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. 

Steps Involved in Transcription

 Transcription is a heterocatalytic function of DNA which involves transfer of coded information from DNA through the synthesis of RNA over the template of DNA. Only one of the strand of DNA called sense strand (master strand) is involved.
  The segment of DNA involved in transcription is cistron. It has a promoter region where initiation begins and a terminator region where transcription ends.

Enzyme involved in transcription is RNA polymerase. It is single in prokaryotes. There are three types of RNA polymerase in eukaryotes,
I for 28s, 18s & 5.8s RNA.
II for mRNA and SnRNA and
III for tRNA, 5s RNA & Sc RNA.

RNA polymerase has five polypeptides — σ, α, β, β' and ω).

σ or sigma factor recognises the promoter region while the remaining or core enzyme takes part in transcription.
A rho or ρ factor is needed for termination of transcription.

Steps involved in transcription

a] Activation of Ribonucleotides :

With the help of phosphorylase, energy and phosphoric acid, ribo- nucleoside monophosphates are changed into triphosphates ATP, GTP, CTP & UTP.

b] Cistron :

It has a promoter region where transcription begins and a terminator region where transcription ends.
Promoter region possesses RNA polymerase recognition as well as RNA-polymerase binding sites.
Sigma factor or RNA polymerase gets attached to promoter region of cistron. The two DNA strands separate with the help of unwindase and helix destabilising proteins.
One of them function as sense strand and takes part is transcription. Transcription proceeds in 5' to 3' direction.

c] Base Pairing :

Ribonucleoside triphosphate come to lie opposite complementary nitrogen bases of sense strand (A opposite T, C Copposite G, U opposite A and G opposite C).
Pyrophosphatase hydrolyses two phosphates from cach activated nucleotide. This releases energy.

d] Strand Formation : 

RNA polymerase establishes phosphodiester bonds between adjacent ribonucleotides.
Sigma factor σ recognises.
Core enzymes moves along the sense strand till it reaches terminator region and separates from DNA template in the presense of rho (ρ) factor.
Terminator region contains a stop signal made of 4-8 A (poly A tail) nucleotides.

e] Separation of RNA :

rho (ρ) factor has ATP ase activity. It helps in separating the newly formed RNA or primary transcript from the sense strand of DNA.

f] Duplex Formation :

With the release of primary transcript the sense and antisense strands of DNA re-establish their hydrogen linkages and form duplex. Unwindase and helix destablising proteins are released.

g] Post-Transcriptional Processing :

Primary transcript is often called heterogenous or hn RNA as it is generally bigger than the functional RNA.
  1. Cleavage : hn RNA is broken to form smaller pieces. Ribonuclease-P (RNA enzyme) separates 5-7 t RNA precursors from primary transcript.
  2. Splicing : Introns or intervening sequences of nonessential nature are removed by nucleases. Ribozyme (RNA enzyme) is one such enzyme.
  3. Terminal Additions : Nucleotides are added at the ends of RNA for specific functions, e.g. CCA at 3'ends of tRNA, cap nucleotides at 5'ends of m RNA or poly A nucleotides at 3'end of m RNA.
  4. Nucleotide Modifications : Certain nucleotides are methylated, ethylated, deaminated, etc. to produce different chemicals like inosine, methyl cytosine, dihydrouracil, pseudouracil, etc.

The promoters of prokaryotic genes are simple. They generally consist of about 40 bp of DNA that contain two elements :

  • a TATA box, located 10 bp upstream from the transcription initiation site and
  • a second element, located 35 bp in the 5' direction upstream.

Eukaryotic promoters are more complex and usually consist of several elements. One set of elements ensures that transcription initiates at the correct site. The elements in this set, which of ten include a TATA box, which are usually found within 100-200 bp of transcription start site. Another set of clements, usually located further 5' from the transcription initiation site, can enhance or silence transcription. These work with, or serve as tissue specific or signal response elements.

Ribosomal RNA is also synthesized as a large 45s precursor molecule that is processed into the mature 28s, 18s and 5.8s fomms.

Metabolism: Definition, Terminology, Feaction & Function

The metabolism applies to the assembly of biochemical reactions which are employed by the organisms for the synthesis of cell materials and for the utilization of energy from their environments.

  Metabolism may be defined as 'the sum total of all the enzyme-catalyzed reactions that occur in an organism'.

  The large number of reactions in a cell are organized into a relatively small number of sequences or pathways. It is a highly coordinated and purposeful cell activity, in which multienzyme systems co-operate. This obviously points out to the fact that the metabolism of even a simple unicellular organism is time variant.

Metabolism in microorganisms and the human beings:

a] Microorganisms like bacteria (e.g., Escherichia coli) can double in number every 40 minutes in a culture medium containing only glucose and inorganic salts, or in 20 minutes in a rich broth. 

  The components of the medium are depleted and very little is added to the medium by the cells. Each cell contains hundreds to thousands of molecules of each of about 2,500 different proteins, about 1,000 types of organic compounds and a variety of nucleic acids. It is, thus, apparent that the bacterial cells participate in a variety of metabolic activities.

b] Human adults maintain a constant weight for about 40 years, during which period a total-~1! about 60 quintals of solid food and 45,000 litres of water are metabolised. And yet boll body weight and body composition remain almost constant.

TERMINOLOGY OF METABOLISM

The various processes constituting metabolism may be divided, somewhat arbitrarily, into catabolism and anabolism. Those processes, whose major function is the generation of chemical energy in forms suitable for the mechanical and chemical processes of the cells, are termed as catabolism; whereas those processes, which utilize the energy generated by catabolism for the biosynthesis of cell components, are termed as anabolism.

The various activities powered by catabolism include mechanical movement, growth, reproduction, accumulation of foods, elimination of waste, generation of electricity, maintenance of temperature etc. The various anabolic activities may be exemplified by food manufacture etc.

Some processes can be either catabolic or anabolic, depending on the energy conditions in the cell. These are referred to as amphibolism.

Catabolism reaction
Fuels(carbohydrates, fats) ➞ CO₂+ H₂O + Useful energy
Anabolic reaction
Useful reaction + Small molecules ➞ Complex molecules

FUNCTION OF METABOLISM

In all cells, metabolism enables the cell to perform its vital functions. Metabolism performs following 4 specific functions:
  1. to obtain chemical energy from the degradation of energy-rich nutrients or from captured solar energy.
  2. to convert nutrient molecules into precursors of cell-macromolecules.
  3. to assemble these precursors into proteins, lipids, polysaccharides, nucleic acids and other cell components.
  4. to form and degrade biomolecules required in specialized functions of cells.

Metabolism are closely interrelated since the synthesis of the molecules, that are a component of cell, requires an input of energy, while at the same time it is obvious that the cell components are needed to provide the energy supply and to control intracellular solute concentrations.

The specialized functions also require biosynthetic processes as well as a supply of energy. Terms catabolism and dissimilation are synonyins and refer to the pathways or routes breaking down food materials into simpler compounds and resulting in the release of energy contained in them.

The processes of anabolism or assimilation utilize food materials and energy to synthesize cell components.

The energy relations of the biological processes, the term excrgonic is used to denote a chemical reaction which liberates chemical-free energy. The term exothermic refers to the total energy liberated including heat.

  As the magnitude of heat energy is small and also that it cannot drive biological reactions, the biochemists are more interested in free energy changes and often use drive term exergonic.

The corresponding energy-consuming term endergonic refers to the processes which require an input of free energy while the term endothermic denotes a total energy requirement (including heat).

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%.

Prokaryotic Ribosome: structural subunits and function

 Ribosomes present in all living cells. Ribosomes are made up of several RNAs and proteins. They act as site of protein synthesis where mRNA translation takes place. Ribosomes link each amino acid in the order specified by the codons on mRNA to form a polypeptide chain.

Bacterial ribosome structure

  • Several ribosomes come together to form polysome during the protein synthesis. The rate of protein synthesis depends on the number of ribosomes.
  • Bacterial cell contains about     10000 ribosomes and they contribute 30% of total dry weight of the cell.
  • In Bacterial cell ribosomes are present in the cytoplasm.
  • More number of ribosomes provide granular appearance to the cytoplasm.
  • Ribosomes are of two types 70s and 80s.
  • The 80s ribosomes are present in eukaryotic cells.
  • Bacterial cell contains 70s ribosomes
  • Here the "s" denotes svedberg units.
  • Svedberg units indicate the rate of sedimentation during ultracentrifugation.
  • The sedimentation rate depends on size, shape and weight of the molecule, for example svedberg value is more for the heavier molecules than the lighter molecules.
  • These units are named after Theodor Svedberg from sweden who discovered the principle of ultracentrifugation. He was awarded the nobel prize in 1926.

Structure of 70s Ribosome

  • The 70s ribosome is made of two subunits a small and a large subunit.
  • Smaller subunit is of 30s and the larger one is a 50s.
  • Similarly the 80s ribosomes in eukaryotic cell consist of a 40s and 60s subunits.

30s Subunit of Ribosome

  • The smaller subunit in 70s ribosome(30s) has 16 sRNA and comprise of 1540 nucleotides bound to 21 proteins.

50s Subunit of Ribosome

  • The larger subunit has 5 sRNA and 23 sRNA.
  • The 5sRNA comprise of 120 nucleotides bound to 31 proteins.
  • The 23sRNA comprise of 2900 nucleotides bound to the 31 proteins.

Protein synthesis by Ribosome

  • During the protein synthesis the mRNA binds to the smaller subunit(30s).
  • The larger subunit(50s) links amino acids by providing the peptide bonds to the polypeptide chain.
  • The two subunits are combined together. The strength of the attachment depends on the concentration of Mg++ ions.

Difference between Laminar air flow and Biosafety cabinets

 Laminar airflow cabinet(hood) and biosafety cabinet(hood) appear to be one and the same but there are many differences between these two cabinets. Of course both of these cabinets are intended to give protection to the user and the environment at different levels.
Let's understand the main function differences and types of these two cabinets.

Laminar Airflow Cabinet

A laminar airflow cabinet is an enclosed unit that creates a microbial free environment.
This microbial free environment is created by high efficiency particulate air filters which are popularly known as hepa filters these HEPA filters.

  These HEPA filters capture all the airborne particles that enter the cabinet. The pore size of HEPA filter is usually 0.3 microns which can retain bacteria, fungal spores and other particles.

  HEPA filters are so constructed that the air reaching out to the filter is passed through two more filters in order to remove the particles that are larger than 0.3 microns.

There are two types of laminar airflow cabinets based on the direction of the airflow

  1. Vertical laminar airflow cabinet
  2. Horizontal laminar airflow cabinet.

  • In the vertical laminar airflow air moves from the top of the cabinet towards the workbench.
  • Similarly in the horizontal laminar airflow the air flows from the behind towards the operator.

Working principle of laminar air flow cabinet

The air from the surrounding environment is pulled by the blower and released into the working area through the HEPA filters. From the workbench most of the air is moved towards the face of the operator and then back to the environment.

  • The laminar airflow cabinets provide a unidirectional airflow with fixed velocity and pressure.
  • The pressure inside the cabinet does not allow the external air entering the cabinet.
  • Some laminar airflow units equipped with UV light to sterilize the interior surfaces and components before the operation.
  • The laminar airflow cabinets are used for culturing of non-infectious organisms as they do not provide protection to the operator.

Biosafety cabinet

Unlike the laminar airflow cabinets the biosafety cabinets not only give protection to the sample but they also protect the operator and the surrounding environment.
Based on the level of protection biosafety cabinets are classified into three classes -
  Class 1
  Class 2
  Class 3

Class 1 Biosafety cabinets

  • Class 1 biosafety cabinets provide only the operator and environment safety. They do not provide product safety that is being handled.
  • The class 1 biosafety cabinets pull the surrounding air from the operator side. The air inside takes the particles away from the operator and then pass through the HEPA filter before it is discharged into the environment.
  • In this case the surrounding air is coming in direct contact with the product on the work surface and therefore increasing the risk of product contamination.
  • In this way the class 1 biosafety cabinets protect the operator and the environment but not the product.

Class 2 Biosafety cabinets

  • In The class 2 biosafety cabinets the air enters into the cabinet through the front grille.
  • In the laminar airflow the airflow is towards the operator whereas in this case the air is pulled from the front grille. Which means the dirty air is pulled from the operator side and thus providing safety to the operator.
  • The contaminated air is then pushed below the workbench and taken upwards to the plenum. From the plenum the air is passed through the HEPA filter.
  • Usually 30% of the air will be sent back to the environment through the HEPA filter the remaining 70% of the air passes through another hepa filter and enters the working surface of the cabinet.

The class 2 biosafety cabinets provide the following
  • a front opening with continuous inward airflow
  • hepa filtered vertical and unidirectional airflow on the working surface
  • HEPA filtered air to the surrounding area

Depending on the exhaust system the airflow velocity and airflow rate the class 2 biosafety cabinets are further classified as 
type A1 A2 type B1 and B2.

  • The most widely used cabinets are belong to the type A2 of class 2 Biosafety cabinets.

Class 3 Biosafety cabinets

  • The class 3 biosafety cabinets are totally enclosed containers and provided with two gloves attached to the system to perform operations.
  • Hence these cabinets are also called as glove boxes the inflow and outflow of air occurs through HEPA filters.
  • These cabinets are also equipped with a transfer cabinet that allows sterilization of material before they leave the glove box.

Class 3 Biosafety cabinet 

  • The exhaust air is either treated with double HEPA filters or single HEPA filter followed by incineration.
  • The class 3 biosafety cabinets provide complete protection to the operator environment and the product.
  • Hence the class 3 is suitable for all biosafety level organisms.

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.

Electronic count by coulter counter

Electronic count by coulter counter

This is a highly sophisticated technique for counting bacterial cells present in the culture. The method is named so after its Inventor. 

The electronic device consists of tube having small orifice of about 10aua30 mm in diameter, which connects two compartments of the counter, which contain conductive medium. 

  The interior and exterior of the tube is connected with electrodes. The conductive medium completes the circuit.

  When, bacteria pass through the small orifice, the conductivity between two compartment drops momentarily.

  This is electronically recordcd in the counter. Thus as the bacteria pass through the orifice from one compartment to the other, each time they give electronic pulse to be counted. Thus the method involves a much rapid and accurate count.

The other modification of the technique is the use of a photodiode. Near the orifice, a photodiode and sensor are placed on the opposite sides. 

  As the organism passes through the orifice, it obstructs the path of light. This is sensed by the sensor and recorded in the counter.

Figure : Electronic coulter current counter method determining bacterial cell numbers. 


  1. Flow of the growth medium through a tube is allowed through a narrow capillary tube with a small orifice. Both ends of the tube are connected to electronic circuit through electrode. 
  2. Through the orifice only one cell is able to pass at a time. Passage of cell through orifice causes momentary drop in conductivity, which is gives electronic pulse, to be counted.

Limitations of electronic count method

1. However, the method has got a major limitation that it cannot distinguish between dead or living organisms as well as dust particle. Every particulate matter can give rise to pulse to be counted as the organisms. 

  2. Many times if the organisms are larger than the diameter of the orifice, they may plug into It. Similarly clumps of bacteria cannot pass through. Hence the specimen should be initially treated to separate the clumps and then used for the count.

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.

Cancer cousing viruses (carcinogenic viruses)

  According to Viral Gene Hypothesis, certain viruses are responsible for the oncogenesis. Virus which are causes cancer called oncogenic virus. First evidence of a relationship between viruses and cancer was obtained by demonstration of transmission of leukemia by cell free filtrate of blood of diseased chickens to healthy chickens. 

  In 1911, Rous also transmitted chicken sarcoma from diseased chicken to healthy one. The viral etiology of various cancers in animals like fishes, dogs, mice, rats, rabbits, frog, horses etc. is clearly evidenced. 

  Many viruses are found to be associated with many cancers in humans also. Viruses that are capable of producing cancer are called 'oncogenic viruses' or 'tumor viruses'.

There are two classes of tumor viruses as DNA and RNA tumor viruses. A common characteristic of both these viruses is that the viral genome is some way becomes integrated into the host cell DNA and replicates along with host cell chromosome.

I] DNA Tumor Viruses :

There are five groups of DNA viruses causing cancer in humans. They include -
  a. Papova
  b. Adeno
  c. Pox
  d. Herpes and
  e. Hepatitis viruses.

Among these, Papova viruses have been received more attention because they are known to cause tumors in a variety of animals.

a) Papova Viruses :

  • Name papova is derived from first two letters of three viruses as Papilloma, Polyoma and Simian vacuolating 40 virus ( SV 40 virus).
  • They belong to the papovaviridiae family.
  • Papovaviridiae family includes two genera - Papiloma viruses and Polyoma viruses
  • SV40 virus is now placed in the genus Polyoma viruses.
  • All papova viruses are small, naked, icosahedral, circular double stranded DNA viruses.
  • They are commonly found in humans and other mammals.
  • Their size ranges from 45nm to 55nm in diameter. 
  • They contain 5 to 7 genes. The icosahedral capsid is constructed from 2 to 3 polypeptides

i) Papilloma virus :

  • Small, non enveloped, icosahedral, circular, ds DNA viruses.
  • They are about 55 nm in diameter.
  • Their capsid contains 72 capsomers.
  • Capsomers made from two types of proteins as L,(Major)and L2(Minor).
  • These are wart causing viruses.
  • Natural infection results in formation of benign warts in humans and other animals.
  • These viruses induce tumor only in natural host.
  • They transform normal cells into cancer cells in vitro very rarely.
  • Warts produced by these viruses are usually benign But they may convert into malignant carcinomas.
  • Also found to be associated with human penile, uterine and cervical carcinomas.
  • They replicate exclusively in the keratinocytes.
  • Keratinocytes form the outermost layer of the skin as well as some mucosal surfaces of the genitals, anus and mouth.

ii) Polyoma Viruses :

  • Name polyoma refers to multiple (poly) tumors (oma) producing viruses.
  • They are also small, naked, circular, ds DNA viruses, about 45nm in diameter.
  • Capsid is made from three proteins as - VP1, VP2 and VP3.
  • These three proteins form pentameric capsomers.
  • There are about 72 pentameric capsomers.
  • These viruses also encode a protein called 'T antigen'. The polyoma viruses do not induce tumors in the natural hosts like monkey and mouse.
  • For example - Simian vacuolating virus (SV 40) replicates in the kidneys of monkeys ( natural host ) without causing any disease.
  • However, the SV 40 virus cause the sarcomas in the hamster. They also transform the cells in vitro.
  • The SV 40 virus is most thoroughly studied oncogenic DNA virus.
  • It is a monkey polyoma virus.
  • It can't induce tumor in monkey (natural host) but induce tumors in experimental hamsters as well as rodents.
  • The two other examples of human polyoma virus are JC and BK virus.

JC Virus - It causes progressive multifocal leukoencephalopathy – damage or inflammation of white matter of brain at multiple locations. Symptoms include- weakness, paralysis, loss of vision, impaired speech, coma and death.

BK virus - Associated with renal diseases in kidney transplanted patients.

II] RNA Tumor Viruses :

  • Oncogenic RNA containing viruses are called "OncoRNA viruses'.
  • They are classified as 'retroviruses' because the viral RNA produces intermediate DNA by using reverse transcriptase enzyme.
  • From this DNA copy, viral RNA are produced which are accommodated in viral capsid while DNA copies are integrated into host genome.
  • Retroviruses have been isolated from many vertebrates like fishes, reptiles, birds and mammals.
  • They cause leukemia and sarcoma in chicken while lymphoma and mammary carcinoma in mice.

Based on the host range and disease type, there are five categories of Oncorna viruses They include -
a) Avian leukosis complex -

  • e.g. Rous sarcoma virus of chickens.

b) Murine leukosis virus -

  • e.g. Murine leukemia & sarcoma virus.

c) Mammary tumor virus of Mice -  

  • e.g. Bittner virus that causes breast cancer and multiplies in mammary glands. It is transmitted to the offsprings through the breast milk. 

d) Leukosis sarcoma virus -

  • It produces sarcoma in the animals such as cat, hamster, guinea pigs and monkey. 

e) Human T cell lymphotrophic virus ( HTLV) -

  • It causes T cell lymphoma.
  • There are two types of HTLV as HTLV type 1 and HTLV type 2.
  • Type Icauses adult T cell lymphoma and Type 2 causes hairy cell leukemia.
  • In hairy cell leukemia - the bone marrow has difficulty in producing enough normal cells like WBCs, RBCs and platelets.

Retrovirus :

  • Retroviruses are icosahedral with envelope. They have diploid genome i.e. they carry two identical segments of RNA genome.

  • RNA molecules are plus sense single stranded. Consists of 3 important genes- gag, env & pol.  
  • Name retroviruses refers to participation of reverse transcriptase in their multiplication. 
  • Multiplication of these viruses requires the transcription of RNA into DNA and its subsequent integration into a host genome. 
  • Only an integrated viral DNA can be transcribed into viral RNA.

Retrovirus replication

  • Replication of positive sense ssRNA occurs as shown in figure.
  • Virus multiplication is not lytic and does not kill the cell. 
  • Integrated genome of the retroviruses replicate along with host genome and therefore, it is present in all cancer cells. 
  • Oncogenic development is due to the expression of a viral gene known as 'src' gene. 
  • Base sequence of viral 'src' gene is similar to that of the host cell gene whose product plays a role in the regulation of growth of the normal cells. 
  • This suggests that the oncogenes of retroviruses are of cellular origin. Thus, the retroviruses cause the cancer as they introduce the new genetic material into host DNA and cause transformation.
  • Three theories have been proposed which are concerned with the oncogenesis by RNA viruses in human beings. 
• These are
  a). Oncogene theory
  b). Protovirus theory and
  c). Provirus theory

a) Oncogene Theory :

Oncogene Theory is Proposed by Huebner & Todaro.

  • According to this, the genetic information for cancer is present in each of the body cell and it is transmitted from parent to offspring through the germ cells.
  • Infection of body cells by RNA viruses has occurred millions of years ago during evolution.
  • Therefore, every cell is assumed to contain oncogene i.e. viral gene responsible for oncogenesis. 
  • This oncogene is normally kept in repressed condition (prevented from functioning by the regulatory genes of the host cell). 
  • When the oncogene becomes derepressed either by a physical, chemical or biological agent, it gets expressed itself by the formation of a transforming protein. 
  • This protein could change the normal cell into a malignant one although no virus could be recovered from it.

b) Protovirus Theory :

Protovirus Theory was Proposed by Temin

  • According to this, certain DNA regions of normal cells are transcribed to RNA & reverse transcribed to DNA by reverse transcriptase enzyme. 
  • Such newly formed DNA segments get back integrated into original DNA either at the same site or at different sites.
  • This leads to the amplification of genetic regions.
  • In this flow of genetic information from DNA to RNA & back to DNA, the genetic information becomes independent of cell genome. 
  • At this stage with or without effect of mutations, the protoviruses may arise which would direct synthesis of RNA tumor viruses or alternatively the integration of newly formed DNA could produce new gene alignment leading to oncogenesis.
  • Thus, this theory postulates that the normal back & forth flow of genetic information during amplification process, with or without the effect of mutation or due to alignment of new gene sequences may generate RNA tumor viruses or genes necessary to induce oncogenesis.

c) Provirus theory :

This theory was Proposed by Spiegelman

  • It states that some cancers are caused by direct infection with oncogenic viruses. 
  • It further states that tumor viruses are transmitted horizontally from individual to individual entering in provirus states. 
  • They may cause the cancer as a result of chance activation of their transforming genes.
  • Occasionally, they may be transmitted vertically i.e. from one generation to other. 
  • Thus, this theory is different from the oncogene and protovirus theory which state that the spontaneous mutation in animals and humans cells may cause oncogenic events. 
  • Among these three hypothesizes, the oncogene hypothesis is consistent with number of observations. They include -
  • Spontaneous intracellular appearance of oncogenic RNA viruses after repeated sub-culturing of cells.
  • This gave no indication of previous virus infection.
  • The appearance of oncogenic RNA viruses upon exposure to oncogenic agents in the cells.
  • The demonstration of the occurrence of oncogenic viral genetic material in the cells by hybridization studies.