learn Microbiology

In 1675 – Antoni Van Leeuwenhoek (1632 – 1723) of Holland first described microbe (bacteria and protozoa) under the microscope. He made his own microscopes, which were superior to any of that time. He is known as the father of bacteriology because it was he who first accurately described the different shapes of bacteria (coccal, bacillary and spiral) and pictured their arrangement in infected material (1683). Prior to 17th century the view of the importance of micro organisms in human health and other areas was of necessity, mostly philosophical rather than scientific. Leeuwenhoek’s findings were transmitted in a series of letter from 1674 - 1723 to the Royal Society in London through which his observations were widely disseminated. In one of the first letters September 7th 1674 he gave detailed description of microbes which he called ‘animalcules’ now known as protozoa. Unfortunately while Leeuwenhoek shared his observations with the scientific community he never revealed the details of his methods of microscope nor how he constructed his hundreds of microscopes. The early microscope used by Hooke and Leeuwenhoek permitted only a fussy view of micro organisms they where observing. Many advances in the art of microscope making and science of microscopy were needed before observations could be made on the fine structure of micro organism. The major advances made in microscope in the late 19th century went hand in hand with the period of great advancement in microbiology. During this period, Ernst Abbe, a German Physicist developed microscope lenses that corrected for aberrations inherent in magnifying lenses which had limited the ability to view micro organisms.

All the main types of unicellular micro organisms protozoa, algae, yeasts and bacteria was described by him as early as in 1676. Leeuwenhoek did not go beyond describing the microbes. He never tried to associate them with their surrounding as causative agents. During the time of Leeuwenhoek and even later, microscopic observations was regarded as an idle hobby with no practical relevance.

Louis Pasteur (1822 – 1895)

Pasteur had been trained as a Chemist, and this training had a marked influence on his approach to scientific questions. As a Chemist, Pasteur was able to separate an optically inactive mixture of two optically active chemical compounds that differ only in spatial orientation such that the individual compounds bend light in opposite directions called a racemic mixture into its two optical active components, thus explaining the riddles concerning the optical activities of liquids and why light passing through a solution was bent sometimes to the left and sometimes to the right.

Discredited the theory of spontaneous generation and established that living microorganisms are responsible for the chemical changes that occur during fermentation.

Pasteur was irritated by the ongoing debate between the spontaneous origin and the non spontaneous origin. In order to settle the matter once for all Pasteur filtered air through cotton and found that objects resembling plant spores has been trapped. If a piece of cotton was placed in a sterile medium after air had been filtered through it, microbial growth appeared. Next he placed nutrient solution, heated their neck in a flame and drew them out into a variety of curves, while keeping the ends of the necks open to the atmosphere. Pasteur then boiled the solutions for a few minutes and allowed them to cool. No growth took place even though the content of the flask was exposed to the air.

Pasteur pointed out that no growth occurred because dust and germs had been trapped on the walls of curved neck. If the necks where broken growth commenced immediately. Pasteur had not only resolved the controversy by 1861 but had shown how to keep solution sterile.

Pasteur began his career as a professor of Chemistry at the University of Lille. A principal industry of France being the manufacture of wines and Pasteur studied the methods and process involved in order to help his neighbours produce a consistently good product. He found that fermentation of fruits and grains resulting in alcohol was brought about by microbes.

Pasteur suggested that the undesirable types of microbes might be removed by heating – not enough to hurt the flavour of the fruit juice, but enough to destroy a very high percentage of microbial population. He found that holding the juice at a temp of 62.8°C (145°F) for ½ an hour would kill microorganisms. Pasteurization, is widely used in fermentation industries, but are most familiar with it in diary industry.

Pasteur’s success is solving the problem of fermentation led the French govt. to request that he investigate pebrine, a silk worm disease, that was ruining an important French industry. He isolated the parasite causing the disease.

Pasteur tackled the problem of anthrax, disease of cattle, sheep and sometimes human beings. He grew the microbe in laboratory flasks after isolating them from the blood of animals that died of the disease. Mean while Robert Koch was busy with the anthrax problem in Germany. Koch grew these bacteria in cultures in his laboratory, examined them microscopically to be sure he had only one kind present and injected them into other animals to see if these become infected and developed clinical symptoms of anthrax.

Pasteur continued to make discoveries concerning the cause and prevention of infectious diseases. About 1880 he isolated the bacterium responsible for chicken cholera and grew it in pure culture. Pasteur made use of fundamental techniques devised by the more theoretical Koch. He inoculated healthy chickens with his pure culture, the chicken failed to get sick and die. Pasteur found that he had accidently used cultures several weeks old instead of the fresh ones grown especially for the demonstration. Some weeks later he repeated the experiment, using two groups of chickens. One of these groups had been inoculated at the first demonstration with the old cultures that had proved in effective and the second had not been previously exposed. Both groups received bacteria from fresh young cultures. This time the chickens in the second group got sick and died but those in the first group remained hale and hearty.

This puzzled Pasteur but he soon found the explanation. In some way bacteria could lose their ability to produce disease that is their virulence after standing and growing old. But these attenuated bacteria still retained their capacity for stimulating the host to produce substances i.e. antibodies that protect against subsequent exposure to virulent organisms.

This demonstration explained the principle involved in Edward Jenner’s successful use of cowpox virus in 1798 to immunize people against smallpox. Pasteur next applied this principle to treating Hydrophobia or Rabies. Pasteur next prepared rabies vaccine by a different approach. The pathogen was attenuated by growing it in an abnormal host, the rabbit. After infected rabbits had died their brains and spinal codes were removed and dried. Injection of a mixture of this material andglycerine stimulated dogs to develop resistance to rabies. Then a 9 year old boy, Joseph Meister who had been bitten by a rabid dog was brought to Pasteur. Since the boy’s death was certain in the absence of treatment, Pasteur agreed to try vaccination Joseph was injected 13 times over the next ten days with increasingly virulent preparations of the attenuated virus. He survived.

In gratitude to Pasteur’s development of vaccines people from around the world contributed to the construction of Pasteur institute in Paris, France. One of the initial tasks of the institute was vaccine production

Chemical agents: Sterilization by Chemical Agents

Ideal anti-septic should have the following properties

I. It should be a wide spectrum

II. Active in the presence ororganic matters ( Carbon compounds)

III. Effective in acid, alkaline medium

IV. It should have speedy effect

V. High penetrative power

VI. Should be stable, should not decompose

VII. Compatible with other antiseptics(should not react and hinder the action of other antiseptics when used together)

VIII. Should not corrode metals

IX,.Should not cause local irritation or sensitization

X,.Should not interfere with healing eg: clotting

XI.Should be cheap and easily available

XII. Safe to use and easy to use.

Different Factors that affect the potency of disinfectants

1. Concentration of the substance

Diluted ones have less potency.

Concentrated ones have more potency.

2. The time of action

3,.H of the medium

4. Temperature:- Increased temperature of the substance should increase the action speedily eg:phenols

5. Nature of the organism

6. Presence of extraneous material

Mode of action

1. Protein coagulation

2. Disruption of the cell membrane(cell lysis and death)

Most of the chemicals act on cell membrane

3.Act on free sulphydryl groups thus activating the enzymes. Enzymes are made up of amino acids. Example of amino acids with ‘S’ (Sulphur): Methionine and Cystine They have ‘SH’ group They are called thiol groups.

Enzymes are proteins. Proteins are made up of amino acids

Chemicals change the sulphur group in the amino acids and remove the free sulphydryl group (SH) which are essential for the functioning for the enzymes. Suppose from a chemical, one part is removed, its whole function is lost.

4. The chemicals competes with substrates. In other words, substrate competition takes place. Naturally, enzyme binds with substrate to give enzyme-substrate complex which in turn gives the product and the free enzymes.

E+S ES P+E

When the chemical competes it binds with the enzymes and prevents the formation of enzyme-substrate complex and even de-activates the enzyme. The enzymes are altered.

1, Alcohols

They are organic solvents. They are good antiseptics eg: Ethyl alcohol CH3CH2OH in concentration between 50 and 90% is effective against vegetative or non-spore forming cells. For practical application, a 70% concentration of alcohol is generally used. Alcohols are protein denaturants and this property to a large extent accounts for the anti-microbial activity. Alcohols are lipid solvents and hence they may damage lipid complexes in cell membrane. They are also de-hydrating agents. It is possible that very high concentration removes so much of water from the cells that the alcohol is unable to penetrate. This severe de-hydration would result in a bacterio-static condition. They are used for surface sterilization,by swabbing using cotton on surfaces.

Eg: Methyl alcohol is less bactericidal than Ethyl alcohol. It is highly poisonous. Even the fumes of Methyl alcohol may produce permanent injury to the eyes and is not generally employed for the destruction of micro-organisms.

Eg: Higher alcohols-propyl, butyl, amyl and others are more germicidal than ethyl alcohol.

Some of the effectiveness of the alcohol is because of cleansing or detergent action, which results in mechanical removal of micro-organisms. They are used as a disinfectant for thermometers.

2, Aldehydes

Among the class of chemicals with a general formula RCHO (aldehydes) several of the low molecular weight compounds are anti-microbial. Two of the most effective are Formaldehyde and gluteraldehyde. Both are highly microbicidal and both have the ability to kill spores (sporicidals).

Eg: Formaldehyde

Formaldehyde is the simplest compound in the aldehyde series. It is a gas that is stable in high concentrations and at elevated temperatures. At room temperatures, it polymerises, forming a solid substance, paraformaldehyde. They are active against nitrogen compounds such as proteins and nucleic acids. Amino acids have amino groups, NH2. It is likely that interaction of formaldehyde with the cellular substances accounts for its anti-microbial actions. Formaldehyde is a fumigative agent, that is used in hospitals.The gas causes irritation. It is used in fumigating operation theatres, laboratories and even laminar airflow.

Eg: Glutaraldehyde

It is a saturated aldehyde with the formula

CHO - CH2-CH2-CH2-CHO

A 2 % solution of this chemical agents exibhits a wide spectrum of anti-microbial activity. It is effective against vegetative bacteria, fungi, bacterial and fungal spores, and viruses. It is used in the medical field for sterilizing urological Instruments, respiratory therapy instruments. It is less toxic. It acts on specific amino group of proteins.

3, Dyes

Two classes of dye compounds are of interest to microbiologists. They are triphenyl methane and acridine dyes.

Eg: Acridine dyes

Two examples of Acridine dyes are acriflavine and tryptoflavine. They act against Staphylococci and Gonococci. They possess little if any, anti-fungal activity. They are used to some extent for the treatment of burns and wounds. Actually they are intercalating agents that intercalate between two bases of the DNA.

Eg: Triphenyl methane dyes

Included in this category are malachite green, brilliant green and crystal violet. Gram-positive organisms are more susceptible to lower concentrations of these compounds than are Gram-negative ones. Crystal violet will inhibit Gram-Positive cocci at a dilution of 1:2,00,000- 1:3,00,000. This general relationship between gram reaction and susceptibility to triphenyl methane dyes has a number of practical applications. They interfere with cellular oxidation processes.

4, Halogens

Iodine, Chlorine

Iodine acts against tubercle bacilli , bacteria viruses and spores. Iodine is one of the oldest and most effective germicidal agent. Pure iodine is readily soluble in alcohol and aqueous solutions of potassium or sodium iodide. It is slightly soluble in water. It is used as a germicidal agent referred to as tincture of iodine. There are several preprations available such as 2% Iodine+ 2% Sodium Iodide diluted in alcohol, 7% Iodine+ 5% Potassium Iodide in 83% alcohol and 5% Iodine and 10% potassium Iodide in Aqueous solution. Iodine is also used in a form of substance known as iodophors. Idophors are mixtures of iodine with surface active agents which act as carriers and solubilizers for the Iodine. One of these agents is polyvinylpyrrolidone (PVP): The complex can be expressed as PVP-I. Iodine is released slowly from this complex.

Mode of Action

Iodine is an oxidizing agent. It affects the surface, that is reduction of surface. Oxidising agents can irreversibly oxidize and thus inactivate essential metabolic compounds such as proteins with sulphydryl group. It has also been suggested that the action may involve the halogenations of tyrosine units of enzymes.

Chlorine

Chlorine either in the form of gas or in certain chemical combinations is used as disinfectant. The compressed gas in liquid form is used for the purification of sewage-contaminated municipal water supplies. Chlorine gas is difficult to handle unless special equipment is available to dispense it.

There are available many compounds of Chlorine which can be handled more conveniently than free chlorine. Eg: Hypochlorites

Eg: Chloramines

Hypochlorites, Calcium hypochlorite, Ca(OCl)2 also known as chlorinated lime and sodium hypochlorite NaOCl.

Chloramines, represent another category of Chlorine compounds used as disinfectants, sanitizing agents, or antiseptics. Chemically, one or more hydrogen atoms in an amino group of a compound are replaced with chlorine. The simplest of these is monochloramine, NH2Cl.

Cl2+H2O HCl + HClO (Hypochlorous acid)

Hypochlorous acid is formed when free chlorine is added to water. Similarly, Hypochlorites and Chloramines undergo hydrolysis with the formation of hypochlorous acid. The hypochlorous acid formed in each instance is further decomposed.

HClO HCl + O

Formed from chlorine, hypochlorites, chloramines

The Oxygen released in this reaction is nascent oxygen. It is a strong oxidizing agent and through its action on cellular constituents, micro-organisms are destroyed. The killing of micro-organisms by Chlorine and its compounds is also due in part to the direct combination of chlorine with proteins of the cell membranes and enzymes.

5, Phenol and phenolic compounds

In 1880, Phenol was used successfully by Joseph Lister, a surgeon to reduce infection of surgical incisions and surgical wounds. Later he developed the practice of spraying phenol in the operating room area to control infection.

Phenol has been used as a standard against which, other disinfectants of a similar chemical structure are compared to determine their antimicrobial activity. The procedure is called Phenol coefficient test.

6, Heavy metals and their compounds

Most of the heavy metals, either alone or in certain compounds exert a detrimental effect upon microorganisms. The most effective are Mercury, Silver and Copper.

Heavy metals and their compounds acts anti-microbially by combining cellular proteins and inactivating them. Eg: In the case of mercuric chloride, the inhibition is directed at enzymes which contain sulphydryl grouping.

High concentration of salts of heavy metals like mercury, copper and silver coagulate cytoplasmic proteins resulting in damage or death to the cell. Salts of heavy metals are also precipitants, and in high concentrations such salts could cause the death of a cell.

S

Enzyme- SH + HgCl2 Enzyme Hg + 2HCl

SH S

Active enzyme Mercuric chloride Inactive enzyme

7, Quaternary Ammonium compounds

Most components of the germicidal cationic detergent class are Quaternary ammonium salts. Their characteristic structure with reference to a common inorganic ammonium salt such as ammonium chloride.

The bactericidal power of quaternaries is exceptionally high against Gram-positive bacteria and they are also quite active against Gram-Negative bacteria. The action of these compounds demonstrate the need to distinguish between static and lethal activity in test procedures for the evaluation of disinfectants. Quaternaries have been shown to be fungicidal as well as destructive to certain of pathogenic protozoa. Viruses appeared to be resistant than Bacteria and fungi.

They are used as skin disinfectants, as a preservation in ophthalmic solution and in cosmetic preprations. Quatenaries are used to control microorganisms on floors, walls and other surfaces in hospitals, nursing homes and other public places. They are used to sanitize food and beverage utensils in restaurants and certain equipments in food-processing plants, with reference to fish and fishery based product ( F & FP) industries.

8, Gaseous Agents.

Certain kinds of medical devices that needs to be available in sterile condition are made of materials that are damaged by heat. Eg; plastic syringes, blood transfusion apparatus and catheterization equipments. Also sterilization of enclosed areas warrant the use of sterilization by the means of gaseous agents. The main agents currently used for gaseous sterilization are Ethylene Oxide, β-propiolactone and formaldehyde

Ethylene Oxide: It is a liquid at temperatures below 10.8 degree celsius (51.4 degree Farenheit). Above this temperature, it vaporizes rapidly. Vapors of this compound in air are highly flammable even in low concentrations. This feature was overcome by preparing ethylene oxide in carbon dioxide or Freon.

The carbon dioxide-ethylene oxide or Freon-ethylene Oxide mixtures are non-flammable and there is no alteration of the microbicidal activity of the ethylene oxide. The carbon dioxide and the Freon merely serve as inert diluents which prevent flammability.

It is used for sterilizing heat or moisture sensitive materials in hospitals, industries, and laboratories. Bacterial spores which are many times more resistant than vegetative cells as measured by other anti-microbial agents, show little resistance to destruction by this agent. It has high penetrative power. It will pass through and sterilize large packages of materials, bundles of cloth and even certain plastics. It must be used with caution although devices are available for its safe routine laboratory use.

Mode of Action

The mode of action of ethylene oxide is believed to be alkylation reactions with organic compounds such as enzymes and other proteins. Alkylation consists in the replacement of an active hydrogen atom in an organic compound. Eg; The hydrogen atom in a free carboxyl, amino or sulphydryl group with an alkyl group. In this reaction, the ring in the Ethylene oxide molecule splits and attaches itself where the Hydrogen was originally. This inactivates an enzyme with the sulphydryl group.

H2C CH2 + Enzyme-SH enzyme SH enzyme S CH2CH2OH

Inactive

O

Β-propiolactone

This compound is colorless liquid at room temperature with a high boiling point 155 degree celsius and has a formula

CH2 CH2

O C O

β propiolactone

It is not flammable like ethylene oxide. It lacks the penetrative power of ethylene oxide but is considerably more active against micro-organisms. It is sporicidal, fungicidal, and Virucidal. β-Propiolactone is very effective in destroying microorganisms on surfaces. However, it has a low power of penetration coupled with its alleged carcinogenic properties has restricted its use as a practical sterilizing agent.

Phenol coefficient method

A specific official test method called the FDA (Food and Drug Administration) method. This procedure is suitable for testing disinfectants which are miscible with water and exerting their antimicrobial action in a manner similar to that of phenol. The test organism employed in this procedure is a specific strain of either Salmonella typhi or Staphylococcus aureus. The temperature at which the test is performed, the manner of making subcultures, the composition of the subculture medium, the size of the test tubes and other details of the test are spelled out in the official procedure.

It should be emphasized that no single microbial test method is suitable for the evaluation of all germicidal chemicals for all applications recommended. Testing of antimicrobial agents often begins with an initial screening test to see if they are effective and at what concentrations. This maybe followed by more realistic in-use testing.

The best known disinfectant screening test is a phenol coefficient test in which the potency of a disinfectant is compared with that of phenol. A series of dilutions of phenol and the experimental disinfectant are inoculated with the test bacteria. Then placed in a 20 or 37 degree celsius water bath. These inoculated disinfectant tubes are next subcultured to regular fresh medium at 5 minutes intervals. And the subcultures are incubated for two or more days. The highest dilutions that killed the bacteria after a 10-minute exposure, but not after 5-minutes, are used to calculate the phenol coefficient. The reciprocal of the appropriate disinfectant dilution is divided by that for phenol to obtain the coefficient. Suppose at the phenol coefficient was 1/90 and maximum effective dilution for disinfectant X was 1/450, the phenol coefficient of X would be 5. The higher the phenol coefficient value, the more effective the disinfectant under these test conditions. A value greater than 1 means that the disinfectant is more effective than phenol.

The phenol coefficient test is a useful initial screening procedure but the phenol coefficient can be misleading if taken as a direct indication of disinfectant potency during normal use. This is because the phenol coefficient is determined under carefully controlled conditions with pure bacterial strains wheras disinfectants are normally used on complex populations in the presence of organic matter and with significant variations in environmental factors like pH, temperature and presence of salts. To more realistically estimate disinfectant effectiveness, other tests are often used. The rates at which selected bacteria are destroyed with various chemical agents maybe experimentally determined and compared.

Stains are biological dyes . They consist of colour giving compound or chromogenic portion that imparts colur and Auxochrome that determines the ionic binding with the specimen or the textile fabric

Use of Arecanut as a therapeutic agent against SARS Coronavirus .

Reena R.Nelson Anthikat , Phd and Ignacimuthu S, Phd, DSC

PSG College of Arts and Science, Coimbatore -640014 . Tamil Nadu.

The study is aimed at the possible certification of arecant extract s efficacy as therapeutic agent against SARS corona virus.Then we could convert their use to a therapeutic use against several respiratory viral germ. It may become prophylactic and/or therapeutic regime for virus infection. Since Corona virus cannot be cultured , we have to resort to straight studies on influenza virus .Both viruses share similar structural properties and symptoms in infection. First based on antimicrobial potential and antioxidant activity of the efficacy of arecanut extract(AE) proved that studies could be done to compare the effect of extract against influenza virus and corona virus. This AEt has a potential antifungal activity against unicellular yeast Candida albicans and mycelia fungi Mucor sp and Aspergillus flavus. In the tube method against bacteria and Candida sp , the test samples Absorbance was read at 530 nm at different time intervals. Disc Diffusion Method was used to evaluate the Zone of clearance suggestive of the zone of inhibition of the AE against fungi by taking Nystatin as standard. Inhibition of Aflatoxin production was checked by Pons Method. 85 % inhibition of the production of aflatoxin by Aspergillus flavus was at,between 100-250 µg/ml of the AE. The AE was shown to inhibit the viral growth and propogation of AVIAN viruses NDV(New castle Disease Virus) and Egg Drop Syndrome Virus( EDS ) in embryonated culture. Invitro antioxidant studies showed that AE could inhibit superoxide radical production, could inhibit hydroxyl radicals and could prevent lipid peroxidation. AE could scavenge DPPH (DI PHENYL-2-PICRYL Hydrazyl ) radicals and also ABTS(Di ammonium salt)(2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). In FRAP(Ferric Reducing Antioxidant Power) assay, the reduction of ferric to ferrous is also seen in a concentration dependant manner. Concentration ranging from 5µg/ml – 100 µg/ml showed the potential of AE to scavenge 50% free radicals. Cytotoxicity studies of AE against Influenza virus previously reported and documented with scientific evidence proved all the more its efficacy in the development of new antiviral agent.

Result The present line of thinking was supported by results to show the effectiveness of AQUEOUS AE as potential therapeutic agent against Influenza virus . Thus probably its use against Corona virus be established with further scientific evidence is the perception. This is based on AE antimicrobial , antioxidant activity. Further, the identification and purification of their effective components leads to the use of AE as traditional cure.

Key words: ARECANUT-ANTIMICROBIAL-ANTIOXIDANT-ANTIVIRAL

Light field Microscopes, Dark Field Microscopes, Phase contrast Microscopes and electron Microscopes. We need to concern ourselves with Light Microscopes and Electron microscopes detailed in the notes narrative.

the power point explains the basic bright field microscope and the remaining are the different types. of microscopes in the next lecture content which is elaborated in detail.

They , the student community will have safety issues adressed in their homes , schools, public transport as they will definitely begin to use more soap and water to disinfect their hands. Soaps destroy viruses. Viruses of Respiratory types eg Rhinovirus spreads common cold.

eg Virus attcks the human and cause viral fever or a flu by breaking down the host immuno machinery and taking over the immune sysytem

A better know how and an idea of the concepts ,,,,creates and spreads awareness of simple steps like hand wash

2) Tetra packed milk and juices once opened ought to be refrigerated or completed as early and quickly as possible as microrganisms gain entry into the sterile packing once the seal is opened. These basic ideas help them dig deep into the wealth and treasure of knowledge pool as to why Pasteurisation  is used as a method of sterilisation, who developed this method...etc for the food industry

3.The cognitive capacity to think through the spread of COVID 19 virus, why vaccination is important and what is "herd immunity"   which is achieved as a result of mass vaccination campaign and real time vaccination...thus emphasising on the discovery of vaccination

They , the student community will have safety issues adressed in their homes , schools, public transport as they will definitely begin to use more soap and water to disinfect their hands. Soaps destroy viruses. Viruses of Respiratory types eg Rhinovirus spreads common cold.

eg Virus attacks the human and cause viral fever or a flu by breaking down the host immuno machinery and taking over the immune system

A better know how and an idea of the concepts ,,,,creates and spreads awareness of simple steps like hand wash

2) Tetra packed milk and juices once opened ought to be refrigerated or completed as early and quickly as possible as microrganisms gain entry into the sterile packing once the seal is opened. These basic ideas help them dig deep into the wealth and treasure of knowledge pool as to why Pasteurisation  is used as a method of sterilisation, who developed this method...etc for the food industry

3.The cognitive capacity to think through the spread of COVID 19 virus, why vaccination is important and what is "herd immunity"   which is achieved as a result of mass vaccination campaign and real time vaccination...thus emphasising on the discovery of vaccination