichrome resistance wires. One loopful of specimen is transferred onto the surface of a well dried agar plate on which it is spread over a small area at the periphery. It is called the primary inoculum. The primary inoculum is then distributed over the plate by streaking it with the loop in a series of parallel lines in different segments of the plate. The loop should be flamed and cooled between the different sets of streaks. At some point in the process, single cells dropped from the loop as it is rubbed along the agar surface would develop into separate colonies on incubation. Growth may be confluent at the site of original inoculation but become progressively thin and well separated colonies are obtained over the final series of streaks. Common streaking methods include T- streaking, Zig-zag streaking,  Continuous streaking and Quadrant streaking (Figure 1).

 

Quadrant Streaking

Zig-zag Streaking

T-Streaking

Radiant streaking

Continuous streaking

Fig 1. Different streaking patterns

A)  The pour plate technique

 

A pour plate can yield isolated colonies and extensively used with the bacteria and fungi. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating (Figure 2). Serial dilution is the method commonly used to dilute the original sample. For serial dilution, a series of tubes containing a definite volume of sterile liquid, usually water or physiological saline is prepared. Suppose we prepare a series of tubes containing 9 ml of sterile distilled water. For carrying out serial dilution of the original sample we can add 1 ml of the original sample into the first tube which contains 9 ml of sterilized distilled water. Now the final volume of the first tube becomes 10. So the dilution of the first tube is 1/10 which can be also written as 10^-1.  To continue the serial dilution,  from the first tube we can add 1 ml to the second tube. This is continued in the following tubes also till we reach the final tube. From the final tube, remove 1ml of the media, so that the volume will be 9 ml in all the tubes. From these diluted tubes small volumes of several diluted samples are mixed with liquid Agar that has been cooled to about 45 degrees celsius. The mixtures are then poured immediately into sterile culture dishes. Most bacteria and fungi are not killed by a brief exposure to the warm Agar. After the agar has hardened, each cell is fixed in place and forms  individual colony. Plates containing between 30 to 300 colonies are counted. The total number of colonies equals the number of viable microorganisms in the diluted sample. Colonies growing on the surface can be used to inoculate fresh medium and to prepare pure cultures. This is considered as a quantitative as well as qualitative method. This is a preferred quantitative method for urine cultures. Surface colonies formed by pour culture technique are circular and subsurface colonies could be lenticular or lens shaped.

Disadvantages

1.    Some of the organisms are trapped beneath the surface of the medium during pour plate technique and therefore both surface and the subsurface colonies develop.  The subsurface colonies can be transferred to fresh media only by first digging them out of the agar with a sterile instrument. So, there is more chance for contamination.

2.    The organisms being isolated must be able to withstand temporary exposure to 45 degree celsius, temperature of the liquid agar medium. So, this method would be unsuitable for isolating psychrophilic bacteria. (Psychrophiles are the bacteria that grow well at 20⁰ C degree and have an optimum growth temperature of 15⁰ C or lower and maximum around 20⁰ C)

Figure- 2. The pour plate technique- The original sample is diluted several times to thin out the population sufficiently. The most diluted samples are then mixed with warm agar and poured into petri dishes. Isolated cells grow into colonies and can be used to establish pure cultures.

CFU/ml (Colony Forming Unit/ml)= (No: of colonies X Dilution factor)  / Volume of sample plated

Dilution factor = Reciprocal of dilution or Final volume/ sample volume

(If 1 ml sample is added to 9 ml, its dilution = 1/10= 10 -1; Dilution factor is 10)

C) Spread plate technique

The spread plate is an easy, direct way of achieving pure cultures. This method is also considered as a quantitative as well as qualitative method.  In this method also, serial dilution has to be conducted initially. The original culture is diluted in a series of tubes containing sterile liquid, usually water or physiological saline. After that, a small volume of dilute microbial mixture containing around 30- 300 cells is transferred to the center of a dry Agar plate. Then it is spread evenly over the surface of agar media with a sterile  glass rod or the L-rode. The dispersed cells develop into isolated colonies. The number of colonies developed on the plates would be equal to the number of viable organisms, as each viable cell develops into a colony on incubation. In contrast to the pour plate technique, only surface colonies develop in the pour plate technique. Moreover the organisms are not required to withstand the temperature of liquid agar as in the case of pour plate technique.

 Preparation of spread plate

●      Pipette a small volume (0.01ml) of sample on to the center of an agar medium plate

●      Dip  the glass spreader into a beaker of ethanol

●      Briefly flame the ethanol soaked spreader and allow it to cool

●      Spread the sample evenly over the agar surface with the sterilized spreader and incubate (Fig 3)

 

Figure 3. Spread Plate Technique

Culture Media Part 4- Classification Based on Function

Based on the application or function media can be divided into Supportive media (General purpose media) and special media. Special media includes a number of media like Enriched media, Enrichment media, Selective media, Differential media, Indicator media, Sugar media and Transport media. However, sometimes a single media may fulfil more than one functions. For example; Blood agar can act as both Enriched and Differential media. Eosin Methylene Blue (EMB) agar can act as both Selective and Differential media. MacConkey (MAC) agar can act as Selective, Indicator and Differential media. Mannitol Salt agar can act as both Selective and Differential media. 1. Supportive media (General purpose media) This media support the growth of most of the organisms and do not contain any added inhibitors. Basal media used in microbiology lab like Peptone water, Nutrient Agar and Nutrient Broth can be considered as supportive media for growing non-fastidious organisms. Media such as Tryptic Soy Broth and Tryptic Soy Agar are also examples of GPM as they sustain the growth of many microorganisms. Blood or other nutrients can be added to the GPM to support the growth of fastidious organisms, then they are called Enriched media. 2. Special Media 2.1. Enriched Media Enriched media contains special ingredients like blood, serum or egg added to the basal media. It is used to cultivate microorganisms that are more exacting in their nutritional needs.ie; to grow fastidious organisms. Eg: Blood agar, Chocolate agar, Egg media and Brain Heart Infusion Broth. a) Blood agar:

Blood agar contains 5% mammalian blood added to the basal media. Blood is added after autoclaving the media. It is enriched medium as it contains many nutrients like protein, carbohydrate, lipid, iron, a number of growth factors and vitamins that can support the growth of many fastidious organisms including aerobic and anaerobic bacteria. Vitamin K, cysteine and hemin in blood support the growth of anaerobic organisms. Blood agar acts as both enriched and differential medium. Blood agar is a differential media as it distinguishes haemolytic and non- haemolytic bacteria. Hemolytic organisms produce hemolysin, a protein that cause hemolysis or breakdown of Red Blood Cells (RBC). Sheep blood is preferred as hemolysis is more clearly defined on it.  Hemolysis appears as a clear zone if there is complete lysis of RBC (β hemolysis) or greenish halo around the colony if there is partial lysis of RBC (α hemolysis). Blood agar also help to differentiate among Gram positive cocci.

Eg: Streptococcus pyogenes and Staphylococcus aureus cause β hemolysis.  Streptococcus viridans cause α hemolysis.

a)                 Chocolate Agar: Used to grow fastidious organisms like Neisseria gonorrhoeae, N. meningitidis, H. influenza and Pneumococci. It is called chocolate agar due to its chocolate brown appearance. The brown colour is the result of heating red blood cells and lysing them before adding to the media. Heating a mixture of sheep blood and nutrient agar releases haemoglobin, hemin or X factor and Nicotinamide adenine dinucleotide (NAD or V factor) which gives the brown colour. 

b)                 Brain heart infusion broth: It is highly nutritious buffered liquid media for cultivating fastidious organisms. It is prepared using non- enzymatic infusion from calf brain and cow heart with added peptone and dextrose.

1.1.  Enrichment Media

In samples containing mixed population of microorganisms the bacteria to be isolated is often overgrown by the unwanted microbes.  Usually non-pathogenic or commensal bacteria tend to overgrow the pathogenic ones. Enrichment media are liquid media containing special nutrients that stimulate the growth of particular organisms that might not be otherwise present in sufficient amount for isolation and identification. For eg: In fecal samples S typhi may not be sufficiently numerous for identification and usually overgrown by E coli. If cultivated on the medium Selenite cystine (SC) broth with trace element selenium greater number of S typhi could be obtained on incubation and selenium is inhibitory to coliforms. Such media which contain substances that have a stimulatory effect on wanted organisms are called enrichment media. Other Egs: Tetrathionate broth to promote growth of Typhoid and Paratyphoid bacilli in samples containing coliforms; Selenite F Broth for Dysentery bacilli.     

1.2.           Selective media

Selective media are solid media that contain nutrients to promote the growth of wanted organisms and also contain substances that inhibit or suppress the growth of unwanted organisms. Incorporation of substances like bile salts or dyes such as basic fuchsin and crystal violet allow the growth of Gram negative organisms while they inhibit the Gram positive ones. MacConkey agar, Eosin Methylene Blue Agar are examples of selective media which are widely used for detection of E coli in water supplies. These media will not allow the growth of Gram positive organisms.

a) MacConkey Agar is made up of peptone, lactose, agar, neutral red, taurocholate, crystal violet and bile salts. Crystal violet and bile salts act as selective agents and inhibit the growth of  G +ive bacteria and allow G –ive growth.

b) Eosin Methylene Blue Agar contains two dyes, Eosin Y and Methylene Blue that inhibit G +ive bacteria.      

c) Mannitol Salt Agar allows the selective growth of halophiles. 7.5% NaCl act as selective agent promoting the growth of halophiles and inhibiting non-halophiles.                                                                                                                           

d) Sulfadiazine and Polymyxin Sulfate (SPS) Agar: It is used to isolate Clostridium botulinum which causes food poisoning. This media promote the growth of  Clostridium botulinum while inhibiting the growth of most other Clostridium species.  It contains the antibiotics sulfadiazine and Polymyxin sulphate as selective agents.

e) Desoxycholate Citrate Agar for isolating Dysentery bacilli

f) Thiosulphate Citrate Bile Sucrose Agar (TCBS) for isolation of Vibrio species from fecal samples. Alkaline pH of the medium promote the growth of Vibrio species and high concentrations of sodium thiosulfate and sodium citrate to inhibit the growth of Enterobacteriaceae.

g) Thayer Martin Media used to isolate N.gonorrhoeae from clinical specimens contain antimicrobials like vancomycin, colistin, nystanin in Chocolate agar. On incubation in the presence of 3-10% CO_2, this media will give colonies of N.gonorrhoeae and inhibit other commensals.

h) Lowenstein-Jensen Media used for the isolation of Mycobacterium species. This media contains mineral salts, asparagine, glycerol, malachite green and hen’s egg.  Malachite green in the medium act as the inhibitor for unwanted organisms.

1.3.           Indicator media

Indicator media contains an indicator that changes its colour when bacterium grows in them. Blood agar can be considered as an indicator media. In some indicator media, a pH indicator will be incorporated. As the organisms grow, pH of the medium will be changed due to acid production and it causes the indicator to change its colour. Example is MacConkey agar. The presence of the pH indicator neutral red makes it an indicator media. The indicator changes the colonies of lactose fermenters into red or deep pink in colour and leaves the non-fermenters colourless or translucent. Lactose fermenters catabolise lactose by fermenting it and release acidic waste products. These acidic waste diffuse into the media, changing the colour of indicator. Neutral indicator is red or orange in acidic pH (<6.8) and colourless when pH is over 6.8. The indicator imparts pink or red colour to colonies of the lactose fermenters.  

Other Examples:

Wilson & Blair Medium for isolation of S typhi. This media contains the indicator Sulphite. S typhi reduces Sulphite to Sulphide in the presence of Glucose and produce colonies with black metallic sheen.

McLeod’s Medium or Potassium Tellurite Agar used to isolate C.diphtheriae. Potassium Tellurite in the medium is reduced to metallic tellurium by C.diphtheriae to produce black colonies.

Thiosulphate Citrate Bile Sucrose Agar (TCBS) for isolation of Vibrio species. Bromothymol blue is the indicator in the medium (Yellow in acidic, green in neutral and blue in alkaline conditions). Organisms that ferment sucrose appear as yellow colonies while the non-fermenters appear as green colonies. Hence act as an indicator medium also.

1.4.           Differential media

Differential media helps to distinguish among different groups of microorganisms and even permit identification of organisms based on their biological characteristics. In some cases, indicator in the differential media changes its colour when a particular biochemical reaction occurs. Many of the media we mentioned yet also falls under differential media.

Blood agar which is an enriched media, indicator media, is an example of differential media too. It helps to differentiate haemolytic and non-haemolytic organisms. ie; they differentiate organisms based on their ability to produce hemolysin.   MacConkey agar is also a differential media as it helps to differentiate between lactose fermenters and non-lactose fermenters. Neutral red indicator changes its colour in acidic pH and impart red colour to lactose fermenters. Most of the commensals of intestine including E coli are good lactose fermenters and appear pink to red in colour whereas most of the pathogens like Salmonella and Shigella are non-lactose fermenters and appear colourless.

Mannitol salt agar, selective media for halophiles act as differential media too. It helps to differentiate pathogenic and non-pathogenic Staphylococci.  Pathogenic ones like Staphylococcus aureus release acidic by-products as they make use of mannitol as carbon or energy source.  Acidic products cause the phenol red indicator in the medium to change into yellow colour (Phenol red indicator: Yellow in acidic condition and red in alkaline conditions).

EMB agar which is a selective media also serve as the differential media. Eosin Y and Methylene blue dyes in EMB agar react with acidic products released by some G –ive bacteria when they use lactose or sucrose in the medium as carbon and energy sources. Fecal bacteria such as E coli produce large amounts of acidic products and have green metallic sheen on EMB agar. Help to differentiate E coli.

SPS (Sulfite Polymyxin Sulfadizine) Agar, selective media for Clostridium botulinum also serve as differential media. Clostridium botulinum produce black colonies on SPS Agar because of the production of hydrogen sulphide by the organism from the sulphur containing additives. 

1.5.           Sugar media

Sugar media contains fermentable sugars in it. It can be monosaccharides like pentoses (eg: Arabinose, Xylose) or hexoses (eg: Dextrose, mannose), disaccharides (eg: Saccharose, lactose), trisaccharides (eg: Raffinose) or polysaccharides (eg Starch). Sugar media has two functions. Primary function is that the media will act as readily available source of energy, provided the organisms are able to utilize the sugar present in the media. Second function is that it is helpful in the identification and classification of organisms.

Sugar media consists of 1% sugar in peptone water along with an indicator. Durham tube is kept inverted in the media to detect gas production. Indicator changes its colour if the inoculated organism ferments the sugar. Fermentation could be with or without gas production. Gas production is indicated as bubble formation in Durham tube. 

1.6.           Transport media

Special media devised to transport the microorganisms are called transport media. They are usually used to transport either fastidious or delicate organisms that cannot withstand the time taken for transport of specimen from the site of collection to the laboratory. Transport media are essentially buffered solutions containing carbohydrates, peptones and other nutrients (excluding the growth factors) which would preserve the viability of bacteria during transport without allowing their multiplication. Examples: Glycerol saline for Enteric bacilli, Stuart’s medium for Gonococci, Venkatraman Ramakrishnan (VR) medium for Vibrio cholera.

 

Examples of  multipurpose media
Blood Agar	Enriched /indicator/ differential media
MacConkey agar	Selective/ indicator/ differential media
Eosin Methylene Blue Agar	Selective /differential media
Mannitol Salt Agar	Selective /differential media
Sulfadiazine and Polymyxin Sulfate (SPS) Agar	Selective/ differential media
Thiosulphate Citrate Bile Sucrose Agar (TCBS)	Selective/ indicator media

LEVELS OF CLASSIFICATION- Dr C R Meera

Taxonomy deals with identification, Classification and Nomenclature of organisms. Objective of taxonomy is to arrange organisms into categories that reflect the similarities of the individuals within the groups.

•   Carolus Linnaeus: 1700’s: Two Kingdoms: Plants and Animals

•   Carolus Linnaeus: 1753’s: Binomial nomenclature --"father of modern taxonomy"

•   Ernst Haekel: 1866: Kingdom Protista

•   R.H. Whittaker: 1969: Five Kingdoms: Monera, Protista, Fungi, Plantae and Animalia

•   Carl Woese: 1990: Three Domains:  archaea, bacteria, and eukaryote domains.

Taxonomic Ranks

 Th.e classication of microbes involves placing them within hierarchial taxonomic levels. Microbes m each level or rank share a common set of specific features. The levels are arranged in a non-overlapping manner so that each level includes not only the traits that define not only the rank above it, but a new set of more restrictive traits. The highest rank is the domain. All prokaryotes belong to the domain Bacteria or the Archaea. Within  each domain, each microbe is assigned to a  phylum, class, order, family , genus and species.  Some prokar·yotes  are also divided into subspecies.                                                                           An example of Taxonomic ranks and name is given below.

Rank	Example
Domain	Bacteria
Phylum	Proteobacteria
Class	Gammaproteobacteria
Order	Enterobacteriales
Family	Enrerobacteriaceae
Genus	Shigella
Species	S. dysenteriae

Order- ends with “ales” and Family with “ceae”

The basic taxonomic group (taxon) is species. Species of higher organisms are groups of potentially interbreeding natural populations that are reproductively isol ated from other groups. This definition is satisfactory for sexually reproducing organisms, but fails wjth many microorganisms as they do not reproduce sexually. So a prokaryotic species is a collection of strains having similar characteristics. In other words, prokaryotic species·is a collection of strains that share many stable properties and differ significantly from other groups of strains. Perhaps a species should be the collection of organisms that share the same sequences in their core housekeeping genes (genes coding for products that are required by all cells and which are usually continually expressed).

A strain is the descendants of a single, pure microbial culture. Bacterial species consist of a special strain called "type strain" together with all other strains that are considered sufficiently similar to the type strain. The type strain is the strain that is designated to be the permanent reference specimen for the species. It is the strain to which all other strains must be compared to see if they resemble it closely enough to belong to the species. So they are special and hence much care is taken to preserve and maintain them by national reference collections such as the ATCC (American Type Culture Collection) in United States or the National Collection of Type Cultures in England.

There are a number of different ways in which strains within a species may be described. Biovars are variant strains characterized by biochemical or physiological differences, morphovars differ morphologically, and serovars  have different antigenic properties.

 Just as bacterial species is composed of a collection of similar strains, a bacterial genus is composed of a collection of similar species. One of the species is designated as the type species and this serves as the permanent example of the genus.

Species	A group of similar Strains
Genus	A group of similar Species
Family	A group of similar Genera
Order	A group of similar Families
Class	A group of similar Orders
Phylum (Division)	A group of similar Classes
Domain (Kingdom)	A group of similar Phylum

Microorganisms are named by using the binomial system introduced by Carl Linnaeus. The Latinized, italized name consists of two parts. The first part, which is capitalized, is the generic name, and the second part is the uncapitalized species name (Eg. Escherichia  coli).