Conventional Bacterial Identification Methods

1) Introduction:

Bacterial identification is the first step in establishing the bacterial etiology of a particular disease. It includes the procedures and techniques used to correctly identify the bacterial pathogens responsible for disease. Bacteriologist employs a wide variety of techniques, based upon known characteristics of specific bacteria, to arrive at the identity of a given specimen. The traditional way of detecting and identifying bacteria from given specimen is based on culturing, enumeration and isolation of presumptive colonies for further identification analysis. The more conventional methods for further subtyping of bacteria include the study of the phenotypic characteristics of the microorganisms. These phenotypic methods include biotyping, serotyping, and phage typing. In biotyping, the biochemical growth requirements, environmental conditions (pH, temperature, antibiotic resistance, bacteriocins susceptibility) and physiological (colony and cell morphology, cell wall composition by microscopy and membrane composition such as by fatty acid analysis) aspects of bacteria are investigated while serological and phage typing concentrate more on the surface structure differences of bacteria.

2) Principles of identification:

Identification of bacteria requires the knowledge of their morphological, biochemical, physiological. and genetic characteristics. Collectively, these characteristics can be grouped as phenotype and genotype. The identification schemes start with broad categorization (e.g., Gram-staining) and then progress to more specific tests. Identification schemes can be classified into one of two categories: those that are based on genotypic characteristics of bacteria and those that are based on phenotypic characteristics. Certain schemes rely on both genotypic and phenotypic characteristics. 

i) Organism identification using genotypic criteria:

It uses molecular techniques to identify bacteria by doing DNA or RNA analysis of the bacterium’s genome . This usually involves detecting the presence of a gene, or a part thereof, or an RNA product that is specific for a particular organism. In principle, the presence of a specific gene or a particular nucleic acid sequence is interpreted as a definitive identification of the organism. The genotypic approach is highly specific and often very sensitive. With the ever-expanding list of molecular techniques being developed, the genetic approach to organism identification will continue to grow and become more integrated into diagnostic microbiology laboratory protocols.

ii) Organism identification using phenotypic criteria:

Phenotypic properties are expressed properties of the organism like shape, size, staining properties and reactions in biochemical tests. They are properties that can be measured without reference to the genome. Phenotypic criteria are based on observable physical or metabolic characteristics of bacteria, that is, identification is through analysis of gene products rather than through the genes themselves. The phenotypic approach is the classic approach to bacterial identification, and most identification strategies are still based on bacterial phenotype. Most of the phenotypic characterizations used in diagnostic bacteriology are based on tests that establish a bacterial isolate’s morphology and metabolic capabilities. The most commonly used phenotypic criteria include:

  •  The range of temperatures within which the organism can grow.
  • The requirement of specific nutrients in order to grow.
  • The ability of the organism to make one or more specific chemicals (such as indole).
  • Production of specific enzymes by the organism (such as coagulase).
  • Fermentation of one or more specific sugars such as lactose (sugar fermentation tests).
  • Sensitivity or resistance to one or more specific antibacterial agents.
  • Presence of one or more specific proteins or antigens on its surface.

Most of the conventional bacterial identification methods are based upon the phynotypic characteristics of bacteria. They can be classified as:

  1. Identification by Bacterial cultivation.
  2. Bacterial identification based upon their enzymatic capabilities.
  3. Tests for presence of metabolic pathways

Identification by Bacterial cultivation 

 

Culture involves placing some of the specimen in conditions where the organism or organisms of interest can grow and multiply. Once grown in culture, most bacterial populations are easily observed without microscopy and are present in sufficient quantities to allow laboratory testing to be performed. The successful transition from the in vivo to the in vitro environment requires that the nutritional and environmental growth requirements of bacterial pathogens be met. The in vivo to in vitro transition is not necessarily easy for bacteria. In vivo they are utilizing various complex metabolic and physiologic pathways developed for survival on or within the human host. Then, relatively suddenly, they are exposed to the artificial in vitro environment of the laboratory. The bacteria must adjust to survive and multiply in vitro. The bacterial cultivation has three main purposes, which are:

■    To grow and isolate all bacteria present in an infection.
■    To determine which of the bacteria that grow are most likely causing infection and which are likely contaminants or colonizers.
■    To obtain sufficient growth of clinically relevant bacteria to allow identification and characterization.

i) General Concepts of Culture Media:

In the laboratory, nutrients are incorporated into culture media on or in which bacteria are grown. If a culture medium meets a bacterial cell’s growth requirements, that cell will multiply to sufficient numbers to allow visualization by the unaided eye. Of course, bacterial growth after inoculation also requires chat the media be placed in optimal environmental conditions.

ii) Phases of Growth Media:

Growth media are used in either of two phases: liquid (broth) or solid (agar). In some instances (e.g., certain blood culture methods), a biphasic medium chat contains both a liquid and a solid phase is used.

Broth media: These are liquid media in which bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of  undisturbed broth. Liquid media tend to be used when a large number of bacteria have to be grown. These are suitable to grow bacteria when the numbers in the inoculum is suspected to be low.

Solid media: These are are made by adding a solidifying agent to the nutrients and water. Agar agar (simply called agar) is the most commonly used solidifying agent. It is an un-branched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesn’t contribute any nutritive property, it is not hydrolyzed by most bacteria and is usually free from growth promoting or growth retarding substances.

3) Classification of culture media:

i) Enrichment media

The basic principle involved here is to control the nutrients and culture conditions in such a way that it suits mainly to a particular specific species. these factors include  temperature, air supply, light, pH etc. This media type is used to enhance the growth of a particular bacterial pathogen from a mixture of organisms by using nutrient specificity. 

ii) Supportive media

It contain nutrients that support growth of most nonfastidious organisms without giving any particular organism a growth advantage.

iii) Selective media

These are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen. They contain one or more agents that are inhibitory to all organisms except those being sought. Inhibitory agents used for this purpose include dyes, bile salts, alcohols, acids, and antibiotics. An example of a selective medium is phenyl-ethyl alcohol agar, which inhibits the growth of aerobic and facultatively anaerobic gram-negative rods and allows gram-positive cocci to grow.

iv) Differential media

Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media are called differential media or indicator media. For example MacConkey’s agar, CLED agar, TCBS agar, XLD agar etc.

4) Factors affecting growth of microbial colonies in culture:

Optimizing the environmental conditions to support the most robust growth of clinically relevant bacteria is as important as meeting the organism’s nutritional needs for in vitro cultivation. The four most critical environmental factors to consider include oxygen and carbon dioxide (CO2) availability, temperature, pH, and moisture content of medium and atmosphere.

Oxygen and Carbon Dioxide Availability:

Most clinically relevant bacteria are aerobic, facultatively anaerobic, or strictly anaerobic. Aerobic bacteria use oxygen as a terminal electron acceptor and grow well in room air. Most clinically significant aerobic organisms are actually facultatively anaerobic, being able to grow in the presence (i.e. aerobically) or absence (i.e., anaerobically) of oxygen. However, some bacteria, such as Pseudomonas spp., members of the Neisseriaceae family, Brucella spp., Bordetella spp., and Francisella spp., are strictly aerobic and cannot grow in the absence of oxygen. Other aerobic bacteria require only low levels of oxygen and are referred to as being microaerophilic, or microaerobic. Anaerobic bacteria are unable to use oxygen as an electron acceptor, but some aerotolerant strains will still grow slowly and poorly in the presence of oxygen. Oxygen is inhibitory or lethal for strictly anaerobic bacteria. In addition to oxygen, the availability of CO2 is important for growth of certain bacteria. Organisms that grow best with higher CO2 concentrations (i.e., 5% to 10% CO2) than is provided in room air are referred to as being capnophilic. For some bacteria a 5% to 10% CO2 concentration is essential for successful cultivation from patient specimens.

Temperature

Bacterial pathogens generally multiply best at temperatures similar to those of internal human host tissues and organs (i.e., 37° C). Therefore, cultivation of most medically important bacteria is done using incubators with temperatures maintained in the 35° to 37° C range. For others, an incubation temperature of 30° C (i.e., the approximate temperature of the body’s surface) may be preferable, but such bacteria are encountered relatively infrequently so that use of this incubation temperature occurs only when dictated by special circumstances. Recovery of certain organisms can be enhanced by incubation at other temperatures. For example, the gastrointestinal pathogen Campylobacter jejuni grows at 42° C, whereas many other pathogens and nonpathogens cannot. Therefore, incubation at this temperature can be used as an enrichment procedure. Other bacteria, such as Listeria monocytogenes and Yersinia enterocolitica, can grow at 0° C, but grow best at temperatures between 20° and 40° C. Cold enrichment has been used to enhance the recovery of these organisms in the laboratory.

pH

Most bacteria grow in the range of neutral pH values (between 5 and 8), although some species have adapted to life at more acidic or alkaline extremes. The pH scale is a measure of the hydrogen ion concentration of an organism’s environment, with a pH value of 7 being neutral. Values less than 7 indicate the environment is acidic; values greater than 7 indicate alkaline conditions. Most clinically relevant bacteria prefer a near neutral pH range, from 6.5 to 7.5. Commercially prepared media are buffered in this range so that checking their pH is rarely necessary.

Moisture

Water is provided as a major constituent of both agar and broth media. However, when media are incubated at the temperatures used for bacterial cultivation, a large portion of water content can be lost by evaporation. Loss of water from media can be deleterious to bacterial growth in two ways: (1) less water is available for essential bacterial metabolic pathways and (2) with a loss of water there is a relative increase in the solute concentration of the media. An increased solute concentration can osmotically shock the bacterial cell and cause lysis. In addition, increased atmospheric humidity enhances the growth of certain bacterial species. For these reasons, measures, such as sealing agar plates to trap moisture or using humidified incubators, are taken to ensure appropriate moisture levels are maintained throughout the incubation period.

Many culture medias are used presently. Some commonly used for routine diagnostic bacteriology are,

  • Brain-heart infusion.
  • Chocolate agar.
  • Columbia CNA with blood.
  • Gram-negative (GN) broth.
  • Hektoen enteric (HE) agar.
  • MacConkey Agar
  • Phenylethylalcohol (PEA) agar.
  • Sheep blood agar
  • Thayer-Martin agar.
  • Thioglycollate broth.
  • Xylose lysine and desoxycholate (XLD) agar.

Bacterial identification based upon their enzymatic capabilities

 

Staining provides valuable information about bacterial morphology, Gram reaction, and presence of such structures as capsules and endospores. Then microscopic observation provides little additional information as to the genus and species of a particular bacterium. For identification of particular bacterial species we depend upon biochemical testing. The types of biochemical reactions each organism undergoes act as a “thumbprint” for its identification. This identification scheme depends upon following facts,

  • Each different species of bacterium has a different molecule of DNA (i.e., DNA with a unique series of nucleotide bases).
  • Since DNA codes for protein synthesis, then different species of bacteria must, by way of their unique DNA, be able to synthesize different protein enzymes.
  • Enzymes catalyze all of the various chemical reactions of which the organism is capable. This, in turn, means that different species of bacteria must carry out different and unique sets of biochemical reactions.

5) Types of Enzyme based tests:  

In diagnostic bacteriology enzyme-based tests are designed to measure the presence of one specific enzyme or a complete metabolic pathway that may contain several different enzymes. 

Several tests are commonly used to determine the presence of a single enzyme. These tests usually provide rapid results because they can be performed on organisms already grown in culture. Of importance, these tests are easy to perform and interpret and often play a key role in the identification scheme. Although most single enzyme tests do not yield sufficient information to provide species identification, they are used extensively to determine which subsequent identification steps should be followed. For example, the catalase test can provide pivotal information and is commonly used in schemes for gram-positive identifications. The oxidise test is of comparable importance in identification schemes for gram-negative bacteria

Catalase Test: 

The aerobic respiration lead to production of  hydrogen ions (H+), which are given off and must be removed by the cell. The electron transport chain takes these hydrogen ions and combines them with half a molecule of oxygen (an oxygen atom) to form water (H2O). However, some cytochromes in the electron transport system form toxic hydrogen peroxide (H2O2) instead of water, and this must be removed. The enzyme catalase catalyzes the release of water and oxygen from hydrogen peroxide (H2O2 + catalase = H2O + O2); its presence is determined by direct analysis of a bacterial culture. The rapid production of bubbles (effervescence) when bacterial growth is mixed with a hydrogen peroxide solution is interpreted as a positive test (i.e., the presence of catalase). Most bacteria are catalase-positive; however, certain genera that don’t carry out aerobic respiration, such as Streptococcus, Lactobacillus, and Clostridium, are catalase-negative.

Oxidise Test:

Cytochrome oxidise participates in electron transport and in the nitrate metabolic pathways of certain bacteria. The test for the presence of oxidase can be performed by flooding bacterial colonies on the agar surface with the reagent 1 % tetramethyl-p-phenylenediamine di-hydrochloride. Alternatively, a sample of the bacterial colony can be rubbed onto filter paper impregnated with the reagent (Kovac’s method) The test is initially used for differentiating between groups of gram-negative bacteria. Among the commonly encountered gram-negative bacilli Enterobacteriaceae, Stenotrophomonas maltophilia, and Acinetobacter spp. are oxidase-negative, whereas many other bacilli, such as Pseudomonas spp. and Aeromonas spp., are positive. The oxidase test is also a key reaction for the identification of Neisseria spp. (oxidase-positive).

Indole Test:

This test is based upon the fact that some bacteria use the enzyme tryptophanase to convert the amino acid tryptophan into molecules of indole, pyruvic acid, and ammonia. Since only a few bacteria contain tryptophanase, the formation of indole from a tryptophan substrate can be another useful diagnostic tool for the identification of an organism.  Indole is detected by combining with an indicator, aldehyde (1% paradimethylaminocinnamaldehyde), that results in a blue color formation. Indole production is a key test for the identification of Escherichia coli.

Urease Test:

Urea is a nitrogen containing compound that is produced during decarboxylation of the amino acid arginine in  the urea cycle.  Some bacteria produce the enzyme urease, which catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Organisms that do not produce this enzyme cannot metabolize urea. Urease hydrolyzes the substrate urea into ammonia, water, and carbon dioxide. The presence of the enzyme is determined by inoculating an organism to broth or agar that contains urea as the primary carbon source and detecting the production of ammonia. Ammonia increases the pH of the medium so its presence is readily detected using a pH indicator. Change in medium pH is a common indicator of metabolic process and, because pH indicators change color with increases (alkalinity) or decreases (acidity) in the medium’s pH, they are commonly used in many identification test schemes. The urease test helps to identify certain species of Enterobacteriaceae, such as Proteus spp., and other important bacteria such as Corynebacterium urealyticum and Helicobacter pylori.

PYR Test:

This test is used for the detection of pyrolidonyl arylamidase (also called pyrolidonyl aminopeptidase) activity in certain groups of bacteria, such as Streptococcus pyogenes (group A strep), Enterococcus spp., some coagulase-negative staphylococci, and some EnterobacteriaceaePYR detects PYRase activity in streptococcal organisms. This is based on the principle that the enzyme L-pyrroglutamyl-aminopeptidase hydrolyzes the substrate L-pyrroglutamyl-β-naphthylamide (PYR) to produce a β-naphthylamine. When the β-naphthylamine combines with a cinnamaldehyde reagent, a bright red color is produced

Hippurate Hydrolysis: 

Hippurate hydrolysis test is used for detection of hippurate hydrolyzing bacteria, mainly Streptococcal species. It is based upon the principle that hippurate acid is hydrolyzed to benzoic acid and glycine by the enzymatic action of hippuricase. The glycine end product is detected by the addition of ninhydrin reagent. The hippurate test is most frequently used in the identification of Streptococcus agalactiae, Campylobacter jejuni, and Listeria monocytogenes.

Tests for presence of metabolic pathways

Several identification schemes are based on determining what metabolic pathways an organism uses and the substrates processed by these pathways. In contrast to single enzyme tests, these pathways may involve several interactive enzymes. The presence of an end product resulting from these interactions is measured in the testing system. Assays for metabolic pathways can be classified into three general categories: carbohydrate oxidation and fermentation, amino acid degradation and single substrate utilizations.

i) Oxidation and fermentation tests: 

Bacteria use various metabolic pathways to produce biochemical building blocks and energy. For most clinically relevant bacteria, this involves utilization of carbohydrates (e.g., sugar or sugar derivatives) and protein substrates. Determining whether substrate utilization is an oxidative or fermentative process is important for the identification of several different bacteriaOxidative processes require oxygen; fermentative ones do not. Facultative anaerobic and anaerobic bacteria are capable of fermentation during which carbohydrates are broken down for energy production. A wide variety of carbohydrates may be fermented by various bacteria in order to obtain energy. The types of carbohydrates which are fermented by a specific organism can serve as a diagnostic tool for the identification of that organism. The clinical laboratory determines how an organism utilizes a substrate by observing whether acid byproducts are produced in the presence or absence of oxygen. In most instances the presence of acid byproducts is detected by a change in the pH indicator incorporated into the medium. The color changes that occur in the presence of acid depend on the type of pH indicator used.

ii) Amino acid degradation: 

Determining the ability of bacteria to produce enzymes that either deaminate, dihydrolyze, or decarboxylate certain amino acids is often used in identification schemes. The amino acid substrates most often tested include lysine, ornithine, arginine, and phenylalanine.  Decarboxylases cleave the carboxyl group from amino acids so that amino acids are converted into amines; lysine is converted to cadaverine, and ornithine is converted to putrescine. Because amines increase medium pH, they are readily detected by color changes in a pH indictor indicative of alkalinity. Decarboxylation is an anaerobic process that requires an acid environment for activation. The most common medium used for this test is Moeller decarboxylase base, whose components, include glucose, the amino acid substrate of interest (i.e., lysine, ornithine, or arginine), and a pH indicator.

iii) Single Substrate Utilization: 

Whether an organism can grow in the presence of a single nutrient or carbon source provides useful identification information. Such tests entail inoculation of organisms to a medium that contains a single source of nutrition (e.g., citrate, malonate, or acetate) and, after incubation, observing the medium for growth. Growth is determined by observing the presence of bacterial colonies or by using a pH indicator to detect end products of metabolic activity.

6) Conclusion:

This was a brief overview of bacterial identification techniques. this knowledge is mandatory for understanding the bacterial etiology of diseases. The bacterial etiology of periodontal diseases is well established. we must have the basic knowledge of bacteriology so that we can understand the etiopathogenesis of periodontal diseases. References are recommended for detailed study of the subject.

References:

  1. Bailey & Scott's Diagnostic Microbiology, 12th Edition.
  2. Medical Microbiology, 4th edition. Edited by Samuel Baron.
  3. Microbiology. Robert Bauman.
  4. Microbiology 5th Edition. Michael Pelczar.

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