Koch's postulates application in Microbiology

 In the world of microbiology, understanding the causes of infectious diseases is crucial for effective prevention and treatment strategies. One of the fundamental principles in this field is Koch's postulates, named after the German physician and microbiologist Robert Koch, who developed them in the late 19th century. Koch's postulates provide a systematic approach to demonstrate the causative relationship between a microorganism and a disease, laying the groundwork for the field of medical microbiology.

The Four Koch's Postulates:

1. The microorganism must be present in every case of the disease but absent from healthy organisms.

   This postulate emphasizes the importance of identifying a specific microorganism consistently associated with a particular disease. Koch recognized the need to isolate and characterize the microorganism responsible for causing the illness.

2. The microorganism must be isolated from the diseased organism and grown in pure culture.

 Once the microorganism is identified in a diseased individual, it must be isolated and cultivated in laboratory conditions. This step ensures that the microorganism can be studied and manipulated independently of its host.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

   To establish a causal relationship, Koch demonstrated that inoculating a healthy organism with the isolated microorganism results in the development of the same disease observed in the original host. This step confirms that the microorganism is indeed responsible for the illness.

4. The microorganism must be re-isolated from the experimentally infected organism.

   Finally, to complete the chain of evidence, Koch reisolated the same microorganism from the experimentally infected organism. This step confirms that the microorganism retrieved from the experimental host is identical to the one initially isolated from the original diseased individual.

Applications and Limitations:

Koch's postulates have been instrumental in identifying the causative agents of numerous infectious diseases, including tuberculosis, cholera, and anthrax. By rigorously applying these criteria, researchers have been able to establish causal relationships between specific pathogens and their associated diseases, paving the way for the development of vaccines, antibiotics, and other treatments.

However, it's important to note that there are limitations to Koch's postulates. For example, some microorganisms cannot be grown in pure culture or do not cause disease when introduced into a healthy host due to complex interactions with the host's immune system or other factors. Additionally, advances in molecular biology and genomics have revealed cases where multiple microorganisms or non-infectious agents contribute to disease pathogenesis, complicating the application of Koch's postulates in certain contexts.

Conclusion:

Despite these limitations, Koch's postulates remain a cornerstone of microbiology and have greatly contributed to our understanding of infectious diseases. They provide a systematic framework for establishing causation and have guided generations of researchers in their quest to unravel the mysteries of microbial pathogenesis. As technology continues to advance, researchers will undoubtedly refine and expand upon Koch's postulates, ensuring their continued relevance in the field of medical microbiology.

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.

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.

Microbial Metabolism

  Every living organism has the fundamental capability to grow and synthesize new cell material. This requires processing of the nutrient molecules taken up by the cell and involves a series of biochemical transformations. The set of biochemical reactions occurring in cell includes degradation, synthesls as well as modification of the molecules.

   These chemical reactions, which operate by the living cells are collectively referred to as metabolic reactions and the phenomenon is called metabolism. Thus, metabolism is defined as the sum total of all biochemical reactions carried out by a living cell.

The metabolic reactions are further categorized as

1. Catabolism
2. Anabolism
3. Primary metabolism
4. Secondary metabolism
5. Intermediary metabolism

Catabolism

Catabolism includes the set of biochemical reactions which involve degradation of the molecules taken up by the cell and generation of substances essential for biosynthesis of cell constituents. The products of catabolism are catabolites.

  They, generally, involve formation of various precursor metabolites, energy rich compounds and reducing power. Hence, these metabolic reactions are often called as fuelling reactions, which provide essential fuels required for cellular synthesis. The precursor metabolites provide basic carbon skeleton for the synthesis of building blocks of the called cellular macromolecules.

  The energy rich compounds are ATP, GTP, CTP, TTP, UTP and acetyl CoA, which provide necessary form of biochemical energy required to drive various energy requiring blochemical reactions. On the hydrolysis of high energy bond of these compounds, necessary free energy is available for the purpose.
 
Reducing power, generated during the catabolism. is in form of reduced pyridine compounds. NADH and NADPH. They provide essential reducing conditions required for several blosynthetic as well as assimilatory reactions of the cell.

Anabolism

Anabolism includes the set of blochemical reactions which involve synthesis cellular molecules.
These include blosynthesis of

1. Building blocks of cellular macromolecules e.g. amino acids, nucleotides, fatty acids, sugars etc.
2. Vitamins and coenzymes, which are essential for driving various enzyme catalyzed reactions.
3. Cellular macromolecules such as proteins, lipids, nucleic acids, polysaccharides as well as synthesis of cell structural compounds.

These anabolic reactions are fuelled by the products of catabolism.

Primary metabolism

  The part of cellular metabolism which is very much essential for cell growth is termed as the primary metabolism. The products of primary metabolism are called primary metabolites.
 
  The primary metabolism includes the metabolisms associated with generation of energy rich compounds, reducing power, precursor metabolites as well as the synthesis of bullding blocks of cellular macromolecules.

The products of primary metabolism include

1. Energy rich compounds such as ATP and others.
2. Organic acids such as lactic acid, citric acid, acetic acid etc.
3. Organic alcohols and solvents such as ethanol, glycerol. acetone, butanol etc.
4. Amino acids, vitamins coenzymes, nucleotides etc.

The primary metabolism, in general, is found operative lin the cell during log phase of the growth.

Secondary metabolism

  The part of cellular metabolism, which is not essential for cellular growth is called secondary metabolism. Products of secondary metabolism are called secondary metabolites.

  It becomes active during late log phase and stationary phase of the growth. It involves utilization of the excess remains of carbon precursors and energy for synthesis of new molecules which may have secondary role in growth of a cell.
e.g. Antibiotic synthesis, which is not essential for growth. but does help the organisms to survive in the environment in the presence of various antagonistic organisms by destroying them.

Intermediary metabolism

The part of cellular metabolism which occurs after the entry of nutrients into the cell, leading to the synthesis of bullding blocks of the cellular macro molecules is generally referred to as the intermediary metabolism.

Central metabolic pathways

The central metabolic pathways are basically catabolic in nature. These pathways Include Glycolysis, pentose phosphate pathway and TCA Cycle.They participate in generation of energy, reducing power and precursor metabolites required for cellular synthesis.

Role of reducing power in metabolism

All elements, except phosphorus, present in cell are in reduced form. Carbon exists in organic form. Nitrogen is present as amino group. sulfur as úSH group etc. Therefore, all these elements must be reduced at the cellular level of reduction, before they are assimilated in to cell during biosynthetic reactions.

  Most of these elements exist in oxidized state in nature. Therefore, before they are assimilated in the cell, they must be reduced. This requires avallability of sultable reducing power. NADPH serves as the principle reducing power in all such assimilatory and biosynthetic reactions of anabolism. In addition to NADPH, flavin coenzyme, FADH2 can also serve as immediate reducing agent in biochemical reactions.

Generation of reducing power :

  Reducing power is generated on oxidation of a suitable electron donor during catabolic reactions of metabolism. These electron donors can be either organic or inorganic or both depending on the type of organism. NADPH acts as the reducing power in all anabolic reactions. In addition to NADPH, flavin coenzyme, FADH2 can also serve as reducing power in certain biochemical reactions.

  NADPH is generated during oxidative pentose phosphate pathway, where glucose 6 PO4 is oxidized to pentose phosphate in all most all living organisms. However in arche bacteria, where pentose phosphate pathway does not function, alternate glycolytic pathway function to generate NADPH.

NADPH can also be generated in cell by transhydrogenase reaction, where reduced NAD will participate in reduction of NADP. This reaction also helps in maintaining adequate cellular levels of NADH / NADPH ratio. NADH mainly participates In ATP formation, by transferring electron through electron transport chain.

Role of precursor metabolites

  Precursor metabolites are the intermediate molecules in the metabolic pathways. They are produced during operation of catabolic pathways. The precursor metabolites can

1. Provide basic carbon skeleton for the synthesis of all the building blocks required to synthesize macromolecules.
2. Undergo oxidation via catabolic pathways to provide ATP and other energy rich compounds that fuel anabolic pathways.

About 150 different low molecular. weight compounds are required for cellular synthesis. They include
1. Building blocks for synthesis of cellular macromolecules. They include

• Amino acids for synthesis of proteins.
• Fatty acids for synthesis of lipids.
• Monosccharides for synthesis of polysaccharides.
• Purines and pyrimidines for synthesis of Nucleic acids

2. Soluble molecules required for cellular metabolic activities. They include vitamins, co-enzymes and polyamines.

There are only 12 compounds, which act as precursor metabolites. They are virtually the same in all living organisms. They include

• Acetyl CoA
• Pyruvate
• Phospho enol pyruvate (PEP)
• 3 phospho glyceraldehydes (3PGAL)
• Dihydroxy acetone phosphate (DHAP)
• Glucose 6 Phosphate
• Fructose 6 Phosphate
• Erythrose 4 Phosphate
• Ribose 5 Phosphate
• Xylulose 5 Phosphate
• Aplha Keto glutaric Acid (Alpha KG)
• Succinate

The precursor metabolites are the intermediates of three Indispensible pathways of catabolism

1. TCA cycle
2. Glycolytic or gluconeogenic pathways
3. Pentose phosphate pathway

Role of energy rich compounds

To perform all cellular activities, a suitable form of blochemical energy is required by the cell. This biochemical energy is obtalned as the energy rich compounds, which possess high energy rich chemical bonds.
  The necessary energy required to drive a blochemical reaction is released on hydrolysis of this energy rich bond. In living cells, a variety of energy rich compounds are formed, which are utilized for general purpose or to drive a specific biochemical set of reactions.

Energy rich compounds of cell

  There are mainly two classes of energy rich compounds formed in the cell, which satisfy need of energy requiring reactions. They are

1. Compounds having high energy anhydrous phosphoester bond.
2. Compounds having high energy thiolester bond.

Compounds having high energy anhydrous phosphoester bond
Most energy rich compounds of the cell belong to this category. They are obtained as nucleoside triphosphate derivatives. These include

1. ATP Adenosine triphosphate
2. GTP Guanosine triphosphate
3. CTP Cytidine triphosphate
4. TTP Thymidine triphosphate
5. UTP Uridine triphosphate

ATP and its role

  ATP is considered as one of the most commonly used energy currency of the cell. It possesses two energy rich anhydrous phosphoester bonds.

Hydrolysis of each of this energy rich bond releases 7.3 Keal energy.

ATP + H₂O ➞ ADP + Pi + 7.3 Kcal.
ADP + H₂O ➞ AMP + Pi + 7.3 Kcal.
AMP + H₂O ➞ Adenosine + Pi+ 4 Kcal.
ATP is most commonly required for

1. Uptake of nutrients
2. Activation of most substrate molecules so that they are able to enter the cell metabolism
3. Biosynthesis of most cellular molecules, nucleic acids, chromosome replication and cell division.

Other energy rich compounds and their role

  Apart from ATP, various other kinds of energy rich compounds are formed in the cell. They have specialized role in cellular metabolism. Their specialized utilization in specific metabolic reactions may be considered useful for adequate supply of energy for the concerned biosynthetic reactions, so that they can operate at optimal level in the cell. These energy rich compounds and their role are summarised in below table.

Energy rich compounds and their role in metabolism.

Bacterial Cell Wall Staining by Chance's Method

  Bacterial cell is consisting of various structural components. Cell wall is one of the most important component. Cell wall present outside of the bacterial cell membrane. It gives rigidity, protection and shape to the bacterial cell.

  Based on the structure of cell wall, all bacteria are divided into two groups as

• Gram positive and
• Gram negative.

Cell wall of Gram positive cell is monolayered while cell wall of gram negative bacteria is bilayered.
Bacterial cell wall can be demonstrated by various special staining methods like Chance's method, Ringer's method & Dyers method. The most common cell wall staining method is Chance's method.

Requirements :

• Clean grease free slide
• Nichrome wireloop
• 24 hrs old culture of bacteria
• 0.5% New fuchsin/Basic fuchsin solution (Basic Stain)
• 0.5 % Condo red solution (Acidic Stain)

Procedure & Steps

• Prepare the smear on the slide under aseptic conditions with the help of wire loop.
• Air dry the smear, but do not heat fix (Because the heat fixation changes the structure of capsule. therefore heat fixation is avoided here).
• Apply 0.5% New Fuchsin for 3 minutes.
• Remove the excess stain (but do not water wash)
• Apply 0.5 % Congo Red for 4 minutes.
• Gentle wash with water
• Air dry and observe under oil immersion objective lens.

Machanism and Principle of chance's cell wall staining method

  New Fuchsin stain is a basic stain. Therefore it is stained cell wall as well as cytoplasm. Cell wall and cytoplasm both are acidic in nature and they are having negative charge on their surface. However here the strong staining of cell wall take place. Because cell wall is more acidic than cytoplasm due to the presence of free carboxylic groups on its surface.

  Congo red is a acidic stain and this acidic stain bind with basic stain which is already present on the cell and removes that basic stain. That means here Congo red acts as a decolorizer. but the  removal of basic stain(New Fuchsin) takes place only from the cytoplasm and not from the cell wall, because it is strongly bound to the cell wall.

  Therefore after water wash cytoplasm becomes colourless while cell wall becomes pink coloured. that means here the role of Congo red is to carry out the decolorization, But the decolorization of only cytoplasm takes place and decolorization of cell wall does not take place.

Microscopic Observation

Spherical and rod shaped bacterial call wall Pink in colour.

 Under the microscope you will observed pink colour cell wall and colourless cytoplasm.

Gram Staining : Principle & Machanism, Steps, Disadvantages and Factors affecting gram Staining

Gram staining is a technique used to differentiate two major groups of bacteria based on their cell wall composition called gram positive and gram negative bacteria.

The technique was named after Hans Christian Gram who developed this method in 1884.
Gram staining is a preliminary test in the bacterial identification process. It plays an important role in clinical microbiology. It helps the medical professionals in the diagnosis of infectious diseases directly from the clinical sample.

The cell wall of gram positive bacteria is completely different from the gram negative bacteria. It is important for them to understand the Gram nature to provide the appropriate treatment for the infection.

Requirements of Gram Staining

• Clean grease free glass slide
• Nichrome wire loop
• Bacterial cell suspension
• 0.5% Crystal violet
• Gram's lodine
• 95% Ethanol
• 0.5% Safranine/basic fuchsin

Steps of Gram Staining

Imagine our sample has a mixed culture with gram positive cocci and gram negative rods

Step-1 (Smear Preparation)

• Prepare a thin smear of culture on a clean glass slide (oil free)
• Allow to air-dry.

Step-2 (Heat Fixation)

• Heat fix by exposing the smear to indirect flame
• Heat fix slides above burner by moving slides through the peak of the flame.
• Never heat fix wet slides !
• Purpose of heat fixing is to kill and immobilize bacteria on slide.

Step-3 (Primary stain)

• Add a few drops of Crystal violet on the smear.
• Wait for a minute.
• Wash the Crystal violet with water.
• At this stage, all cells appear in purple colour.

Step-4 (Mordant)

• Add plenty of Gram's lodine to the smear.
• Wait for 1 minute.
• Rinse with water.
• Cells appear to be purple yet.

Step-5 (Decolourising)

• Decolourise with 95% Ethyl Alcohol for not more than 30 seconds.
• Wash with water.
• At this stage, gram positive cells appear in purple colour And gram negative appear as colourless.

Step-6 (Counter stain)

• Add counter stain Safranin to the smear.
• Wait for 1 minute.
• Wash with water.

Step-7 (Microscopic Observation)

• Dry the slide and put a drop of immersion oil on the smear & Observe under 100X lens.

• Gram positive cells retain the purple colour
• Gram negative cells appear in pink colour.

Machanism of Gram Staining (Principle) :

The widely accepted theory is based on the differences in the cell wall composition between the gram-positive and gram-negative bacteria.

When crystal violet and iodine are added to the smear both will penetrate through the cell wall. And form a large crystal violet iodine complex (CVI complex) within the inner and outer layers of gram positive and gram negative bacteria.

The cell wall of gram-negative bacteria is thin and made of one or two layers of peptidoglycan. In addition to this it has got an outer lipopolysaccharide layer surrounding the cell wall.

  When a decolorizer like alcohol or acetone is added. The outer lipopolysaccharide layer will be completely dissolved leaving the thin peptidoglycan layer exposed.
The effective alcohol makes the peptidoglycan layer become perforated.

  In the decolorization step the gram-negative bacteria failed to retain the CVI complex and become colourless as the complex is washed away.

On contrary to this thick and multi-layered peptidoglycan in gram positive bacteria will be dehydrated by the addition of alcohol. Because of the thick peptidoglycan layer and dehydration by the alcohol treatment the CVI complex gets trapped in the cell.

  Therefore after the decolorization step the gram positive bacteria appear purple in color and the gram negative bacteria become colorless in the last step.

  When a counter stain likes a safranine is added the gram negative bacteria easily absorbed the stain and gives pink color to the cells. The gram positive bacteria remain purple in color due to the retention of CVI complex which is darker than Safranine.

Factors affecting Gram staining

• Age of culture.
• Excessive heat fixation.
• Thick smear or overcrowding of cells.
• Old staining reagents.
• Air drying.
• Over decolourization.

Disadvantages of Gram Staining

Athough the gram staining is used as primary test in the identification process this method will not be able to identify the bacteria to the species level. It will be used in combination of other modern and traditional identification tests
  The CVI complex may get lost from the gram positive bacteria due to over decolorization, which might lead to misinterpretation.
  There were evidences where some old gram positive cultures were not able to retain purple color and therefore observe as gram negative. 

The Five Kingdom Classification by Robert Whittaker & It's Limitations

 Robert Whittaker (1969), proposed the first popular classification system called five-kingdom system, which was accepted widely. The classification system is based on three criteria.

1. Cell types

1. Prokaryotie (cells with primitive nucleus and lacking membrane enclosed organelles) and
2. Eukaryotic (cells with well-developed nucleus and membrane-enclosed organelles.

2. Level of cellular organization 

1. Unicellular or
2. Multicellular.

3. Mode of nutrition

1. Photoautotrophic nutrition, which is concerned with use of sunlight as energy source and CO2 as source of carbon.
2. Heterotrophic nutrition, which is concerned with use of organie compounds as source of energy and carbon. It may be absorptive (which means absorption of nutrients by body wall) and
3. Ingestive (which means intake of solid food particles ).

  This system consists of one prokaryotic kingdom of Monera (which are prokaryotes) and four eukaryotic kingdoms - Protista, Fungi, Plantae and Animalia.

The Five Kingdom Classification  by Robert Whittaker

I] Kingdom Monera (Prokaryotes)

• It includes bacteria, cyanobacteria and archaebacteria.
• Unicellular, microscopic, solitary or colonial forms.
• Can respire aerobically or anaerobically and reproduce by asexual, sexual or vegetative methods.
• Act as decomposers and mineralisers and some may be concerned with nitrogen fixation also.
• No membrane bound organelles like mitochondria and Golgi complex. No nuclear membrane.
• Mode of nutrition: Nutritionally. these organisms exhibit great diversity from autotroph to heterotroph, phototroph to chemotroph and organotroph to lithotroph.

II] Kingdom Protista (Unicellular Eukaryotes)

• Usually (phytoplanktons or zooplanktons).
• Organisation ranges from unicellular to multicellular colonial forms. aquatic pue planktonic
• Some are commensals and some are parasites (heterotrophic or absorptive mode of nutrition)
• Some coloured algae contain various types of accessory pigments.
• Reproduction is by asexual as well as sexual modes and both haploid as well as diploid forms exist.
• Locomotion pseudopodial, ciliary or flagellar depending on the type of locomotary appendages present.
• The phytoplanktons are producers whereas the zooplanktons are consumers (holozoic nutrition).

III] Kingdom Fungi (Multicellular Decomposers)

• Include non-green plants that are the important decomposers and mineralizers.
• Hyphae constitute an entangled cottony mass of filaments termed mycelium that may be aseptate coenocytic or septate multicellular.
• Possess a cell wall made up of chitin or fungal cellulose and membrane bound organelles.
• May be solitary unicellular types or multicellular filamentous types called hyphae.
• Plastids are absent.
• Mode of nutrition heterotrophic (absorption and extracellular digestion), saprophytic or parasitic.
• Non-motile in forms but some may produce motile zoospores.
• Embryo formation does not occur but various fruiting bodies are formed.
• Most members pathogenic on plants as well as animals and cause various diseases as rust, smut and mildew (in higher plants).
• Useful fungi include yeast, Penicillium etc. used in bakery, brewery and antibiotic industries.
• Intercellular mycelia give out haustoria that absorb the nutrients from host cells.
• Asexual and sexual reproduction observed: members with no sexual reproduction known fungi imperfecti.
• Edible fungi: mushrooms and morels.

IV] Kingdom Plantae (Multicellular Producers)

• Members consist of muiticellular, green, photosynthetic, primary producers of the biosphere.
• Aquatic as well as terrestrial, large-bodied and non-motile (except bryophytes which produce motile zoospores).
• Cells bound by cellulosic cell wall and contain the photosynthetic pigments or chloroplast.
• Higher plants have complex cellular organization and vascular tissues (tracheophytes).
• Reproduction: by vegetative and sexual methods.
• All plants produce seeds except bryophytes and pteridophytes.
• Nutrition: autotrophic, absorptive (insectivorous plants) or parasitic (totally parasitic e.g. Cuscuta or partial parasite, e.g. Santalum).
• Lower plants possess simple thalloid organization and lack vascular tissue (bryophytes).

V] Kingdom Animalia (Multicellular Consumers)

• Consist of multicellular, eukaryotic organisms, lacking cell wall, plastids and photosynthetic pigments.
• Nutrition: by ingestion and subsequent digestion within the gastric cavity some possess absorption type of nutrition (parasites).
• Reproduction: asexual or sexual in lower forms while predominantly sexual in higher forms.
• Animals: main consumers in the food chain and may be primary consumers (herbivores) or secondary consumers (carnivores).
• Bear locomotory appendages and are, therefore, motile.
• Characterized by well developed muscular and nervous systems.

Limitations of Five Kingdom System

Most microbiologists do not accept the five-kingdom system for following reasons.

1. It includes prokaryotes in Monera, but it does not distinguish between Bacteria and Archaea.
2. There is a great diversity in kingdom Protista. Protozoa are heterotrophs with animal like cell organization. Algae are photoautotrophs with plant like cell organization.
3. Boundaries between Protista, Plantae and Fungi are poorly defined. For example, the red algae is not closely related to plants, still it is included in Plantae.
4. It does not explain about evolution and phylogenetic relationship.

How Does 70% Alcohol-based Hand Sanitizers Kill Microbes ?

Hand sanitizers often contain alcohol such as ethyl alcohol as an active ingredient other ingredients may include water and glycerin. Hand sanitizer has reduced the levels of microorganisms by killing them, just like other disinfectants do. 

  The effectiveness of hand sanitizers depend on quantity used duration of exposure and frequency of use. Usually 70% alcohol-based hand sanitizers, if rubbed thoroughly over finger and hand surfaces for 30 seconds followed by complete air drying can effectively reduce populations of bacteria, fungi and some viruses.

How alcohol-based hand sanitizers kill microorganisms ?

• Alcohols disturb the normal arrangement of lipids structure of bacterial plasma membrane. The plasma membrane of the bacteria is formed by several phospholipid molecules.
• Phospholipid is composed of a phosphate group and a lipid. The phosphate group is hydrophilic and the lipid is hydrophobic.
• The phospholipid bilayer of plasma membrane which is formed by two layers of phospholipids is arranged in such a way that the hydrophilic parts are directly exposed to the internal and external environments of the cell. 

Plasma membrane of microorganisms

• The reason being is that the internal and external environments of the cell are occupied with water.
• In most cases the hydrophilic ends are always in contact with water. The hydrophilic nature provides the membrane more stability in the aqueous solutions.
• When phospholipids come in contact with alcohol, the lipid molecules move out of place and break the arrangement as the hydrophilic ends chase for water. Thus it increases the membrane fluidity.
• This alcohol stressed condition changes the shape of proteins that are present on the membrane.
• When the proteins are not in normal shape they cannot perform their routine job. Therefore the microorganisms cannot survive.

Hand sanitizers are commonly used in pharmaceutical companies, hospitals and clean rooms.
Particularly alcohol-based hand sanitizers will be used in combination of water.

Why Alcohol solution with 70% alcohol and 30% of water effectively kills bacteria ?

• Alcohol in combination of water evaporates slowly, therefore increases the contact time of the sanitizer and thus works effectively.
• Water acts as a catalyst and plays an important role in denaturing the proteins.
• The 100% alcohol coagulates the proteins rapidly and forms a protein layer. This will prevent coagulation of other protein molecules. This is the reason why the 70% alcohol is found to be more effective than 100%.

Behaviour of bacteria towards stimull - Taxis or Tactic response

 The motile prokaryotes enjoy a selective advantage over their non-motile counter parts under certain environmental conditions. When they encounter gradient of physical and chemical agents in nature, the flagellar machinery functions to develop a response towards the stimulus.

• In a positive response, bacteria move towards the stimulus e.g. nutrients, light. temperature, etc.
• In a negative response the bacteria move away from the stimulus.

The movement of prokaryotes towards or away from the stimulus or the environment is called taxis or tactic response. There are several types of tactic responses.

1]. Chemotaxis

• Movement of an organism towards the chemical attractant or away from the repellants is called chemo taxis.
• Prokaryotic cell surface possesses special proteins, called chemo receptors.
• Chemo receptors may be located either in the periplasmic space or the plasma membrane.
• The tactic responses develop due to gradients of concentration of various chemicals.

2]. Phototaxis

• The tactic responses exhibited by phototrophic (photosynthetic) prokaryotes to a gradient of light Intensity is called phototaxis.
• They accumulate towards intense allowing most efficient photosynthesis.
• Like the chemo receptors, bacteria possess photoreceptor.

3]. Magnetotaxis

• The movement of bacteria towards the Earth's magnetic field or to local magnetie fields (magnets placed near the bacterial culture) is called magnetotaxis.
• It has been observed in Aquaspirillum magnetotacticum.
• It is due to the presence of a chain of magnetite (Fe3O4) inclusions (or magnetosomes) within the cell, which facilitates the positioning of bacteria as a magnetic dipole.
• Magnetotactic bacteria move toward the oxygen-deficient sediments in the aquatic environment.

4]. Osmotaxis 

• Movement of prokaryotes in response to the osmotic gradient is called osmotaxis.
• It is related to the concentration gradient of disolved solutes. 

Determination of Cell Mass for Measurement of microbial Growth

 The growth results into synthesis of new cellular material. Therefore, it results into the increase in cell mass. Hence, methods used to determine cell mass can be useful to measure the growth.

  Further, every cell possesses a specific amount of cell mass. Hence, this can be also useful to find out probable number of organisms present in the growth culture.

Criteria used to determine cell mass

Every cell possesses a specifie chemical composition, dry weight as well as the volume. Hence various methods based on determination of chemical constituents of cell, volume and dry weight are applied to measure cell mass.

1]. Determination of cellular nitrogen and protein content

• Every cell possesses a fixed proportion of protein and nitrogen. To determine them, cells are first harvested, washed and then used for quantitative determination.
• Commonly micro Kjeldahl method is used to measure cellular nitrogen. This method is much laborious.
• Nitrogenous matter of the medium is also estimated together. Therefore this method is not used for routine cell mass determination.

2] Determination of cellular nucleic acid content

• Like nitrogen and protein, each bacterial cell possesses a fixed proportion of RNA and DNA. Hence their estimfuion can also be a useful criterion to measure cell mass.

3] Determination of cellular phosphorus and sulphur contents

• Phosphorus and sulfur are other major elemental constituents of cell, apart from carbon and nitrogen. They also occur in a specific proportion in cell.
•  Hence, chemical estimation of cellular phosphorus and sulfur can be useful to measure cell mass.

4] Determination of dry weight of cell

• This is one of the highly useful methods to measure growth. However, it can be used only when cell population is much dense in the culture.
• The cells are separated from culture by centrifugation or filtration, washed thoroughly and then dried in oven at 50-55°C. Then their dry weight is determined.
• However in some instances, dry weight of cells may not be indicative of living matter.
• E.g. Bacteria can accumulate large amount of poly B hydroxy butyric acid as reserve material during late log phase without causing any increase in other cellular constituents. It may account for 70% of total dry weight in some instances.

5] Determination of packed cell volume (PCV)

• Each bacterial cell belonging to a particular species has a specific volume. Increase in cell mass and cell number results in to overall increase in cell volume.
• Hence, methods determining cell volume of bacteria can be used to measure growth.
• This is achieved by centrifugation of a fixed volume of growth culture and then the settled cell mass is measured as packed cell volume.

6] Determination by turbidimetric method

• Another method of measurement of cell mass is an optical method, by determining turbidity resulting due to growth in broth cultures.
• When, a beam of light is allowed to pass through a suspension of bacteria, the bacterial particles coming in the path of light cause scattering and absorption of light depending upon the cell density as per Beer-Lambert's law.
• Therefore amount of light transmitted through the culture suspension is reduced.
• The reduction in the amount of transmitted light, therefore, depends on the cell density or cell mass in the culture suspension.
• It is measured as % transmittance or optical density (O.D.) by the use of photoelectric colorimeter or spectrophotometer or densitometer.
• Usually the O.D. is measured at 540 nm wavelength of light. This is because at this wavelength light absorption by medium will be maximum and hence it will give more correct value of O.D. of the bacterial suspension.
• Another method of measuring cell density by applying same principle is through use of Nephelometer.
• Nephelometer does not measure transmitted light, passing through the culture suspension, but it measures intensity of light scattered by bacterial particles.
• The diagrammatic presentation of measurement of cell density by both photoelectric colorimeter and nephelometer is shown in  figure below.

Diagrammatic presentation of measurement of cell density by photoelectric colorimeter.

• a] Adjustment of instrument to read zero optical density by use of uninocculated culture medium as "blank" tube. 
• b] Measurement of optical density of the growth tube, more the turbidity, greater the optical density.

Advantages and disadvantages of methods for determination of cell mass

1. Of all the methods, cell mass determination by turbidimetric method is most convenient and reliable.
2. Other methods especially involving determination of cell constituents such as cellular nitrogen, protein nucleic acids, phosphorous or sulfur has only academic value. In routine, these methods are complex, and impractical. Media constituents may interfere with estimation.
3. The methods provide information about total cell mass; both living and non-living.

Microbiology of Air: Sources of Micro-organisms in Air

  Air is the mixture of gases that makes up the Earth's atmosphere. It is a mixture of about
- 78% Nitrogen,
- 21% Oxygen,
- 0.9% Argon,
- 0.04% Carbon dioxide and very small amounts of other gases.
Air also contains the variable amount of water vapours.

  Air microbiology is concerned with the study of living microbes in the air. They are usually referred to as bioaerosols. Atmosphere is actually unsuitable for growth of microorganisms due to extreme temperature variations, solar radiations, lack of nutrients, low amount of available water etc.

  Therefore, the number of microorganisms in air is less than in the other natural environments. However, there is still a large enough number that they can affect the atmosphere.

Microbes in air have an opportunity to travel long distances with the help of wind. They are ecologically significant because they can be associated with disease in humans, animals and plants.

  Most of the organisms are found in the lower region of atmosphere. Atmosphere is usually occupied by those forms of microorganisms that are resistant to adverse conditions in the air. They can be bacterial spores or cysts, capsulated bacteria, fungal spores, enveloped viruses etc.

Sources of Micro-organisms in Air

• Entry of microorganisms in air takes place through various ways.   
• Natural sources such as soil, lakes, oceans, animals and humans.
• Unnatural sources such as sewage treatment plants, animal rendering, fermentation processes and agricultural activities.
• Soil is one of the source.
• Wind blow disturbs soil surface and liberates the soil microorganisms into the air.
• These microorganisms remain suspended in air for long time.
• Manmade actions like plugging and digging.
• Plant and animal surfaces.
• Large water bodies like oceans and bays.
• Water droplets or aerosols produced by wind or tidal actions at surface of these water bodies.
•  Most droplets are produced from top 0.1 mm water surfaces called as 'micro layer'.
•  It consists of many more micro-organisms than from deep layers.
• Bubbles from surface add thousands of microbes in air.
• Most significant source is the man himself. Organisms from oral, nasal, rectal passages of man and animals come in air.
• Human activities like coughing, sneezing, laughing and even talking.
• Such biological aerosoles may spread bacteria upto distance of about 15 feet.
• Infected persons release pathogens.
• Respiratory pathogens are drought resistant and can survive in air for long period.
• Air in the hospital is a site where microbial load is always very high.
• Many human infections are caused by microorganisms from atmosphere.
• For example - Epidemics of Legionnaires disease (a severe form of pneumonia caused by bacterium Legionella) were due to aerosols generated from contaminated water of air conditioning equipments.
• Aerosols formed by high speed drills in dental clinics add a large population of bacteria.
• Various industrial, agricultural, municipal are responsible for the generation of bioareosols.
• For example - Sprinkler irrigation of crops and forest land with effluent from sewage treatment plants, trickling filter beds in sewage treatment plants, slaughter houses, spray washing processes etc.

Infectious dust, Droplets & Droplet Nuclei

(i) Infectious dust:

• Dust in air arises from sand, soil, ash, lint from bedding, clothing, carpets.
• It usually contains all saprophytic organisms.
• Dust loaded with saprophytic organisms is harmless for human. But if it is loaded with air borne pathogens, it is called as 'infectious dust.
• Air borne pathogens are added to the dust by body secretions.
• When secretions are dried , pathogens remain in dust and form infectious dust.
• Nasal or throat secretions on the handkerchief eventually dry and leave some residual material.
• During sneezing or coughing, large aerosol droplets get expelled and settle on floor or bed clothes.
• Moisture content of these droplets evaporates leaving behind residue.
• These residues are then disturbed during the handling of handkerchiefs, sweeping of floor, bed making This makes the dust infectious.
• If it is inhaled by healthy persons, they may get infected.

(ii) Droplets:

• Sneezing or coughing activity of respiratory tract infection persons expels (exhales) millions of micro droplets of saliva and mucus along with varying number of micro-organisms.
•  Such particles are called as droplets. Thus, droplets contain thousands of living micro- organisms along with saliva and mucus.
• Most of the droplets are larger in size (0.1 mm in diameter).
•  Such droplets settle rapidly, within shorter distance from their source of origin before drying.
• Even if such droplets are inhaled by a healthy person before their settling, they are immediately trapped in nasal baffles and in the nasopharynx material and are prevented from reaching upto lungs.
• Thus, droplets are not that much dangerous as far as the spread of infection is concerned.

(iii) Droplet Nuclei :

•  In warm and dry atmosphere, small droplets are dried due to evaporation of moisture content leaving behind dried mucous with live bacteria.
• They are called as 'droplet nuclei'. They are very much small in size with diameter less than 0.1 mm.
• Such droplet nuclei can remain in air for longer period of time (upto hours to days).
•  The dried mucus protects the organisms present at the center.
• They are also carried away to a longer distance.
• If these are inhaled by health persons, they directly enter into the lungs, without capturing in mechanical traps of nasal baffles and mucus of nasopharynx.
•  The organisms from droplet nuclei directly settle in the alveoli of lungs.
• Chances of spread of infection via droplet nuclei increases when people are crowded together.
• Frequency of air borne infections is more in winter when people prefer to live in crowd places.

   In modem days, the chances of spreading infection are increased because of modern urban areas like buses, aero planes, restaurants and bars which are always crowded.
 The diseases caused by Klebsiella pneumoniae, Streptococcus pneumoniae and Mycobacterium tuberculosis may spread through droplet nuclei.

Sampling Methods for Microbiological Examination of Air

Sampling Methods for Microbiological Examination of Air

Microorganisms in air cause a problem of contamination in food industries, milk processing plants, industrial processes, pathological and research laboratories and mainly in operation theatres.

   Therefore, measurement of no. of organisms in air at such places is important. Microorganisms in air may be present in free form or in the form of clumps or chains.

  They may adhere to the dust particles or to saliva or to mucus of man. Several methods have been developed for the microbiological examination of air.

  Simplest method is exposure of sterile nutrient medium plates to air for different time periods The plates are then incubated during which the settled organisms grow and form colonies.

  The number of organisms can be calculated from the number of colonies. But this gives a rough idea about the numbers and kinds of microorganisms in air. Because the volume of air coming in contact with plate is not known.

  Therefore, the advanced techniques like solid impaction, liquid impingement, filtration, sedimentation, centrifugation, electrostatic precipitation, thermal precipitation etc. are used. Among these, solid impaction and liquid impingement techniques are most commonly used.

1. Solid Impaction (Sieve Device) :

• Number of sampling devices which work on the principle of impaction of airborne particles.
• In these, an aspirator sucks a known volume of air which collides with nutrient agar in plate.
• This causes the microbes in the air to stick to the surface of an agar medium.
• The plates are then incubated at specific temperature for a specific time to form the colonies.
• These are the most often used methods of detection and enumeration of microbes in air.
• The most widely known device that is based on the solid impaction is the Sieve Device.
• The sieve device is of two  types : Single Stage, Multistage Device

a). Single Stage Device :

• It is mechanically very simple.
• It consists of a metal box with a metal cover.
• The cover has a large number of evenly spaced small holes.
• Under this a petriplate containing agar medium can be kept.
• A measured volume of air is drawn through these hole. The particles containing microorganisms are allowed to settle on the agar surface.
• Plates are then incubated for specific time. Then microbial colonies on agar surface are counted.

Andersen's Air sampler & Single Stage Sieve Device

b). Multistage Device :

• It consists of perforated plates with progressively smaller holes at each stage.
• Allows particles to be separated according to size.
• Best known multi-stage samplers is Andersen's air sampler
• In this, air is drawn through a series of six circular plates each perforated with 400 holes.
• The plates in series have progressively smaller holes.
• Therefore, the largest particles being deposited in the first while the smallest in last petri dish.
• Viable particles can be collected on a variety of bacteriological agar media.
• Incubated for counting and identification.
• All the particles collected are sized aerodynamically and can be directly related to human lung deposition.

Advantages of Sieve Device

1. The volume of air to be analyzed can be measured.
2. An air is drawn with high speed. Therefore, the particles of smaller size that might be escaped can also be settled on agar surface.
3. There will be even distribution of air sample on agar surfaces that results in formation of isolated colonies.
4. This is helpful to take a colony count.
5. The methods are very simple and efficient.
6. Therefore, they are most widely used.

2. Liquid Impingement Device :

• In this, a known volume of air is passed in a sterile broth medium, where micro-organisms from air are trapped.
• The aliquots of broth are then used for plating.
• The plates are incubated at room temperature for specific time and the colony count is taken to determine the number of micro-organism.
• There are several devices, among which the most important one is Bead Bubbler Device.

Bead Bubbler Device

• It consists of 250 ml capacity suction flask with side arm linked to suction pump and flow meter.
• The flask is filled with sterile nutrient broth.
• The sterile glass beads of size 5 mm in diameter are also placed in the flask.
• A glass bubbler is fitted in the flask with rubber stopper.
• Diameter of glass bubbler is approximately 14 mm.
• It has many small openings at the base with3 mm diameter.
• A suction flask is added with sterile nutrient broth medium along with sterile glass beads.

 

• The bubbler is fitted with rubber stopper.
• With help of suction pump and flow meter, known volume of air is allowed to pass through medium.
• Air enters into broth medium in the form of bubbles.
• Because of glass beads bubbles are formed and the clumps or chains of organisms are broken down.
• Aliquots of medium are used to culture on solid agar medium & plates are incubated at room temperature.
• Colony count is taken & number of organisms is determined.

Advantages

1. Volume of air to be analyzed can be measured
2. Because of glass beads and bubble formed, the micro-organisms from the clumps and chains are separated from each other.
3. Thus exact count can be drawn.