Bergey's Manual of Systemic Bacteriology

 Bergey's manual is an accepted reference on the identification of bacteria. It has undergone gradual transformations and expansion since the time of its first publication. 

 The American Society of Microbiology published first edition of Bergey's Manual of Determinative Bacteriology in 1923. Professor David H. Bergey (Chair Person) and other four colleagues acted as the members of the editorial board. Afterwards, there was a sequel of eight editions, an abridged version and several supplements. At present, ninth edition (published in 1994) is available. It is used to classify bacteria based on their structural and functional attributes by arranging them into specific familial orders. However, this process has become more empirical in recent years.

Bergey's Manual of Systematic Bacteriology  Manual of Systematic Bacteriology

It is the main resource for determining the identity of prokaryotic organisms, emphasizing bacterial species, using every characterizing aspect. First edition of this manual consists of four volumes. It's first volume was published in 1984, the second in 1986 and final two volumes in 1989. This manual has much broader scope. It includes all information of earlier manuals. In addition it includes the information on taxonomy, ecology, cultivation, maintenance and the preservation of organisms. It includes many kinds of information such as...
  1. Descriptions and photographs of species,
  2. Test of distinguish to distinguish among genera and species,
  3. DNA relatedness among organisms and
  4. Various taxonomic studies.
There are four divisions of kingdom Procaryotae according to Bergey's Manual of Systematic Bacteriology.

Division I : Gracilicutes

Includes prokaryotes with thin cell walls e.g. Gram negative bacteria.

Division II : Firmicutes

Includes prokaryotes with thick cell wall e.g. Gram positive bacteria

Division III : Tenericutes

Includes the prokaryotes that lack cell wall.

Division IV : Mendosicutes

Includes the prokaryotes lacking peptidoglycan in their cell wall.

Second edition of Bergey's Manual of systematic Bacteriology consists of five volumes. Volume I was published in 2001, Volume II in 2005 and the other three volumes in 2007. In comparison to the first edition, second edition has certain changes. e.g .,

  1. Volume - I includes The Archaea and The Deeply Branching And Phototrophic bacteria.
  2. Volume II includes the Gram-negative Proteobacteria. It includes medically important genera are Escherichia, Neisseria, Pseudomonas, Rhizobium, Rickettsiae, Salmonella and Vibrio.
  3. Volume - III includes the Gram- positive bacteria with low G + C content in their DNA. They are the members of phylum Firmicutes . It includes rods and cocci and also pleomorphic Mycoplasma. They may form endospores. Its classes are Clostridia, Mollicutes and Bacilli.
  4. Volume - IV includes the Gram - positive bacteria with high G + C content in their DNA. They have more than 50- 50 % G + C content. 
  5. Volume - V includes ten phyla. They are located here for convenience. Includes morphologically diverse gram - negative organisms. They may not be related. Organisms included are Plantomycetes, Chlamydia, Spirochaetes, Bacteroilds, Fusobacteria, Chlamydiae, Acidobacteria, Verrucomicrobia and Pictyoglomus.

General Nature and Basic Structure of Enzyme

 A living cell is capable of performing a multitude of  biochemical reactions in order to survive grow and multiply. This involves

  • Degradation of complex nutrients into simpler forms so that they can be absorbed by the cell.
  • Uptake of these simple nutrients.
  • Chemical transformation of these simple nutrient molecules into various precursor metabolites, so that they are available for biosynthesis.
  • Generation of ATP and other bio reactive molecules, which co-operate in cellular biochemical reactions.
  • Biosynthesis of cellular molecules and structural components of the cell etc.

All these diverse types of chemical reactions can occur with a high degree of specificity and rate, under normal growth conditions for the organism. These chemical changes are brought about by the living cell through the role and participation of a special class of molecules known as enzymes . Enzymes function in the cell by acting as a biocatalyst.

Discovery of Enzymes

The term enzyme was coined by Kuhne in 1878, suggesting in yeast (en = in, zyme = yeast). It was examined that cell free extract from yeasts is capable of causing conversion of sugar into alcohol. This lead to the understanding that the molecules present inside the cells of yeasts are responsible for these chemical transformations. Kuhne called them as enzymes.

Later on, it was established that all biochemical activities of a living cell are attributed to these magic molecules called enzymes. The first enzyme to be discovered was 'amylase'. Its presence in malt extract was detected in 1833 by two French chemists Pain and Persuses.

General Nature of Enzymes

Enzymes are regarded as organic biocatalysts which are capable of functioning both extra cellular and intra cellular. Following are the major characteristics of enzymes. 

1] With exception of a few catalytic RNA molecules or ribozymes, all enzymes are protein in nature. Over 90 % enzymes are globular proteins. Many of them are conjugated proteins, having non protein component. 

Enzymes may be monomeric or multimeric proteins. As they are proteins, they share all major characteristic of proteins .

  • They are macromolecules with high MW.
  • They are non dialyzable and are unable to pass through semi permeable membrane.
  • They are amphoteric in nature. i.e. they possess both types of lonizable groups : -NH₂ and -COOH. which on ionization . yield positively charged ammonium (NH4+) ion and negatively charged carboxyl (COO-) ion.
  • As they are amphoteric, they possess specific isoelectric pH. At this pH, both -NH₂ and -COOH groups get equally ionized such that their net electrical charge becomes minimum.
  • They show electrophoretic mobility. If the enzyme solution is at pH, above isoelectric value, they acquire negative charge and hence move towards anode and vice versa.
  • They are colloidal in nature.
  • They can be salted out by salts like ammonium sulfate
  • They can be precipitated out by protein denaturing solvents like acetone and alcohol.
  • They can absorb maximum UV light at 280 um wavelength.

2]. Enzymes are mostly thermo labile and get denatured at high temperature, usually above 60°C. However, some enzymes are found thermo stable and can withstand high temperature up to 70°C - 80°C or even more.

3]. Enzymes are highly specific in action. They can act on a specific substrate molecule to bring about specific biochemical reaction .

Catalytic RNA - Ribozyme

  Apart from proteins, a few RNA have also been recognized to have catalytic activity. They are called ribozymes or catalytic RNA. They were first recognized by Thomas Cech in 1982. The ribozymes have two types of common roles :
  1. RNA processing, where the RNA is involved in RNA splicing, RNA ligation and RNA replication.  
  2. Peptide bond formation during protein synthesis. In ribosomes, they function as a part of rRNA and participate in peptide bond formation.

Basic structure of Enzymes

In general, enzyme molecule consists of two components.
  1. Apo enzyme and
  2. Prosthetic group or cofactor

Apo enzyme is protein part of enzyme.
Prosthetic group or cofactors are non protein part of enzymes. They consist of vitamin or their derivatives or metal lons. (If these non protein components are bound loosely to protein, they are called cofactors) and if they are bound firmly (covalently), are called prosthetic groups. Apo enzyme and prosthetic group form complex to form active form of enzyme, known as Holoenzyme.

Classification of Enzymes

pH Effect on Microbial growth

 The self-ionization of water is a continuous process when it is in pure form. In this process, when there is collision of two H₂O molecules they dissociate into H+ and OH-  ions. There is no free hydrogen ions in the water. Because the free hydrogen ions have tendency to attach to the H₂O molecule to form hydronium ion. This means, you will always find  H₃O⁺ or hydronium ions in water, instead of free H+ ions.

What is pH ?

pH stands for potential of hydrogen, or power of hydrogen. pH can be defined as measurement of hydrogen ion concentration in a solution, or mathematically it can be defined as negative logarithm of hydrogen ion concentration.

Formula of pH : -log [H+]

pH is applied only to aqueous solutions. That means where there is water, there is pH. pH was first described by Sorensen in 1909. pH reveals the acidity or basicity of water.
  The pH scale ranges from 0 to 14 where the value 7 indicates the neutral pH. pH less than 7 indicate acidic conditions, and greater than seven indicate basic or alkaline. In other word pH is about calculating the free hydronium ions and free hydroxyl ions in a given solution.

A solution with more H+ ions is acidic and gives a pH value less than 7 Similarly, a solution with more OH- ions, is basic and gives a value greater than seven.

Importance of pH

Certain reactions in our body require certain value of pH. Anything higher or lower value may cause damage. For example, if too much of hydrochloric acid is produced in the stomach, the patient will be advised to take antacids like magnesium hydroxide in order to neutralize the excess pH.
  In the same way plants cannot grow if there is continuous rising and falling of pH of soil. Animals in the river cannot survive when acid rains fall on the river.
  Similarly, pH plays an important role on microbial growth.

There were cases where microbes did not show growth in the absence of right pH conditions, even they were supplied with all the required nutrients.
  The pH value where a microbe can grow its best, is called the optimum pH.
Most bacteria grow best at a pH value near to 7, which means, most bacteria are neutrophils.
Some bacteria can grow at a pH range between 3 and 4. These are called acedophiles.
Alkaliphiles are the bacteria that can tolerate pH between 8 and 11.

Impact of pH on Microbial growth

The bacterial cell consists of several protein molecules, lipids, and nucleic acids. And the cell hosts several biochemical reactions. All these activities are regulated when there is optimum pH.
In lower pH conditions, the increased hydrogen ions break the weak hydrogen bonds of protein side chains and finally change the shape of the protein.
  When a protein is not in its original shape, it cannot perform its routine function, and ultimately the bacteria cannot survive.

There are two methods to measure pH in a microbiology lab. The first one is using pH paper. The pH paper changes its color when it is dipped into a solution. This color change is based on acidity and basicity of the sample solution.
Later, the color of the paper will be compared with the color chart provided along with the paper. These papers are coated with a pigment called Flavin, which is extracted from red cabbage. Flavin has ability to change color when it comes in contact with an acid or base. This method will not provide the exact pH value. However, this will provide a value which is closer to the actual pH value.
The other method to measure pH that gives an accurate value is using a pH meter.

INTRODUCTION TO BIOGECHEMICAL TRANSFORMATIONS IN SOIL : MINERALIZATION AND IMMOBILIZATION OF ELEMENTS

 Erth is a closed system, where the over all quantity of matter remains constant. Microorganisms need electron, energy and nutrients to grow. They are responsible for cyclic transformation of compounds, and therefore they are called biogeochemical agents. They carryout transformation of carbon, nitrogen, sulphur, phosphorus, iron etc. This cycling of elements is called biogeochemical cycling. Both biological and chemical process are involved in biogeochemical cycling.

The oxidation reduction reactions are mainly responsible for biogeochemical cycling of compounds. This changes the chemical and physical characteristics of different compounds. These cyclic turnover of elements are brought about by different types of microorganisms resulting into continuous change in chemical states of matter.

The life of earth depends on cyclic conversion of chemicals from inorganic state to organic (complex state) to the elemental state. The break in the cycle at any point would dramatically affect all life forms. Various processes carrying out these transformation include :

Mineralization : 

It is a process of conversion of complex organic compounds to simple inorganic forms. Many heteroprophic microbes play role in mineralization.
The resultant simple compounds are made available to plants and microbes. Energy is released in the process. The process of mineralization is very important as it increases soil fertility.

Carbon mineralization :

  • Organic carbon is mineralized to inorganic state.
  • Under aerobic condition the main products of carbon mineralization are CO₂ and water.
  • In absence of O₂ organic carbon is incompletey metabolized to produce organic acids, alcohols and gases.

Assimilation :

  • It is the process of conversion of substrate elements to protoplasmic elements. 
  • Microbes take up the simple materials from the environment (soil) and convert them into cellular materials. It is known as assimiliation or biosynthesis.
  • In this process synthesis of energy and cellular material takes place. Through assimilation microbes store the excess of simple inorganic chemicals, and prevent their loss due to erosion.

Immobilization :

It is a process in which the quantity of plant available nutrients are reduced in soil by microorganisms. The nutrient assimilation is an important method of immobilization.
The uptake of various elements like carbon, nitrogen, phosphorus, sulphur, etc. cause immobilization.

  Intermediary substances accumulate abundant quantities of CH4 and smaller amount of H₂ is evolved. The factors affecting mineralization are level of organic matter, temperature, moisture, pH, depth and aeration.
  All these factors affect the growth and metabolism of microbes and the process of mineralization.

  • In nitrogen mineralization ammonium, nitrite and nitrates are accumulated from organic nitrogenous compounds like proteins, nucleic acids, etc.
  • In phosphorus mineralization organic phosphorus present in nucleic acid, phytin, lecithin, etc. are converted to inorganic phosphorus.
  • Sulfur mineralization involves aerobic breakdown of sulfur containing amino acids : cystine, cysteine, methionine, and vitamins, thiamine, biotin, thioctic acid releasing sulfates. Whereas in absence of oxygen , H₂S and odoriferous mercaptans accumulate in soil.

Lytic cycle: Multiplication of T4 Bacteriophage

T4 coliphage is a phage that infect coliform bacteria especially E.coli. T4 is a double stranded DNA phage, it has a contractile sheath and an unique base 5-hydroxyl methyl cytosine (5-HMC). It is the best known member of large virulent phages.

The lytic cycle of T4 phage involve following steps: ADSORPTION
PENETRATION
BIOSYNTHESIS
ASSEMBLY
RELEASE

1]. Adsorption :

  • The lytic cycle begins when a bacteriophage comes in contact with a susceptible host cell by random collision.
  • Phage possess an adsorption organs or anti-receptors and host cells possess receptors.
  • Host cell surface components -Flagella, Pilli , Teichoic acids, Proteins, Carbohydrates , LPs and Lipopolysaccharides serves as receptors.
  • Phage components such as tail fibers, tail proteins and spikes serves as adsorption organs or anti-receptors.
  • Each phage has its specific receptor to which it adsorbs.
  • Adsorption takes place only when the anti-receptor is chemically complementary to the receptor.
  • T4 phage possess tail fibers that serves as an adsorption organ or anti-receptor.
  • Normally, tail fibers are present in folded form around the tail.
  • Whiskers hold the tail fibers in folded form.
  • When phage come in contact with host, tail fibers unfold.
  • Unfolding of fibers requires tryptophan & co-factors - Mg++ & Ca++.
  • Thus, the phage and host binding is favoured by ionic environment.
  • T4 host E.coli possess outer membrane protein C (OmpC) – lipopolysaccharide complex as receptor.
  • Initial attachment occurs when tail fibers attach to the OmpC- lipopolysaccharide complex.
  • Initial adsorption is weak and reversible.
  • It becomes irreversible when tail pins attach to lipopolysaccharide.

2]. Penetration :

  • Once attached, the bacteriophage injects DNA into the bacterium.
  • Bacteria possess rigid cell wall and therefore the phages directly cannot penetrate into the bacterial cells.
  • They inject only their nucleic acids inside the host cell.
  • In the T-even phage, irreversible binding of the phage to host results in the contraction of the sheath and the hollow tail tube is inserted through host cell wall.
  • Some phages have enzymes like lysozyme that digest the cell wall components of the bacterial cell.
  • The penetration of T4 phage DNA occurs when -
  1. There is irreversible attachment of phage to host cell,
  2. Contraction of sheath, pushing tail tube through cell envelope
  3. Injection of DNA into cell like injection of vaccine/drug by a syringe

3]. Biosynthesis :

Biosynthesis divided into three steps:
  1. Formation of immediate early and delayed early protein
  2. Replication of phage genome
  3. Formation of late proteins

i) Formation of immediate early and delayed early protein :

  • Part of phage DNA is immediately transcribed by host RNA polymerase to form immediate early m-RNAS.
  • These early m-RNA translate to following enzyme proteins -
  • a) Nucleases - Breaks down host DNA & make nucleotides available for its own synthesis.
  • b) α-subunit modifying enzyme - modifies α-subunit of host RNA polymerase.
  • Modified host RNA polymerase transcribes part of viral genome to delayed early m-RNAS.
Delayed early mRNAs are translated to following enzymes-

  •  a) Phage enzymes that produce 5-hydroxyl methyl cytosine (5-HMC), a unique base in phage DNA
  • b) Polymerases and ligases - that play role in phage DNA replication and recombination.
  • c) Glucosylation enzyme-adds glucose to HMC & protects phage DNA from host restriction endonuclease
  • d) σ-subunit modifying enzyme - modifies σ-factor of RNA polymerase so that is transcribes late mRNAs.

ii) Replication of Phage Genome :

  Two modes have been proposed for the replication of T4 phage DNA.
By bi-directional mode - at early stage
By rolling circle mode - at later stage

  • Initial replication is bi-directional and semi-discontinuous.
  • Leading strand is synthesized continuously and lagging strand is synthesized discontinuously leading to the formation of eye structure.
  • Bi-directional replication is initiated at several origins along the DNA and is catalyzed by phage coded enzymes.
  • In the rolling circle mode of replication, a cut is made in one of the DNA strands by a specific endonuclease and 3'end is made free.
  • DNA polymerase extends the free 3'OH end by adding complementary bases.
  • Intact strand serves as template for addition of complementary bases.
  • Due to extension of 3'OH end, the 5'end is displaced.
  • Displaced strand is synthesized discontinuously by adding Okazaki fragments.
  • This mechanism produces multi-genome length molecules.
  • Such molecules are referred to as concatemers.
  • The concatemers are later cleaved to head sized molecules by headful cutting mechanism.
III] Formation of late proteins 
  • Soon after the replication of phage DNA, transcription of late m-RNAs occur.

  • These late m-RNAs translate to different proteins.
  • These proteins include the structural proteins.
  • They are proteins involved in phage assembly and an enzyme lysozyme that degrades the peptidoglycan layer of bacterial cell wall.
  • For example - head (capsid) proteins, tail tube protein, sheath proteins, collar, whiskers, base plate, tail fiber, tail pins, lysozyme etc.

4. Assembly of Phages :

  • Assembly of new phage particles begins after accumulation of structural proteins and nucleic acid molecules in the cell.
  • Process of assembling phage particles is known as known as maturation.
  • There are four different pathways that lead to the formation of phage particles.
  • These include base plate, tail tube & tail sheath, tail fibers and head.
  • About 50 genes take part in the morphogenesis of T4 phage.
  • Subunits of base plate assemble to form a base plate.
  • Then tail tube and sheath subunits polymerize on base plate to form mature tail.
  • The subunits of head assemble together to form prohead and then DNA is inserted in the prohead to form complete head.

5. Release :

  • The release of newly synthesized phages occurs by sudden explosion or bursting (lysis) of bacterial cell.
  • Lysis begins after about 22 minutes.
  • One of the gene products involved in the process include lysozyme.
  • It cleaves glycosidic bonds in the peptidoglycan making the cell wall susceptible to the rupture.
  • There is another protein termed as holin that make holes in the cell membrane and makes the way for lysozyme action.

Eijkman Test Principle and Procedure

 The IMViC test has two drawbacks. The first, It has many controversial procedures and second is test results do not give satisfactory differentiation between fecal and non-fecal coliforms. In 1904 Eijkman proposed another test to differentiate fecal and non-fecal coliform.

Principle of Eijkman Test

  • Only fecal coliforms of warm blooded animals grow at 46°C and ferment lactose with Acid & Gas production
  • Most strains of fecal E.coli can ferment lactose in a special buffered broth when incubated at 45°C, where as very few or less frequently the Enterobacter aerogenes do so.
  • The test is called as Eijkman test or elevated temperature test.

Procedure of Eijkman Test

  • A buffered tryptose lactose broth in tubes with inverted Durham's tube is inoculated with a culture of coliforms.
  • It is then incubated in water jacketed incubator at 45°C for 48 hours.
  • Gas production after incubation constitutes a positive test for fecal coliforms.
  • Another method, instead of tryptone lactose broth, buffered boric acid lactose broth (BALB) medium is used.
  • The advantage is that, the medium used is selective for fecal Escherichia.
  • It selectively inhibits growth and gas production by Enterobacter and other intermediate members of coliforms.
  • Sterile medium is first warmed to 37°C and then inoculated with culture & incubated at 45°C for 48 hrs.
  • Gas production indicates positive test.
  • Eijkman test gives better result than IMViC tests.
  • Therefore, it is generally preferred in water examination.

IMViC Biochemical Tests: Principles Procedures and Results

IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. In this the first test is Indole test, the second one is Methyl Red, the third one is Voges Proskauer and the fourth one is Citrate Utilization test.

  • In the quantitative test for coliforms if completed test is positive, further testing is essential to differentiate fecal and nonfecal coliforms.
  • Escherichia coli and Enterobacter aerogens are the important contaminants of water respectively.
  • Escherichia coli is a fecal coliform as it is mainly found in human feces while Enterobacter aerogenes is considered as non fecal as it also occurs in soil & plant material.
  • They closely resemble each other in their morphological and cultural characteristics. 
  • Therefore, the biochemical tests are performed to differentiate them.
  • Tests are collectively designated as the IMViC tests.
  • The name was coined by Parr from the first letters of the four tests namely - I for Indole, M for Methyl Red, V for Voges Proskauer and C for Citrate Utilization test.
  • The letter i between V and C is added solely for euphony.

Indole Test

  • Indole Test is used to detect indole production from amino acid tryptophan.
  • E. coli has the ability to breakdown the tryptophan by enzyme tryptophanase with release of indole, pyruvic acid and ammonia.
  • Enterobacter doesn't produce enzyme tryptophanase. Therefore, they are not producing indol from an amino acid tryptophan.
  • Test is performed by inoculating the test organism in 1 % tryptone water or 2 % peptone water, incubation at 37°C for 24 hrs.
  • Indole production can be detected by adding few drops of xylene and Kovac's or Ehrlich's reagent which contains p-dimethyl aminobenzaldehyde.
  • This p-dimethyl aminobenzaldehyde reacts with the indole and produce a cherry red(pink) coloured compound. This is a reduction type of reaction.
  • Xylene extracts the indole in upper layer of the medium.

Methyl Red Test

  • Methyl Red Test is carried out to detect acid production ability of test organism from glucose.
  • It is performed by inoculating test organism in glucose phosphate broth tube and incubating at 37°C for 24 hrs
  • Methyl red indicator is then added to detect acid production which gives red colour in positive reaction and yellow in negative.
  • Escherichia coli rapidly ferments glucose with production of acids and reduce the pH to about 5.0
  • This pH / acidity prevents further growth of E.coli in glucose phosphate broth tube.
  • Enterobacter aerogens initially produce acids but later on it is converted to non acid products such as ethanol, acetyl-methyl-carbinol (acetoin) and 2, 3- butane-di-ol ( reduction product of acetoin).
  • Due to this, E.aerogens continues to grow without producing its limiting pH.
  • Thus, Escherichia coli gives positive methyl red test while Enterobacter aerogeys gives negative test.
  • Methyl red is a pH indicator which is red at pH 4.4 while yellow at pH 6.2

Voges Proskauer

  • The test used for the detection of acetyl methyl carbinol (acetoin) production from glucose, by the test organisms.
  • It is also performed by inoculating the test organisms in glucose phosphate broth medium and incubating at 37°C for 24 hrs.
  • This is followed by addition of 40 % potassium hydroxide and 5 % a-naphthol solution with the shaking of the tube.
  • For the Detection of acetoin requires its further oxidation to diacetyl, In presence of catalyst α-naphthol, alkali(KOH) and air, acetoin is further oxidised to diacetyl.
  • Diacetyl in presence of peptone, gives a red colour.
  • The constituent of peptone responsible for red colour is guanidine nucleus of the amino acid arginine.
  • Thus, a positive test is indicated by development of red colour.
  • Enterobacter aerogenes produces acetoin from pyruvic acid(Positive test) while Escherichia coli doesn't produce it(Negative test).

IMViC tests Results

Citrate Utilization Test

  • Citrate Utilization test is carried out to detect the ability of organism to utilize citrate as a sole source of carbon and energy.
  • The utilization of citrate depends upon the enzyme citrate permease that facilitates citrate transport into the cell.
  • E. aerogenes produce citrate permease and are able to utilize citrate as the sole source of carbon while E.coli do not produce
  • The test is performed by inoculating the test organisms in Koser's citrate medium which sodium citrate as the sole source of carbon; and incubating at 37°C for 24 hrs.
  • Ability to use citrate is indicated by the development of turbidity in medium.
  • Citrate Utilization test can also be performed by inoculating the test organisms on the Simmon's citrate agar slant and incubating at 37°C for 24 hrs.
  • The Simmon's citrate agar is the modified processed agar media which contains bromothymol blue as a pH indicator
  • Enterobacter converts citrate to oxaloacetate and acetic acid by enzyme citrase.
  • These products are further converted to pyruvic acid and CO2.
  • The CO2 reacts with sodium and water to form sodium carbonate
  • Sodium carbonate is an alkaline product and it raises the pH of medium Bromothymol blue (pH indicator) is blue in alkaline and green in acidic condition.
  • The change in colour of slant from green to blue indicates positive test.

 Results :


The IMViC test has two drawbacks as- 
  1. It has many controversial procedures 
  2. Test results do not give satisfactory differentiation between fecal and non-fecal coliforms.