Microbiology (Flash Cards)

Microbiology flash cards expose the user to everything ranging from an introduction to microorganisms to a clear understanding of microbial diseases. These cards are useful in distinguishing the differences between viruses, bacteria, protozoa, and fungi as well as creating an understanding about the cause of the diseases and the methods to control them. ISBN: 1878576100 13-DIGIT ISBN: 9781878576101 CARDS: 163 DIMENSIONS: 4.25" x 5.5"

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Lambda Phage as a Model Organism [VHS]

This videotape has been designed to help the teacher explain the key concepts of basic immunology to an audience whose training entails a significant exposure to the subject.

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Dr. Mary's Monkey: How the Unsolved Murder of a Doctor, a Secret Laboratory in New Orleans and Cancer-Causing Monkey Viruses are Linked to Lee Harvey Oswald, ... Assassination and Emerging Global Epidemics

The 1964 murder of a nationally known cancer researcher sets the stage for this gripping exposé of medical professionals enmeshed in covert government operations over the course of three decades. Following a trail of police records, FBI files, cancer statistics, and medical journals, this revealing book presents evidence of a web of medical secret-keeping that began with the handling of evidence in the JFK assassination and continued apace, sweeping doctors into coverups of cancer outbreaks, contaminated polio vaccine, the arrival of the AIDS virus, and biological weapon research using infected monkeys.

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Key to Successful "in Situ" Pcr [VHS]

This practical, hands-on video training programme is designed for pathologists and laboratory scientists who are beginning to use "in situ" polymerase chain reaction (PCR) - a diagnostic technique for infectious diseases and cancer. This video presents a step-by-step demonstration that shows viewers how to succesfully use the technique in their own laboratories. Part 1 of the video reviews key preparatory steps and includes information on the protease, fixative, hot start manoeuvre and reagents. Part 2 demostrates the actual techniques of "in situ" PCR and "in situ" hybridization. The third part of the video goes on to explain the basics of molecular biology and histology so that viewers can interpret "in situ" PCR findings.

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Immunology in Focus [VHS]

This videotape has been designed to help the teacher explain the key concepts of basic immunology to an audience whose training entails a significant exposure to the subject.

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Immunology in Focus [VHS]

This videotape has been designed to help the teacher explain the key concepts of basic immunology to an audience whose training entails a significant exposure to the subject.

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2000 and Beyond: Confronting the Microbe Menace (Four Volumes)

4 VHS tapes with lectures by Donald Ganem, professor of medicine and immunology and microbiology at the U of Cal.-San Francisco; and B. Brett Finlay, professor of biochemistry and molecular biiology, microbiology and immunology, and biotechnology at the U of British Columbia.
Tape 1: Microbe Hunters: Tracking Infectious Agents

Tape 2: The Microbes Strike Back

Tape 3: Outwitting Bacteria's Wily Ways

Tape 4: Emerging Infections: How Epidemics Arise

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PAL Video:HIV & AIDS Set [VHS]

This practical, hands-on video training programme is designed for pathologists and laboratory scientists who are beginning to use "in situ" polymerase chain reaction (PCR) - a diagnostic technique for infectious diseases and cancer. This video presents a step-by-step demonstration that shows viewers how to succesfully use the technique in their own laboratories. Part 1 of the video reviews key preparatory steps and includes information on the protease, fixative, hot start manoeuvre and reagents. Part 2 demostrates the actual techniques of "in situ" PCR and "in situ" hybridization. The third part of the video goes on to explain the basics of molecular biology and histology so that viewers can interpret "in situ" PCR findings.

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People's Century : 1952 Living Longer ; PBS ; Medical Science ; Disease

VHS

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Microcards: Review Cards for Medical Students

These flashcards will help medical students organize and recall medical microbiology information for course exams and USMLE Step 1. The cards are grouped into sections on gram-positive, gram-negative, and other bacteria; RNA and DNA viruses; fungi; protozoa; and helminths. Each section begins with charts summarizing key information on the group of microorganisms, followed by cards for each individual microbe, which include clinical presentation, pathobiology, diagnosis/treatment, quick facts, and a case study. Schematic illustrations show the morphology and pathogenesis of different microorganisms.

A companion Website provides 70 USMLE-style questions and answers.

Price: $36.95

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Clinical Microbiology Made Ridiculously Simple (Medmaster)

A brief, clear, thorough, and highly enjoyable approach to clinical microbiology, brimming with mnemonics, humor, summary charts and illustrations, from AIDS to "flesh-eating bacteria" to ebola, mad cow disease, hantavirus, anthrax, smallpox, botulism, etc. Excellent Board review.

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Gram Staining

A differential staining technique was introduced by Hans Christian Gram in 1884, which is now known as Gram Stain technique. The technique comprises of a primary stain (typically crystal violet), a mordant (Gram’s Iodine), a decoloriser (ethyl alcohol) and a counterstain (dil. carbol fuschin). This technique exploits fundamental physiological differences between gram positive bacteria and gram negative bacteria. Once stained by primary stained and fixed by a mordant Gram positive bacteria resists decolorisation by alcohol and remain violet at the end of staining. Gram negative bacteria gets decolourised and must be stained by a counterstain and appear pink in colour at the end of staining. Although the original method devised by Christian Gram comprised of Gentian violet, Lugol’s Iodine, absolute alcohol and Bismarck brown, there are various modifications of Gram stain in practice. These modifications include Jensen’s modification, Kopeloff & Beerman’s modification, Weigert’s modification and Preston & Morrell’s modification.

The procedure adopted in our institution is as follows:
The smear is covered with few drops of crystal violet solution and allowed to act for one minute. The slide is then washed with gentle stream of running tap water. The smear is then covered with few drops of Gram’s Iodine and allowed to act for a minute and then washed in tap water. The smear is then decolourised by alcohol by until no more violet colour comes off the slide. This process is completed within 30 seconds to prevent overdecolourisation. The slide is washed in water and counterstained using dilute carbol fuchsin for 30 seconds. The slide is then washed in water and dried with blotting paper and observed under oil immersion objective.

For more information and commonly asked questions, visit www.microrao.com/staining.htm

Photo of Gram stained smear showing gram positive cocci and gram negative bacilli


For a detailed description, visit my recent post here or type http://medimicro.blogspot.com/2008/10/gram-stain.html.

For PDf version, click here or type www.microrao.com/micronotes/pg/Gram stain.pdf

Ziehl Neelsen staining

This differential staining method was introduced by Ehrlich in 1882 and was subsequently modified by Ziehl and Neelsen independently. This staining method is useful in staining Mycobacteria in clinical specimens. Mycobacterial cell wall is made up of a waxy material (mycolic acid) that normally does not allow ordinary stains to enter the cell. The staining technique comprises of a primary stain, a decolouriser and a counterstain. The primary stain, which is typically concentrated (strong) carbol fuchsin is made by dissolving the dye basic fuchsin in phenol. Basic fuchsin dissolves better in phenol than in water. Heating the slide softens the waxy material of cell wall and phenolised dye readily enters the cell. Once stained by this method, these bacteria do not readily decolorize by weak mineral acids. Such bacteria are called acid fast bacteria. The non acid fast structures in the smear are then visualized by counterstaining with methylene blue solution. The acid fast bacilli appear pink in colour.

The procedure adopted in our institution is as follows:
The smear is flooded entirely with concentrated carbol fuchsin solution and heated using a spirit lamp from beneath. The heating should be intermittent and should not be intense to boil the solution or dry it completely. Typically, flaming must be stopped once fumes arise and allowed to cool. The solution is then poured off and washed in gentle stream of running tap water. The smear is then covered with few drops of 20% sulfuric acid and allowed to act for 1-2 minutes and then washed in tap water. The process of decolourisation may be repeated until the smear is faintly pink or almost colourless. The smear is then washed in water and counterstained with methylene blue solution and allowed to act for 30 seconds. The slide is then washed in water and dried with blotting paper and observed under oil immersion objective.

A positive sputum sample typically contain pink coloured, rod shaped bacteria that are slightly curved, sometimes branching, sometimes beaded in appearance, present singly or in small clumps against a blue background of pus cells and epithelial cells.

For more information and commonly asked questions, visit www.microrao.com/staining.htm

Photo of acid fast bacilli in sputum smear

Antibiotic susceptibility testing

The isolate to be tested should be obtained in pure culture and 3-5 identical colonies are picked up using a sterile straight wire and inoculated into nutient broth (or Mueller Hinton broth) and incubated at 37^oC for 2-4 hours or until sufficient turbidity is achieved. Alternatively, for testing the fastidious organisms such as Haemophilus spp., N. gonorrhoeae, and streptococci, and for testing staphylococci for methicillin resistance, a direct broth or saline suspension of isolated colonies on 18- to 24-hour agar plate can be made. Either way, the turbidity must be matched to that of 0.5 McFarland turbidity prior to testing. This is done by visually comparing the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines. The turbidity of the broth culture is adjusted with sterile broth to obtain a turbidity comparable to that of the 0.5 McFarland standard.This results in a suspension containing approximately 1 to 2 x 10⁸ CFU/ml for E.coli ATCC 25922.

Within fifteen minutes of standardization of inoculum, a sterile cotton swab is dipped inside the tube and squeezed against the sides of test tube to remove excess of fluid. The moist swab is rubbed on the entire surface of a dry Mueller Hinton agar. This procedure is repeated by streaking two more times, rotating the plate approximately 60^o each time so as to obtain an uniform lawn growth. Finally the circular rim of the plate must be swabbed.

The surface of the medium is allowed to dry and filter paper disks containing known antibiotic is placed on the surface and pressed gently to ensure proper contact. Once placed, the discs should not me moved or relocated since diffusion of antibiotic starts instantly. The interdisc distence should be 24 mm to prevent zone overlapping. Ordinarily, no more than 12 discs should be placed on one 150 mm plate or more than 5 discs on a 100 mm plate. Within fifteen minutes of application of discs, the plate is inverted and kept in incubator. The plates are incubated in ambient air or CO2, as required.



After 18-24 hours of incubation, the plates are removed from the incubator and observed for good growth and zones of inhibition around the discs. The diameter of zone of inhibition is measured to the nearest mm and read from the Kirby-Bauer Chart and is reported accordingly as susceptible, moderately susceptible or resistant.

For more information, visit www.microrao.com/micronotes/susceptibility.htm

Bacterial spore

In poor growth conditions some bacteria such as Bacillus and Clostridium produce resistant survival forms termed endospores. This process is known as sporulation. Bacterial spores are endospores in contrast to fungal spores, which are usually exospores. Unlike the spores of fungi, bacterial spores do not serve reproductive function. They are resistant to extreme environmental conditions such as high temperatures, dryness, toxic chemicals (disinfectants, antibiotics), and UV radiation. Once the endospore is formed, the vegetative portion of the bacterium is degraded and the dormant endospore is released. The endospore is able to survive for long periods of time until environmental conditions again become favorable for growth. The endospore then germinates, producing a single vegetative bacterium. Spores can be killed by sterilization methods such as autoclave and hot air oven. Some chemical disinfectants such as formaldehyde and ethylene oxide can also kill spores.

Mechanism of sporulation:
First the DNA replicates and the cell divides asymmetrically. A cytoplasmic membrane septum forms at one end of the cell. A second layer of cytoplasmic membrane then forms around one of the DNA molecules (the one that will become part of the endospore) to form a forespore. Both of these membrane layers then synthesize peptidoglycan in the space between them to form the cortex. Calcium dipocolinate is also incorporated into the forming endospore. A spore coat composed of a keratin-like protein then forms around the cortex. Sometimes an outer membrane composed of lipid and protein and called an exosporium is also formed. Finally, the remainder of the bacterium is degraded and the endospore is released. There is no metabolic activity until the spore is ready to germinate. Single vegetative cell gives rise to a single spore. Sporulation generally takes around 15 hours.

Germination:
Favorable growth conditions signal the process of endospore germination. Germination of a spore results in a break in the spore wall and the outgrowing of a new vegetative cell. The newly formed vegetative cell is capable of growth and reproduction. A single spore upon germination forms a single vegetative cell. Germination occurs in following steps:

Activation: Even in the presence of favorable conditions, the spore will not germinate until its protective spore coat is not damaged. Conditions such as heat, acidity, abrasion or compounds containing free sulphydryl groups activate the spore to germinate.
Initiation: once activated, the spore will germinate provided the environment is suitable. Different signaling effectors exist for different species. Binding of effector stimulates autolytic enzymes that degrade the peptidoglycan of cortex. Water is absorbed and calcium dipicolinate is released.
Outgrowth: once the cortex and outer layers is degraded, a new vegetative cell consisting of spore protoplast and its wall emerges. This is followed by active biosynthetic activity and process terminates with cell division.

The impermeability of the spore coat is thought to be responsible for the endospore's resistance to chemicals. The resistance of endospores is due to a variety of factors:
Calcium-dipicolinate, abundant within the endospore, may stabilize and protect the endospore's DNA. Specialized DNA-binding proteins saturate the endospore's DNA and protect it from heat, drying, chemicals, and radiation. The cortex may osmotically remove water from the interior of the endospore and the dehydration that results is thought to be very important in the endospore's resistance to heat and radiation. DNA repair enzymes contained within the endospore are able to repair damaged DNA during germination.

For more information on anatomy of bacterial cell, visit

www.microrao.com/micronotes/anatomy.pdf

Chemical methods of disinfection

Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. Some chemical have very narrow spectrum of activity and some have very wide. Those chemicals that can sterilize are called chemisterilants. Those chemicals that can be safely applied over skin and mucus membranes are called antiseptics.

ALCOHOLS:
Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Application: A 70% aqueous solution is more effective at killing microbes than absolute alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to ethanol. It can also be used to disinfect surfaces. It is used to disinfect clinical thermometers. Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods.
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

ALDEHYDES:
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably damages nucleic acids. It kills all microorganisms, including spores.
Examples: Formaldehyde, Gluteraldehyde
Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc. Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with potassium permanganate. It also sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. An exposure of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at 40^oC for 20 minutes is used to disinfect wool and 0.25% at 60^oC for six hours to disinfect animal hair and bristles.
Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration, leaves non-volatile residue, activity is reduced in the presence of protein. Gluteraldehyde requires alkaline pH and only those articles that are wettable can be sterilized.

PHENOL:
Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of enzymes.
Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene, chlorhexidine, chloroxylenol (Dettol)
Applications: Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar derivatives. They act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses. They are not readily inactivated by organic matter. The corrosive phenolics are used for disinfection of ward floors, in discarding jars in laboratories and disinfection of bedpans. Chlorhexidine can be used in an isopropanol solution for skin disinfection, or as an aqueous solution for wound irrigation. It is often used as an antiseptic hand wash. 20% Chlorhexidine gluconate solution is used for pre-operative hand and skin preparation and for general skin disinfection. Chlorhexidine gluconate is also mixed with quaternary ammonium compounds such as cetrimide to get stronger and broader antimicrobial effects (eg. Savlon). Chloroxylenols are less irritant and can be used for topical purposes and are more effective against gram positive bacteria than gram negative bacteria. Hexachlorophene is chlorinated diphenyl and is much less irritant. It has marked effect over gram positive bacteria but poor effect over gram negative bacteria, mycobacteria, fungi and viruses. Triclosan is an organic phenyl ether with good activity against gram positive bacteria and effective to some extent against many gram negative bacteria including Pseudomonas. It also has fair activity on fungi and viruses.
Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is inactivated by anionic soaps. Chloroxylenol is inactivated by hard water.

HALOGENS:
Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl groups of enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal.
Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture iodine, iodophores)
Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be combined with neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores such as povidone-iodine. Iodophores permit slow release and reduce the irritation of the antiseptic. For hand washing iodophores are diluted in 50% alcohol. 10% Povidone Iodine is used undiluted in pre and postoperative skin disinfection. Chlorine gas is used to bleach water. Household bleach can be used to disinfect floors. Household bleach used in a stock dilution of 1:10. In higher concentrations chlorine is used to disinfect swimming pools. 0.5% sodium hypochlorite is used in serology and virology. Used at a dilution of 1:10 in econtamination of spillage of infectious material. Mercuric chloride is used as a disinfectant.
Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is corrosive and staining. Bleach solution is corrosive and will corrode stainless steel surfaces.

HEAVY METALS
Mode of action: Act by precipitation of proteins and oxidation of sulfydryl groups. They are bacteriostatic.
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts (e.g., mercurochrome, merthiolate)
Applications: 1% silver nitrate solution can be applied on eyes as treatment for opthalmia neonatorum (Crede’s method). This procedure is no longer followed. Silver sulphadiazine is used topically to help to prevent colonization and infection of burn tissues. Mercurials are active against viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used in preservation of serum. Copper salts are used as a fungicide.
Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

SURFACE ACTIVE AGENTS:
Mode of actions: They have the property of concentrating at interfaces between lipid containing membrane of bacterial cell and surrounding aqueous medium. These compounds have long chain hydrocarbons that are fat soluble and charged ions that are water-soluble. Since they contain both of these, they concentrate on the surface of membranes. They disrupt membrane resulting in leakage of cell constituents.
Examples: These are soaps or detergents. Detergents can be anionic or cationic. Detergents containing negatively charged long chain hydrocarbon are called anionic detergents. These include soaps and bile salts. If the fat-soluble part is made to have a positive charge by combining with a quaternary nitrogen atom, it is called cationic detergents. Cationic detergents are known as quaternary ammonium compounds (or quat). Cetrimide and benzalkonium chloride act as cationic detergents.
Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals.
Disadvantages: Their activity is reduced by hard water, anionic detergents and organic matter. Pseudomonas can metabolise cetrimide, using them as a carbon, nitrogen and energy source.

DYES
Mode of action: Acridine dyes are bactericidal because of their interaction with bacterial nucleic acids.
Examples: Aniline dyes such as crystal violet, malachite green and brilliant green. Acridine dyes such as acriflavin and aminacrine. Acriflavine is a mixture of proflavine and euflavine. Only euflavine has effective antimicrobial properties. A related dye, ethidium bromide, is also germicidal. It intercalates between base pairs in DNA. They are more effective against gram positive bacteria than gram negative bacteria and are more bacteriostatic in action.
Applications: They may be used topically as antiseptics to treat mild burns.
They are used as paint on the skin to treat bacterial skin infections. The dyes are used as selective agents in certain selective media.

HYDROGEN PEROXIDE (H₂O₂)
Mode of action: It acts on the microorganisms through its release of nascent oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages proteins and DNA.
Application: It is used at 6% concentration to decontaminate the instruments, equipments such as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal.
Disadvantages: Decomposes in light, broken down by catalase, proteinaceous organic matter drastically reduces its activity.

ETHYLENE OXIDE
Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-, amino-, carboxyl- and hydroxyl- groups.
Properties: It is a cyclic molecule, which is a colorless liquid at room temperature. It has a sweet ethereal odor, readily polymerizes and is flammable.
Application: It is a highly effective chemisterilant, capable of killing spores rapidly. Since it is highly flammable, it is usually combined with CO₂ (10% CO₂+ 90% EO) or dichlorodifluoromethane. It requires presence of humidity. It has good penetration and is well absorbed by porous material. It is used to sterilize heat labile articles such as bedding, textiles, rubber, plastics, syringes, disposable petri dishes, complex apparatus like heart-lung machine, respiratory and dental equipments.
Efficiency testing is done using Bacillus subtilis var niger.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and carcinogenic.

BETA-PROPIOLACTONE (BPL)
Mode of action: It is an alkylating agent and acts through alkylation of carboxyl- and hydroxyl- groups.
Properties: It is a colorless liquid with pungent to slightly sweetish smell. It is a condensation product of ketane with formaldehyde.
Application: It is an effective sporicidal agent, and has broad-spectrum activity. 0.2% is used to sterilize biological products. It is more efficient in fumigation that formaldehyde. It is used to sterilize vaccines, tissue grafts, surgical instruments and enzymes.
Disadvantages: It has poor penetrating power and is a carcinogen.

Fore more information on sterilization and disinfection, visit www.microrao.com/micronotes/sterilization.pdf

Culture media photos

Here is a slideshow with captions of culture media photos shot by me.

Transduction

Transduction is the transfer of genetic material among bacteria that is mediated through bacteriophage.

When a lytic bacteriophage infects a susceptible bacterium, it injects its DNA inside the cell. Following this, the phage DNA initiates its own replication begining with DNA replication and followed by sysnthesis of phage capsid components. At the same time, the host (bacterial) chromosome suffers damage and gets broken down to smaller pieces.

Normally, while packaging, phage DNA is incorporated inside phage capsid however on occasion chromosome DNA (roughly of same size)gets incorporated inside the phage. Following lysis of the infected all the phages are released, with few containing bacterial DNA.

When those phages that have chromosomal DNA instead of its own DNA infect another susceptible bacterium, they introduce bacterial DNA.

Such DNA recombines with bacterial chromosome at the region of homology and result in exchange of genes via recombination, thus a property originally possessed by one bacterium is introduced into another bacterium.

See flash video for understanding:

Transformation

Transformation involves the uptake of free or naked DNA released by donor by a recipient.
It was the first example of genetic exchange in bacteria to have been discovered. This was first demonstrated in an experiment conducted by Griffith in 1928. The presence of a capsule around some strains of pneumococci gives the colonies a glistening, smooth (S) appearance while pneumococci lacking capsules have produce rough (R) colonies. Strains of pneumococci with a capsule (type I) are virulent and can kill a mouse whereas strains lacking it (type II) are harmless. Griffith found that mice died when they were injected with a mixture of live non capsulated (R, type II) strains and heat killed capsulated (S, type I) strains. Neither of these two when injected alone could kill the mice, only the mixture of two proved fatal. Live S strains with capsule were isolated from the blood of the animal suggesting that some factor from the dead S cells converted the R strains into S type. The factor that transformed the other strain was found to be DNA by Avery, McLeod and McCarty in 1944.

Transformation is gene transfer resulting from the uptake by a recipient cell of naked DNA from a donor cell. Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria, Pneumococcus) can take up DNA from the environment and the DNA that is taken up can be incorporated into the recipient's chromosome.

The steps involved in transformation are:
1. A donor bacterium dies and is degraded.
2. A fragment of DNA (usually about 20 genes long) from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium.
3. Nuclease enzymes then cut the bound DNA into fragments.
4. One strand is destroyed and the other penetrates the recipient bacterium.
3. The Rec A protein promotes genetic exchange (recombination) between a fragment of the donor's DNA and the recipient's DNA.

Some bacteria are able to take up DNA naturally. However, these bacteria only take up DNA a particular time in their growth cycle (log phase) when they produce a specific protein called a competence factor. Uptake of DNA by Gram positive and Gram negative bacteria differs. In Gram positive bacteria the DNA is taken up as a single stranded molecule and the complementary strand is made in the recipient. In contrast, Gram negative bacteria take up double stranded DNA.

Flash animation of conjugation in gram positive bacterium


Significance: Transformation occurs in nature and it can lead to increased virulence. In addition transformation is widely used in recombinant DNA technology.

For more information, visit www.microrao.com/micronotes/genetics.pdf

Conjugation

In 1946 Joshua Lederberg and Tatum discovered that some bacteria can transfer genetic information to other bacteria through a process known as conjugation. Bacterial conjugation is the transfer of DNA from a living donor bacterium to a recipient bacterium.

Plasmids are small autonomously replicating circular pieces of double-stranded circular DNA. Conjugation involves the transfer of plasmids from donor bacterium to recipient bacterium. Plasmid transfer in Gram-negative bacteria occurs only between strains of the same species or closely related species. Some plasmids are designated as F factor (F plasmid, fertility factor or sex factor) because they carry genes that mediate their own transfer. The F factor can replicate autonomously in the cell. These genes code for the production of the sex pilus and enzymes necessary for conjugation. Cells possessing F plasmids are F+ (male) and act as donors. Those cells lacking this plasmid are F- (female) and act as recipient. All those plasmids, which confer on their host cells to act as donors in conjugation are called transfer factor.

Each Gram negative F+ bacterium has 1 to 3 sex pili that bind to a specific outer membrane protein on recipient bacteria to initiate mating. The sex pilus then retracts, bringing the two bacteria in contact and the two cells become bound together at a point of direct envelope-to-envelope contact. In Gram-positive bacteria sticky surface molecules are produced which bring the two bacteria into contact. Gram-positive donor bacteria produce adhesins that cause them to aggregate with recipient cells, but sex pili are not involved. DNA is then transferred from the donor to the recipient. Plasmid-mediated conjugation occurs in Bacillus subtilis, Streptococcus lactis, and Enterococcus faecalis but is not found as commonly in the Gram-positive bacteria as compared to the Gram-negative bacteria.

F+ conjugation:
This results in the transfer of an F+ plasmid (coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium to a female recipient bacterium. The two strands of the plasmid separate. One strand enters the recipient bacterium progressing in the 5' to 3' direction while one strand remains in the donor. The complementary strands are synthesized in both donor and recipient cells. The recipient then becomes an F+ male and can make a sex pilus. During conjugation, no cytoplasm or cell material except DNA passes from donor to recipient. The mating pairs can be separated by shear forces and conjugation can be interrupted. Consequently, the mating pairs remain associated for only a short time. After conjugation, the cells break apart. Following successful conjugation the recipient becomes F+ and the donor remains F+.

Hfr (high frequency recombinant) conjugation:
Plasmids may integrate into the bacterial chromosome by a recombination event depending upon the extent of DNA homology between the two. After integration, both plasmid and chromosome will replicate as a single unit. A plasmid that is capable of integrating into the chromosome is called an episome. If the F plasmid is integrated into the chromosome it is called an Hfr cell. After integration, both chromosome and plasmid can be conjugally transferred to a recipient cell. Hfr cells are called so because they are able to transfer chromosomal genes to recipient cells with high frequency.

The DNA is nicked at the origin of transfer and is replicated. One DNA strand begins to passes through a cytoplasmic bridge to the F- cell, where its complementary strand is synthesized. Along with the portion of integrated plasmid, the chromosome is also transmitted to the F- cell. The bacterial connection usually breaks before the transfer of the entire chromosome is completed so the remainder of the F+ plasmid rarely enters the recipient. Usually only a part of the Hfr chromosome as well as the plasmid is transferred during conjugation and the recipient cell does not receive complete F factor. After conjugation the Hfr cell remains Hfr but the F- cell does not become F+ and continues to remain F-. However the transferred chromosome fragment recombines with the chromosome of F- cell thereby transferring some new property to the recipient cell.

Flash animation of Hfr conjugation:


The integration of episome into the chromosome is not stable and the episomes are known to revert back to free state. While doing so, the episomes sometimes carry fragments of chromosomal genes along with it. Such an F factor that incorporates some chromosomal genes is called F prime (F') factor. When such a F' cell mates with F- recipient cell, it not only transfers the F factor but also the host genes that it carried with it. This process of transfer of chromosomal genes along with F factor is known is sexduction.

View flash animation of sexduction:


Significance: Among the Gram negative bacteria this is the major way that bacterial genes are transferred. Transfer can occur between different species of bacteria. Transfer of multiple antibiotic resistance by conjugation has become a major problem in the treatment of certain bacterial diseases. Since the recipient cell becomes a donor after transfer of a plasmid, an antibiotic resistance gene carried on a plasmid can quickly convert a sensitive population of cells to a resistant one.

For more information, visit www.microrao.com/micronotes/genetics.pdf

Mutation

The term “mutation” was coined by Hugo de Vries, which is derived from Latin word meaning “to change”.
Mutations are heritable changes in genotype that can occur spontaneously or be induced by chemical or physical treatments. (Organisms selected as reference strains are called wild type, and their progeny with mutations are called mutants.) The process of mutation is called mutagenesis and the agent inducing mutations is called mutagen. Changes in the sequence of template DNA (mutations) can drastically affect the type of protein end product produced. For a particular bacterial strain under defined growth conditions, the mutation rate for any specific gene is constant and is expressed as the probability of mutation per cell division.
Spontaneous mutation occurs naturally about one in every million to one in every billion divisions. Mutation rates of individual genes in bacteria range from 10-2 to 10-10 per bacterium per division. Most spontaneous mutations occur during DNA replication.

Mechanisms of mutation
a. Substitution of a nucleotide: Base substitution, also called point mutation, involves the changing of single base in the DNA sequence. This mistake is copied during replication to produce a permanent change. If one purine [A or G] or pyrimidine [C or T] is replaced by the other, the substitution is called a transition. If a purine is replaced by a pyrimidine or vice-versa, the substitution is called a transversion. This is the most common mechanism of mutation.
b. Deletion or addition of nucleotides: deletion or addition of a nucleotide during DNA replication. When a transposon (jumping gene) inserts itself into a gene, it leads to disruption of gene and is called insertional mutation.

Results of mutation
a. Missense mutation: Missense mutations are DNA mutations which lead to changes in the amino acid sequence (one wrong codon and one wrong amino acid) of the protein product. This could be caused by a single point mutation or a series of mutations.
b. Nonsense mutation: A mutation that leads to the formation of a stop codon is called a nonsense mutation. Since these codon cause the termination of protein synthesis, a nonsense mutation leads to incomplete protein products.
c. Silent mutation: Sometimes a single substitution mutation change in the DNA base sequence results in a new codon still coding for the same amino acid. Since there is no change in the product, such mutations are called silent.
d. Frameshift mutation: Frameshift mutations involve the addition or deletion of base pairs causing a shift in the “reading frame” of the gene. This causes a reading frame shift and all of the codons and all of the amino acids after that mutation are usually wrong. Since the addition of amino acids to the protein chain is determined by the three base codons, when the overall sequence of the gene is altered, the amino acid sequence may be altered as well.
e. Lethal mutation: Sometimes some mutations affect vital functions and the bacterial cell become nonviable. Hence those mutations that can kill the cell are called lethal mutation.
f. Suppressor mutation: It is a reversal of a mutant phenotype by another mutation at a position on the DNA distinct from that of original mutation. True reversion or back mutation results in reversion of a mutant to original form, which occurs as a result of mutation occurring at the same spot once again.
g. Conditional lethal mutation: Sometimes a mutation may affect an organism in such a way that the mutant can survive only in certain environmental condition. Example; a temperature sensitive mutant can survive at permissive temperature of 35oC but not at restrictive temperature of 39oC.
h. Inversion mutation: If a segment of DNA is removed and reinserted in a reverse direction, it is called inversion mutation.

Based on extent of base pair changes, mutations can be of two types; microlesion and macrolesion. Microlesions are basically point mutations (affecting single base pairs) whereas macrolesions involve addition, deletion, inversion or duplication of several base pairs.

The mutations in DNA can occur spontaneously or can be caused by an external force or substance called a mutagen. Mutagens can be chemicals such as nitrous acid, which alters adenine to pair with cytosine instead of thymine. Other chemical mutagens include acridine dyes, nucleoside analogs that are similar in structure to nitrogenous bases, benzpyrene (from smoke and soot) and aflatoxin. Radiation can also be a cause of DNA mutations. High energy light waves such as X-rays, gamma rays, and ultraviolet light have been shown to damage DNA. UV light is responsible for the formation of thymine dimers in which covalent links are established between the thymine molecules. These links change the physical shape of the DNA preventing transcription and replication.

Significance of mutation:
• Discovery of a mutation in a gene can help in identifying the function of that gene.
• Mutations can be induced at a desired region to create a suitable mutant, especially to produce vaccines.
• Spontaneous mutations can result in emergence of antibiotic resistance in bacteria.
• Mutations can result in change in phenotype such as appearance of novel surface antigen, alternation in physiological properties, change in colony morphology, nutritional requirements, biochemical reactions, growth characteristics, virulence and host range.

Tests to detect or select mutations:
• Replica plating
• Penicilin enrichment
• Fluctuation test
• Ames test

Genetic Engineering part 1

Genetic engineering is altering the genes in a living organism to produce a genetically modified organism with a new genotype. Recombinant DNA (rDNA) technology is a technique by which the gene (or segment of DNA) is excised from one organism and introduced into another organism. The process of introducing the foreign gene into another organism (or vector) is also called cloning. rDNA technology is a part of vast field of genetic engineering but some authors consider them synonymously. Foreign DNA sequences can be introduced into bacteria, yeast, viruses, plant and animal cells.

Genetic engineering is altering the genes in a living organism to produce a genetically modified organism with a new genotype. Recombinant DNA (rDNA) technology is a technique by which the gene (or segment of DNA) is excised from one organism and introduced into another organism. The process of introducing the foreign gene into another organism (or vector) is also called cloning. rDNA technology is a part of vast field of genetic engineering but some authors consider them synonymously. Foreign DNA sequences can be introduced into bacteria, yeast, viruses, plant and animal cells.

The genes are identified by various methods, these include
Polysome precipitation
Prediction based on DNA sequence
Alignment with known mRNAs
Homology to known genes
Identification of Start and Stop codons
Finding a gene by using a functional assay
Find a gene by where it is located
Once identified, it is convenient to maintain a gene library.

A gene library is a population of organisms, each of which carries a DNA molecule that was inserted into a cloning vector. Ideally, all of the cloned DNA molecules represent the entire genome of the organism. A gene library is also called gene bank. This term also represents the collection of all of the vector molecules, each carrying piece of the chromosomal DNA of the organism, prior to the insertion of these molecules into the population of the host cell. Since there is no way to locate a gene by visibly looking at all of the DNA, scientists make gene libraries to catalogue the organism's DNA and then select the gene of interest. A cDNA library consists of sum total of all actively transcribed genes of a tissue, inserted into a population of bacterial cells. A total mRNA preparation is reverse-transcribed and inserted into plasmids all at once so that every possible cDNA sequence will be carried by at least one bacterium in the culture.

Isolation of the gene (DNA sequence)
The technique involved in recombinant DNA technology is to slice (cut) the desired DNA segment and introduce it into a vector (e.g., plasmid). This is achieved using a specific bacterial enzyme called restriction enzymes or restriction endonucleases. Will Porter and John Darms received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction endonucleases. These enzymes function as endonuclease, which can cleave a DNA sequence at a specific site. These enzymes are named with three letters based on the species where it was isolated. For example EcoRI is isolated from E. coli.

Each restriction enzymes cleaves DNA strand at a specific site called recognition sequence or restriction site. For example, Eco RI recognizes the sequence GAATTC and cleaves it between G and A (G↓A).
Enzyme Source Target sequence

BamHI Bacillus amyloliquefaciens G↓GATCC
EcoRI Escherichia coli G↓AATTC
HaeIII Haemophilus aegyptius GG↓CC
HhaI Haemophilus hemolyticus GGG↓C
HindIII Haemophilus influenzae A↓AGCTT
HpaII Haemophilus parainfluenzae C↓CGG
KpnI Klebsiella pneumoniae GGTAC↓C
PvuII Proteus vulgaris CAG↓CTG
TaqI Thermus aquaticus T↓CGA

Sometimes, the restriction sequence occurs on both the strands but in reverse direction. Such a segment of DNA with identical sequences but opposite in direction is called a palindrome. A palindrome site is a sequence of base pairs in double stranded DNA that reads the same backwards and forward across the double strand.

A palindrome



When a restriction enzyme acts on palindrome, it cleaves both the strands of DNA molecule. While some enzymes cut the two strands symmetrically, others cut them asymmetrically. AluI, EcoRV and HaeIII generates blunt ends when they act on their restriction sites. Only those enzymes that cut the DNA asymmetrically are useful in rDNA technology. When such enzymes cleave DNA, they leave single stranded “sticky ends” on both strands. Same restriction enzymes are used to cleave the DNA molecule to be transferred and the vector. The circular structure of the plasmid is broken by the restriction enzyme, this process leaves a “sticky end” at either strand. The strand of DNA to be transferred must have two restriction sites; one on either side of the DNA segment of interest. When it is acted upon by restriction enzyme, it generates two sticky ends, one at either side of the segment. Since these sticky ends are generated by the same enzyme, they are complementary and hence are cohesive.

click to view the picture


Continuation of Genetic engineering: (part 2) Vector

Genetic engineering part 2

Continuation of part 1

Vector
Bacterial plasmid is the most commonly used vector. Plasmids used in genetic engineering are said to be under relaxed control, their replication is totally independent of chromosomal replication. These plasmids may be present in copies of 10-700 per cell.

The most popular plasmid is pUC18. Under certain culture conditions, plasmids can be induced to replicate to produce multiple copies within a single cell. Bacterial plasmids can not accept DNA strands larger than 5000 base pairs, hence they are restricted to cloning DNA ≤5000 base pairs. Some plasmids can carry DNA segments that are 10Kb long.

Specially developed bacteriophage lambda chromosome can incorporate up to 15-16 kilobases of DNA segment. A central one-third of its genome is normally not required for phage infection and therefore can be replaced by foreign DNA. The chimeric phage DNA can be introduced into the host cells by infecting them with phages.

Cosmids are recombinant vectors that combine features of both plasmids and bacteriophage chromosome. It can accommodate DNA segments up to 50 kilobases.
cos site is segment of DNA, which is 14 base pair long sequence and is located at either ends of phage chromosome.These ends have be separated by

36-51 kilobases of DNA strand. Only those segments of DNA that have two cos sides at either end and are separated by 36-51 kb of DNA are packaged into the phage capsid. If two cos sites are placed 36-51 Kb apart on a functional plasmid vector, it becomes a cosmid. Since cosmids have no phage DNA, upon introduction into a host cell via phage infection, they reproduce as plasmids.

Yeast artificial chromosome (YAC) is a specially constructed yeast chromosome that can incorporate DNA strands up to 1 million base pairs. YACs are liner DNA segments that have all the information required for replication in a yeast cell. Several hundred kb of foreign DNA can be cloned into YACs.

Continuation of Genetic engineering: (part 3) Cloning technique

Genetic engineering part 3

Continuation of genetic engineering (part 2): Vector

The sticky ends are generated by the same enzyme on vector as well as the target DNA are complementary and hence are cohesive. The sticky ends of the cleaved DNA segment cohere with those of the vector, thus the cut DNA sequence can now be introduced into the plasmid. The cut ends are joined by DNA ligase enzyme and the introduced gene becomes a part of the plasmid. Ligase is an enzyme that covalently joins the sugar-phosphate backbone of bases together. Ligase will join either "sticky" ends or "blunt" ends, but it is more efficient at closing sticky ends. The process of introducing foreign gene into a vector is called as cloning and the plasmid containing a cloned gene is called chimera.

Illustration of cloning click to enlarge


If the foreign DNA and the cloning vector does not have a common restriction site at the required position, they may still be spliced through the use of terminal deoxynucleotidyl transferase enzyme. This mammalian enzyme adds nucleotides to 3’-terminal OH group of a DNA chain. It is the only known DNA polymerase that does not require a template. Using this enzyme and dTTP, long poly(dT) tails are build up at the 3’ end of DNA sequence to be cloned. The cloning vector is also enzymatically cleaved at a specific site and 3’ ends of the cleavage sites are extended with poly (dA) tails. The complimentary homopolymer tails are annealed and the strands are joined by DNA ligase. Since the foreign DNA lacks any restriction site, it becomes difficult to recover the insert from the vector.

Illustration on using terminal deoxynucleotidyl transferase enzyme Click to enlarge



Another method to over this problem is to use specially designed palindromic “linker” that are appended to either ends of the DNA insert. This linker is a chemically synthesized DNA fragment that has the same restriction site present in the vector. The linkers are attached to inserts by blunt end ligation with T4 ligase. They are then cleaved with appropriate restriction enzyme resulting in generation of sticky ends at either sides of the insert. The sticky ends of the vector and those of target DNA sequence (with linker) cohere. The strands are annealed and ligated by DNA ligase enzyme.

Illustration on using palindromic linker Click to enlarge


The wild λ bacteriophage has a genome of 48.5 kb, of which the central 1/3rd is not essential for infectivity. Genetically engineered λ phage variants contain restriction sites that flank the dispensable central third of genome. This segment may be cleaved by specific restriction enzyme and replaced be a foreign DNA segment of almost same length. The foreign DNA segment is annealed to the nicked phage DNA and ligated. Only those DNA segments that have length similar to the wild phages gets packaged into the heads. Those phages that have incorporated the “chimeric” chromosome become infectious.

Illustration on using λ phage Click to enlarge


Sometimes when human DNA is inserted into bacterial plasmid, it may not get expressed despite the presence of promoter. This is because bacteria RNA polymerase may not recognize promoter of human origin. This problem can be overcome by replacing human promoter region with bacterial promoter upstream of the gene. Such a vector containing bacterial promoter region (that result in expression of foreign gene) is called expression vector.

The DNA sequence that has been inserted into the vector is also called an “insert”.

Next: continuation of genetic engineering part 4 Transfer of chimeric DNA

Genetic engineering part 4

Continuation of Genetic engineering part 3: Cloning

The chimera is then introduced into its host (e.g.,a bacterium) by various methods. Vectors carrying the genes must be incorporated into the living cells so that they can be expressed or replicated. The cells receiving the vector is called the host cell and once the vector is successfully incorporated into the host cell, the host cell is said to be “transformed”.

Illustration of cloning in bacteria


Foreign DNA cannot be readily sent across the membrane, following are few methods.
Heat shock: The chimera plasmids are placed in a solution containing cold calcium chloride and normal host bacteria. On heating suddenly to 42°C for 2-5 minutes the host bacterial membranes become permeable to plasmid chimeras, which pass into the cell.
Electroporation: The host cells are subjected to a high voltage pulse which temporarily disrupts the membrane and allows the vector to enter the cell.
Viruses: Since viruses have mechanism to infect susceptible cell and replicate themselves, a genetically engineered virus can deliver desired DNA sequence into the target host cell.
Gene gun: Gold particles coated with foreign DNA segments are fired into the host cell.
Microinjection: A cell in held in place with a pipette under a microscope and foreign DNA is injected directly into the nucleus using fine needle.
Liposome: Vectors can be enclosed in a liposome, which are small membrane bound vesicles. The liposomes fuse with the cell membrane (or nuclear membrane) and deliver the DNA into the cytoplasm/nucleus.

Selection of transformed cells
A pUC18 plasmid containing gene (lacZ’) coding for galactosidase activity is inserted with a foreign DNA. The plasmid also codes for ampicillin resistance. Due to the insertion, the gene gets interrupted and the bacterium transformed with this plasmid lacks galactosidase activity. Bacteria lacking this plasmid as well as those transformed by the chimeric plasmid lack galactosidase activity. When grown on medium containing a chromogenic substrate, bacteria containing chimeric plasmid produce colourless colonies.
Bacteria containing plasmid without the insert produce blue colonies and the bacteria not transformed by plasmid also produce colourless colonies. If ampicillin is also incorporated in the medium, bacteria not transformed with plasmid do not produce colonies. Thus, on this medium the colourless colonies indicate bacteria that have received chimeric plasmid.
Other methods to detect successful transfer of DNA include DNA hybridization and PCR.

Gene mapping
It involves determining the locations of genes within specific chromosomes. It is a critical step in the understanding of genetic diseases. There are two types of gene mapping, genetic mapping and physical mapping. Genetic mapping is used to determine the relative position of genes within a chromosome. This is measured by whether or not two genes are "linked". If both genes are inherited together they are considered linked. By determining which genes are linked, the relative positions of genes can be worked out. Physical mapping involves determining the exact position of a specific gene within a chromosome. There are multiple techniques for accomplishing this, including somatic cell hybridization and Fluorescent In Situ Hybridization (FISH).

Next: Continuation of genetic engineering part 5: Application of genetic engineering

Genetic engineering part 5

Continuation of genetic engineering part 4

Applications of Genetic Engineering
Genetic engineering has wide, applications in modem biotechnology. For various industrial processes, this technique may be used in microorganisms as well as with higher organisms. Since microbial cells have a much higher metabolic rate, genes of desired enzymes could be introduced into plasmid of bacteria. Among the medical applications of genetic engineering are the production of hormones, vaccines, interferon, enzymes, antibodies, and vitamins, and in gene therapy for some hereditary diseases.

The bacterial insulin, humulin was prepared by cloning the DNA from chromosome number 11 of human cells in bacteria.

In 1977 Herbert Boyer created E.coli capable of synthesizing somatostatin, the human growth hormone of the brain hypothalamus.

The hormone thymosin alpha-I, as well as Beta-endorphin has been produced by genetically engineered microorganisms.

Subunit vaccines can be prepared by cloning the DNA coding for the antigenic protein present on a pathogen. E.g, Hepatitis B, Foot & mouth disease, malaria etc.
Plants can be made to express antigenically important microbial proteins (edible vaccine).

Weissmann and his associates have produced alpha-interferon by recombinant DNA methods.

The enzyme urokinase, which is used to dissolve blood clots, has been produced by genetically engineered microorganisms.

Chimeric monoclonal antibodies with human Fc region can be made using this technology.

This technology has been applied to treat some of the genetic diseases (gene therapy), but they are mainly in the experimental stages.

AAT (alpha-1 antitrypsin- used in treating emphysema), tissue plasminogen activator, factor VIII, antithrombin, erythropoeitin etc are some of the other proteins produced using this technology.

Transgenic animals with required characteristics can be created by this technique.
A genetically engineered protein called visilizumab has long-lasting clinical benefits for people with the most severe form of ulcerative colitis. Visilizumab specifically binds to CD3 proteins that are expressed on T cells and thus has an inhibiting effect.

Use of recombinant human bone morphogenetic protein, or rhBMP-2, has been approved for certain types of spine fusion surgery. RhBMP-2 is a genetically engineered version of a naturally occurring protein that helps to stimulate bone growth.
Human keratin has been genetically engineered with the aim of incorporating the compound into skin and hair care products.

Gram stain

The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853 –1938) who originally devised it in 1882 (but published in 1884), to discriminate between pneumococci and Klebsiella pneumoniae bacteria in lung tissue. It is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. This reaction divides the eubacteria into two fundamental groups according to their stainability and is one of the basic foundations on which bacterial identification is built. Gram staining is not used to classify archaea, since these microorganisms give very variable responses.

Gram staining consists of four components:
Primary stain (Crystal violet, methyl violet or Gentian violet)
Mordant (Gram's Iodine)
Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture)
Counterstain (Dilute carbol fuchsin, safranin or neutral red)

The original description of staining technique by Christian Gram in a publication titled "The differential staining of Schizomycetes in tissue sections and in dried preparations" in Fortschitte der Medicin; 1884, Vol. 2, pages 185-189 was slightly different from what we use today. The primary stain used was aniline gentian violet, mordant was Lugol's iodine (iodine-potassium iodide in water), decolorizer was absolute alcohol and bismark brown was the counterstain.

Procedure:

The smear on a glass slide is covered with few drops of one of the primary stains. Gentian violet is a mixture of methyl violet and crystal violet. The primary stain renders all the bacteria uniformly violet. After a minute of exposure to the staining solution, the slide is washed in water.

The smear is treated with few drop of Gram's Iodine and allowed to act for a minute. This results in formation of a dye-iodine complex in the cytoplasm. Gram's iodine serves as a mordant.

The slide is again washed in water and then decolorized in absolute ethyl alcohol or acetone. A mixture of ecetone-ethyl alcohol (1:1) can also be used for decolorization. The process of decolorization is fairly quick and should not exceed 30 seconds for thin smears. Acetone is a potent decolorizer and when used alone can decolorize the smear in 2-3 seconds. A mixture of ethanol and acetone acts more slowly than pure acetone. Decolorization is the most crucial part of Gram staining and errors can occur here. Prolonged decolorization can lead to over-decolorized smear and a very short decolorization period may lead to under-decolorized smear.

After the smear is decolorized, it is washed in water without any delay. The smear is finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or safranin.

The slide is washed in water; excess water is removed using a blotting paper, dried in air and heat fixed before observing under microscope.

From bacteriology

Those bacteria that hold on to primary dye-iodine complex and remain violet are called Gram positive and those which get decolorized and subsequently take up counterstain (pink/red) are called Gram negative.

Basic fuchsin (present in dilute carbol fuchsin) stains many Gram negative bacteria more intensely than does safranin, making them easier to see. Some bacteria which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic fuchsin.

In order to ascertain if the staining procedure was satisfactorily conducted, a control smear of known Gram positive organism (e.g., Staphylococcus aureus) and a known gram negative organism (Escherichia coli) must be stained simultaneously. While the fibrin in a clinical specimen may appear gram positive, the pus cells and epithelial cells are always gram negative.

Mechanism of Gram reaction:

Various theories have been proposed to explain why some bacteria retain the dye and some don't. Theories such as differences in cytoplasmic pH (2 in case of Gram positive bacteria and 3 in case of Gram negative bacteria), and presence of Magnesium ribonucleate in Gram positive bacteria and its absence in Gram negative bacteria have not received widespread acceptance. The thickness of Gram positive cell wall and presence of more lipids in Gram negative cell walls have been more acceptable reasons for Gram stain reactions.

It is believed that the positively charged crystal violet pass through the cell wall and cell membrane and binds to negatively charged components inside the cell. Addition of negatively charged iodine (in the mordant) binds to the positively charged dye and forms a large dye-iodine complex within the cell. Crystal violet (hexamethyl-para-rosaniline chloride) interacts with aqueous KI-I2 via a simple anion exchange to produce a chemical precipitate. The small chloride anion is replaced by the bulkier iodide, and the complex thus formed becomes insoluble in water. During decolorization, alcohol dissolves the lipid present in the outer membrane of Gram negative bacteria and it leaches the dye-iodine complex out of the cell. A thin layer of peptidoglycan does not offer much resistance either. The dye-iodine complexes are washed from the Gram negative cell along with the outer membrane. Hence Gram negative cells readily get decolorized. On the other hand Gram positive cells become dehydrated from the ethanol treatment, closing the pores as the cell wall shrinks during dehydration. The dye-iodine complex gets trapped inside the thick peptidoglycan layer and does not get decolorized.

Limitations of Gram staining:

Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). When over-decolorized, even Gram positive bacteria may appear pink and when under-decolorized gram negative bacteria may appear Gram positive.

The Gram reaction also depends on the age of the cell. Old cultures of Gram positive bacteria (where cell walls may be weakened) may readily get decolorized. Gram positive cells affected by cell wall active agents such as lysozyme or antibiotics may become Gram negative. Gram-positive bacteria such Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these cells. In cultures of Bacillus, and Clostridium a decrease in peptidoglycan thickness during cell growth may cause some of them to appear Gram negative.

Certain group of bacteria can display variable response to the stain, which can be due to growth stress (e.g., unsuitable nutrients, temperatures, pHs, or electrolytes) that results in a number of nonviable, gram-negative cells in a gram positive culture, but certain bacterial species are known for their gram variability even under optimal growth conditions. Some bacteria tend to appear Gram negative when grown in acidic medium.

Loss of cell walls in Gram positive bacteria may render them Gram negative (L-forms). Bacteria totally devoid of cell wall (Mycoplasma) are always Gram negative. Bacteria such as Mycobacterium that have extra waxy content in their cell wall are difficult to stain. Small and slender bacteria such as Treponema, Chlamydia, Rickettsia are often difficult to stain by Gram's method. Gram positive bacteria that have been phagocytosed by polymorphs may also appear Gram negative.

Modifications of Gram stain:

There have been several modifications of Gram's stain. These are:
1. Kopeloff and Beerman's modification: Primary stain solution consists of freshly constituted methyl violet with sodium bicarbonate in distilled water. Mordant consists of iodine dissolved in 4% NaOH solution. Decolorization is either using acetone alone or a mixture of acetone and ethanol. Basic fuchsin is used to counterstain the smear. This method may be modified to stain tissue sections.

2. Jensen's modification: This method involves use to methyl violet as primary stain, iodine and potassium iodide in water as mordant, absolute alcohol as decolorizer and neutral red as counterstain. For Neisseria spp, Sandiford's counterstain is useful.

3. Weigert's modification: This modification is particularly useful for staining tissue sections. The primary stain carbol gentian violet is prepared using saturate alcoholic solution of gentian violet and 5% phenol solution. Gram's iodine is used as a mordant and aniline-xylol is used as a decolorizer. The counterstain carmalum (carminic acid and potassium alum in water), however is used ahead of primary stain. This method may be used to stain Pneumocystis cysts.

4) Preston and Morrell's modification: The primary stain used in this modification is ammonium oxalate-crystal violet. The smear is washed in Lugol's iodine and further treated with iodine solution. The smear is decolorized using iodine-acetone decolorizer and counterstained using dilute carbol fuchsin solution. This method has been further modified to overcome the irritating iodine in aerosols by reducing the iodine concentration to one-tenth and shortening the duration of decolorization to ten seconds.

Applications of Gram staining:

Differentiation of bacteria into Gram positive and Gram negative is the first step towards classification of bacteria.
It also the first step towards identification of bacteria in cultures.
Observation of bacteria in clinical specimens provides a vital clue in the diagnosis of infectious diseases.
Useful in estimation of total count of bacteria.
Empirical choice of antibiotics can be made on the basis of Gram stain’s report.
Choice of culture media for inoculation can be made empirically based on Gram’s stain report.

Miscellanea:

Although Gram stain is useful in staining bacteria, certain fungi such as Candida and Cryptococcus are observed as Gram positive yeasts.
Half-Gram stain refers to modified staining technique, where the smear is neither decolorized nor counterstained. It is useful to stain a known Gram positive bacterium.
Rapid Gram stain refers to quickened technique where the smear is exposed to only 30 seconds instead of one minute.
In specimen such as sputum, capsulated bacteria may stand out as clear spaces between the bacterium and the pink (mucus) background.
The spores may stand out as clear, unstained region in sporing bacteria.

Bacterial culture media

Introduction

There are various reasons why bacteria have to be grown (cultured) in the laboratory on artificial culture media. One of the most important reasons being its utility in diagnosing infectious diseases. Isolating a bacterium from sites in body normally known to be sterile is an indication of its role in the disease process. Indeed, isolating an organism from the clinical specimen is the first step in proving its role as an etiologic agent. Culturing bacteria is also the initial step in studying its morphology and its identification. Bacteria have to be cultured in order to obtain antigens from developing serological assays or vaccines. Certain genetic studies and manipulations of the cells also need that bacteria be cultured in vitro. Culturing bacteria also provide a reliable way estimating their numbers (viable count). Culturing on solid media is another convenient way of separating bacteria in mixtures.

Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs. When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. Hence, an artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat. Most often, a culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. Besides these, optimum pH, oxygen tension and osmolarity too have to be taken into consideration.

Ingredients
Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract. While tap water is suitable for culture media, it must not be used if it contains high amount of minerals. In such situations, distilled or demineralised water should be used. Peptone is a byproduct of protein (plant or animal) digestion. Proteins are often obtained from heart muscle, casein, fibrin or soya flour and is digested using proteolytic enzymes such as pepsin, trypsin or papain. The final product contains peptones, proteoses and amino acids besides a variety of inorganic salts including phosphates, potassium and magnesium. Casein hydrolysate is obtained from hydrolysis of milk protein casein using HCl or trypsin. Meat extract is obtained by hot water extraction of lean beef and then concentrated by evaporation. Meat extract contains gelatin, albumoses, peptrones, proteoses, amino acids, creatinine, purines, and accessory growth factors. Yeast extract is prepared from washed cells of bakers’ yeast and contains wide range of amino acids, growth factors and inorganic salts. Malt extract is prepared by extracting soluble materials from sprouted barley in water at 55oC and concentrated by evaporation. It contains maltose, starch, dextrin, glucose and small amounts of protein and protein breakdown products and growth factors.

Brief history
Initially, culture media were very simple; Louis Pasteur used simple broths made up of urine or meat extracts. Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media. Before the use of agar, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems; it existed as liquid at normal incubating temperatures (35-37oC) and was digested by certain bacteria.

Classification
Bacterial culture media can be classified in at least three ways; Based on consistency, based on nutritional component and based on its functional use.
Classification based on consistency:
Culture media are liquid, semi-solid or solid. Liquid media are sometimes referred as “broths” (e.g nutrient broth).
Liquid media are available for use in test-tubes, bottles or flasks. In liquid medium, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen in penumococci. Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits but with a clear medium. Culturing bacteria in liquid media has some drawbacks. Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected. Liquid media tend to be used when a large number of bacteria have to be grown. Culture media are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods).

Solid media:
Any liquid medium can be rendered by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. The word "agar" comes from the Malay word agar agar (meaning jelly). It is also known as kanten, China grass, or Japanese isinglass. Agar is chiefly used as an ingredient in desserts throughout Japan. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). Commercially it is derived primarily from Gelidium amansii. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesn’t contribute any nutritive property, it is not hydrolysed by most bacteria and is usually free from growth promoting or growth retarding substances. However, it may be a source of calcium & organic ions. Most commonly, it is used at concentration of 1-3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibres (shreds) or as powders.

For preparing agar in Petri plates, 3% agar (by weight) is added to the broth and autoclaved, when the medium is at ~50oC, it is poured on to sterile Petri plates and allowed to set. For preparing agar containing media in test-tubes, the culture medium is mixed with 3% agar and heated with stirring to melt. This ensures that all the tubes get equal amounts of agar. These tubes can then be sterilized by autoclaving.

Semi-solid media
Reducing the amount of agar to 0.2-0.5% renders a medium semi-solid. Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains (U-tube and Cragie’s tube). Certain transport media such as Stuart’s and Amies media are semi-solid in consistency. Hugh & Leifson’s oxidation fermentation test medium as well as mannitol motility medium are also semi-solid.

Biphasic media
Sometimes, a culture system comprises of both liquid and solid medium in the same bottle. This is known as biphasic medium (Castaneda system for blood culture). The inoculum is added to the liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium. This obviates the need for frequent opening of the culture bottle to subculture.
Biphasic medium

Other solidifying agents
Besides agar, egg yolk and serum too can be used to solidify culture media. While serum and egg yolk are normally liquid, they can be rendered solid by coagulation using heat. Serum containing medium such as Loeffler’s serum slope and egg containing media such as Lowenstein Jensen medium and Dorset egg medium are solidified as well as disinfected by a process of inspissation.

Classification based on nutritional component:
Media can be classified as simple, complex and synthetic (or defined). While most of the nutritional components are constant across various media, some bacteria need extra nutrients. Those bacteria that are able to grow with minimal requirements are said to non-fastidious and those that require extra nutrients are said to be fastidious. Simple media such as peptone water, nutrient agar can support most non-fastidious bacteria. Complex media such as blood agar have ingredients whose exact components are difficult to estimate. Synthetic or defined media such as Davis & Mingioli medium are specially prepared media for research purposes where the composition of every component is well known.

Classification based on functional use or application:
These include basal media, enriched media, selective/enrichment media, indicator/differential media, transport media and holding media.
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar considered basal medium
Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media.
Blood agar is preparing by adding 5-10% (by volume) to a basal medium such as nutrient agar or other blood agar bases. Since blood can not be sterilized, it has to be collected aseptically from the animal. Animals have to be bled and the blood is collected in sterile containers with anticoagulant or glass beads. While sheep blood is preferred, blood from rabbit, horse and ox can also be collected. Human blood must be avoided since it may contain inhibitory substances including antibiotics. After the blood agar base is autoclaved, blood is added to the medium at temperature just above the solidifying point of agar. The mixture is then poured on to the plates and allowed to solidify. Blood agar is useful in demonstrating hemolytic properties of certain bacteria. Two major types of hemolysis are often seen on blood agar; beta and alpha hemolysis. Beta hemolysis is the complete lysis of RBC resulting in clearing around the colonies whereas alpha hemolysis is the partial lysis of RBC resulting in greenish discolouration around the colonies. Gamma hemolysis is a misnomer and it indicates non-hemolytic colonies. Chocolate agar is also known as heated blood agar or lysed blood agar. The procedure is similar to that of blood agar preparation except that the blood is added while the molten blood agar base is still hot. This lyses the blood cells and releases their contents into the medium. This process turns the medium brown, hence the name. This medium is especially useful in growing Hemophilus and Neisseria.
Serum for medium can be obtained from animal blood but must be filtered through membrane or seitz filter before use.

Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen. Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these. Thayer Martin Agar used to recover N.gonorrhoeae contains Vancomycin, Colistin and Nystatin. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contain 10% NaCl. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% Potassium tellurite. McConkey’s Agar used for Enterobacteriaceae members contains Bile salt that inhibits most gram positive bacteria. Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa contains cetrimide. Crystal Violet Blood Agar used to recover S.pyogenes contains 0.0002% crystal violet. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating Malachite green. Wilson & Blair’s Agar for recovering S.typhi is rendered selective by the addition of dye Brilliant green. Selective media such as TCBS Agar and Monsur’s Tellurite Taurocholate Gelatin Agar used for isolating V. cholerae from fecal specimens have elevated pH (8.5-5.6), which inhibits most other bacteria.

Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone water are used to recover pathogens from fecal specimens.

Differential/Indicator media:
Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media are called differential media or indicator media. When a particular carbohydrate is incorporated into a medium and a mixture of bacteria inoculated on it, only that bacterium that can ferment it produces acid. This change in pH is detected by using a pH indicator incorporated in the medium and the bacterium that can ferment the sugar appears in a different colour. This approach is used in MacConkey’s agar, CLED agar, TCBS agar, XLD agar etc. MacConkey’s agar is the most commonly used media to culture and identify gram negative bacilli (especially enterobacteriaceae members). It contains bile salts (selective agent), lactose (sugar), peptone and neutral red (pH indicator), agar and water. Those bacteria that can ferment lactose produce pink coloured colonies where non-lactose fermenting colonies produce colourless colonies. Similarly, Vibrio cholerae produces yellow coloured colonies on sucrose containing TCBS medium.
Reduction of potassium tellurite to metallic tellurium by Corynebacterium diphtheriae results in production of black coloured colonies on PT agar. Production of H2S by Salmonella typhi results in production of black coloured colonies on Wilson & Blair’s medium. Enterococcus fecalis produces black coloured colonies on bile esculin agar due to reduction of esculin to esculetin. Detection of hemolysis on blood agar can be considered as an indicator property of Blood agar.

Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors. Cary Blair medium and Venkatraman Ramakrishnan medium are used to transport feces from suspected cholera patients. Sach’s buffered glycerol saline is used to transport feces from patients suspected to be suffering from bacillary dysentery. Pike’s medium is used to transport streptococci from throat specimens.

Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation –reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. Robertson cooked meat that is commonly used to grow Clostridium spps medium contain a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin. Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate. Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium. Under reduced condition, methylene blue is colourless.

Preparation and storage:
Care must be taken to adjust the pH of the medium before autoclaving. Various pH indicators that are in use include phenol red, neutral red, bromothymol blue, bromocresol purple etc. Dehydrated media are commercially available and must be reconstituted as per manufacturers’ recommendation. Most culture media are sterililized by autoclaving. Certain media that contain heat labile components like glucose, antibiotics, urea, serum, blood are not autoclaved. These components are filtered and may be added separately after the medium is autoclaved. Certain highly selective media such as Wilson and Blair’s medium and TCBS agar need not be sterilized. It is imperative that a representation from each lot be tested for performance and contamination before use. Once prepared, media may be held at 4-5oC in the refrigerator for 1-2 weeks. Certain liquid media in screw capped bottles or tubes or cotton plugged can be held at room temperature for weeks.

Monoclonal antibody production by hybridoma technique explained

I shall explain the hybridoma technique used in production of monoclonal antibodies in a simplified way.

An antigen (unless it is a small peptide) is a complex molecule with several antigenic determinants (or epitopes). When the immune system encounters such an antigen, it is usually processed to result in several fragments. Humoral (antibody-mediated) response may occur against some of these fragments. There are multiple clones of B cell, each against a specific epitope; resulting in production of antibodies against several epitopes. Such a response is said to be polyclonal. This is what that happens when our body encounters a microbial antigen following infection or immunization.

There are situations when it becomes necessary to have antibodies against a single antigenic determinant produced by a single clone of B cell. Such a response is said to be monoclonal. In order to produce monoclonal antibody, it is necessary to possess a purified antigen.

Hybridoma technique was developed by Georges Kolher and Cesar Milstein in 1975, for which they were awarded the Nobel Prize. The chief participants of this technique are the B cells and the myeloma cells. The B cells are obtained from the mouse which has been immunized with the antigen of choice. Myeloma cells are malignant B cells that are immortal and multiply continuously. Myeloma cells that have lost the ability to produce antibodies are chosen for this technique. In addition, these cells lack the ability to produce hypoxanthine-guanine phosphoribosyl transferase (HGPRT-) and thymidine kinase (TK-) enzymes through an induced mutation. Normal B cells have functional enzymes (HGPRT+ and TK+) and are able to produce antibodies. However, their life span is not beyond two weeks. Hybridoma technique involves physical fusion of both these cells so that the resulting hybrid (called hybridoma) has the features of both these cell types.

The first step towards the production of monoclonal antibodies is the immunization of the animal by antigen of choice. Mouse is the commonly used animal, but rat or hamster too can be used. Following repeated immunization (booster doses) the blood of the animal is tested for antibodies against the immunized antigen. Once it is determined that the animal has produced sufficient antibodies, it is killed and its spleen removed. The spleen is rich in B cells and would contain B cells specific to the immunized antigen among B cells of other specificities. The B-cells are separated from other cells and cultured. They are then mixed with cultured myeloma cells and allowed to fuse. Fusions of the cells are aided by polyethylene glycol (PEG). Not all cells fuse; present in the reaction mixture are unfused B cells, unfused myeloma cells and fused hybridoma cells. The next step involves separation of hybridoma cells from the unfused cells using a special selective medium.

In order to understand the functioning of selective medium, one must be aware of the following facts. Multiplying cells need to produce their DNA. Most cells produce their purines nucleotides and thymidylate (both precursors of DNA) utilizing tetrahydrofolates by a De-Novo pathway. This can be blocked using anti-folate drugs such as Aminopterin. The cells can then adopt Salvage pathway to synthesize DNA if hypoxanthine and thymidine are exogenously supplied. Purine nucleotides are produced from hypoxanthine using hypoxanthine-guanine phosphoribosyl transferase enzyme and thymidylate is produced from thymidine using thymidine kinase. The selective medium (HAT medium) used to select the hybridoma contains aminopterin, hypoxanthine and thymidine. Amiopterin inhibits the De-Novo pathway and presence of hypoxanthine and thymidine facilitates salvage pathway.

Normal unfused B cells can’t produce DNA by De-Novo pathway because of aminopterin but are able to undertake salvage pathway. This is because they contain functional enzymes (HGPRT+ and TK+). However, since they are mortal, they die after few multiplications. Unfused myeloma cells too can’t use De-Novo pathway because of aminopterin. They are unable to utilize the salvage pathway either because of deficient enzymes (HGPRT- and TK-). These cells die despite being immortal. However, fused hybridoma cells receive (HGPRT+ and TK+) trait from normal B cells and immortality from myeloma cells. These cells can utilize salvage pathway for DNA synthesis and yet be immortal. After two weeks, only the hybridoma cells survive in the selective medium.

The surviving hybridoma cells would have formed against different epitopes. The next step is to select the hybridoma produced against the desired antigen. The cultures are diluted to such an extent that only a single cell gets transferred to the wells of microtitre plate. The cells are allowed to multiply. These cells produce antibodies that can be readily detected in their supernatant fluids. Supernatant fluids from all the wells are tested for antibodies against the antigen of choice and the well that contains desired antibodies is selected and the rest may be discarded. Finally, a hybridoma cell producing antibodies against the epitope of choice is available.

These hybridoma cells may be lyophilized, cultured in vitro or injected intra-peritoneally into a mouse and monoclonal antibodies raised whenever required.