BSc 2nd Year Control of Microorganisms Notes Study Material

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Control of Microorganisms Notes Study Material

The control of microorganisms is necessary for good health. In this post we shall briefly consider some of the established methods of their control. There are three principal kinds of agents used in control : (i) physical agents, that are used exclusively on objects outside the body, (ii) chemical agents, which are used on inanimate objects as well as on the body surface and (iii) chemotherapeutic agents, which are most often used inside the living body.

Physical Control

The chief agent used in this type of control is heat, applied in different forms. In addition, there are some other methods also, in which different types of time physical agents are used.

1. Heat. The aim is often sterilisation. This term implies the removal of all life forms and this is an absolute term. Following are the common ways in which heat is used in control.

(a) Direct flame. This method is used in the process of incineration, employed in the laboratory to sterilise the bacteriological loop and needle before removing a sample from a culture tube and after preparing a smear. The tip of tube is also flamed to destroy microbes which may contact it.

(b) Hot-air oven. This instrument utilises dry heat for sterilisation. The material is put at 160°C for a period of two hours to kill the bacterial spores and other microbial structures. This method is oftenly used to sterilise dry powders, oily substances and glasswares.

(c) Boiling water. Here the objects are immersed in boiling water. The moist heat sterilises the objects. This heat penetrates better and rapidly than dry heat. Since temperature is 100°C, bacterial spores may require two hours exposure for destruction. Moist heat kills microorganisms by coagulating and denaturing their proteins. The object must be completely immersed in boiling water.

(d) Autoclave. The principle used here is to increase the pressure of steam (gas) in a closed system that increases its temperature. The water molecules become more aggregated that increases their penetration considerably. This principle is used to reduce sterilising time in the laboratory instrument called the autoclave. It contains a sterilising chamber into which articles are placed and a steam jacket where steam is maintained. A special valve increases the pressure to 15 lb/sq. inch above normal atmosphere pressure. The temperature rises to 121.5°C and superheated water molecules rapidly conduct heat into the microorganisms within about 15 mts. Culture media, glassware, metalware etc.

(e) Fractional sterilisation. It is also called tyndallisation (after its developer, John Tyndall), and intermittent sterilization as it is a stop-and-start operation.

Sterilisation is achieved by a series of events. During first day objects are exposed to free-flowing steam at 100°C for 30 min. which kills all organisms except bacterial spores. When left overnight the spores germinate into vegetative cells. These are killed during the second day’s exposure to steam for 30 min. Again, the material is cooled, and the few remaining spores germinate, which are killed on the third day exposure. This method is particularly important in modern microbiology, for sterilising those objects which could not be sterilised by autoclave. The instrument used is Arnold steriliser.

(f) Pasteurization. This is not supposed to provide sterilization. Here the bacterial population of a liquid as milk is merely reduced, and the organisms which may cause human diseases are destroyed. Spores are not affected. One method for milk pasteurization is holding method. It involves heating at 62.9^o C for 30 min. It kills both tubercle bacterium, Mycobacterium tuberculosis and the Q fever agent. Other methods are the flash pasteurization at 71.0°C for 15 seconds, and the ultrapasteurization at 82°C for 3 seconds.

(g) Hot oil. Some physicians and dentists use hot oil at 160°C for one hour for sterilisation of the instruments.

2. Other methods. Physical methods other than heat, are as follows:

(a) Filtration. It is a mechanical device for removing microorganisms from a solution. The organisms are trapped in the pores of the filter, and the filtrate is decontaminated or possibly sterilised. There are several types of filters used in microbiology laboratory. Inorganic filters are typified by the Seitz filter, which consists of a pad of asbestos mounted in a filter flask. Porcelain and ground glass may also be used. There are organic filters also. The organic molecules of the filter attract the organic components of the microorganisms. An example, the Berkefeld filter, utilises the substance-diatomaceous earth, composed of the skeletal remains of marine alga called diatoms. Third type of filter is membrane filter which received general acceptance. It consists of a pad of organic compound such as cellulose acetate or polycarbonate mounted in a holding device. Membrane filters are available in various pore sizes according to the microorganisms to be trapped. This method can be used for quantitative estimation of microbes in a given sample. The filter pad is put on nutrient culture medium where cells grow.(Control of Microorganisms Notes Study Material)

(b) Ultraviolet light. UV light has a wavelength between 100 and 400 nm, and the energy at about 265 nm is most destructive to bacteria. Exposure to UV damages the DNA. UV radiations are used to reduce air contamination.

(c) Other radiations. The spectrum of energies with wavelengths less than that of UV includes—X-rays and gamma rays which can also be used to destroy bacteria. These rays are called ionizing radiations as they eject electrons out of organic molecules, thus creating ions.

(d) Ultrasonic vibrations. These are high-frequency sound waves. If propagated in fluids, sound waves cause microscopic bubbles to form, and the water appears to boil (also called cold boiling). The bubbles rapidly collapse, giving tiny cavities and sending out shock waves. Microorganisms in the fluid are rapidly disintegrated by the external pressures. A device called cavitron is used by dentists to clean teeth. There are ultrasonic machines to clean dental plates coins, jewellery etc.(Control of Microorganisms Notes Study Material)

(e) Preservation methods. A number of physical methods are used for controlling microorganisms in foods. Thus food spoilage is checked. These are as follows:

i. Drying. It is used for preservation of meat, cereal, fish and other foods.

ii. Salting. It is based on principle of osmotic concentration. In presence of salt, water comes out of cells due to exosmosis, causing ultimate death. This method is used in preservation of syrups, jams, jellies, etc. from bacterial contamination.

iii. Low temperature. In the refrigerator and freezer, low temperatures retard spoilage by reducing the metabolic rates of microorganisms.

Chemical Control

Chemical methods are expected to remove the pathogenic organisms from an object (or body). These methods rarely achieve sterilization as in physical methods. The process of removal is called disinfection. If the object is non-living, the chemical is known as disinfectant. However, if the object is living, as a tissue of human body, then the chemical is an antiseptic. Antiseptics and disinfectants may be either bactericidal or bacteriostatic. The former agent kills the microbes whereas the latter temporarily prevents their further multiplication.

The effectiveness of a chemical agent is based on phenol coefficient (PC). This number indicates the ability of a particular antiseptic or disinfectant as compared to phenol under identical conditions. A PC higher than one indicates that the chemical is more effective than phenol.

Following are some of the commonly used chemicals to control microorganisms.

1. Halogens. Two halogens, chlorine and iodine are most commonly used. The halogens are highly reactive elements. Chlorine is available either as gas or as organic or inorganic compounds. It is used in municipal water supplies, keeping bacterial population at low levels. The residue used is about 0.2-1 ppm of free chlorine. One ppm is equivalent to 0.0001 per cent. Chlorine is also useful as sodium hypochlorite (NaOCl) or as calcium hypochlorite [Ca (oCl₂.)]. Other forms of chlorine are the chloramines. Iodine is used generally in the form of tincture of iodine, as antiseptic for wounds. It has 2% iodine plus sodium iodide in ethanol. Iodophors are complexes of iodine and detergents that release the iodine over a long period of time.(Control of Microorganisms Notes Study Material)

2. Phenolic compounds. Phenol is the standard disinfectant, which cogulates the proteins, particularly cell membrane enzymes. Phenol is especially useful against Gram-positive bacteria. An alternative of phenol, cresol has become more popular in modern medicine as it is cheaper than phenol. Cresols are used as wood preservatives. Bisphenols—a combination of two phenol molecules is also prominent in modern disinfection and antisepsis. For example, orthophenyIphenol is used in lysol, osyl, staphene etc. Another bisphenol, hexachlorophene was used extensively in 1950’s and 1960’s in toothpastes, underarm deodorants, and bath soaps. A phenol derivative, hexylresorcinol is used in a mouthwash, topical antiseptic and in throat lozenges.

3. Heavy metals. The activity of heavy metals on microorganisms is termed oligodynamic action. Metals as silver, mercury and copper are used, Mercury is an older antiseptic, used as mercuric chloride (HgCl₂). In products like mercurochrome, merthiolate and metaphen; mercury is combined with organiccarrier compounds, that reduces its toxicity to skin. Copper is particularly active against algae. It is used as copper sulphate in swimming pools and municipal water supplies, Silver, as silver nitrate is used as an antiseptic and disinfectant. One drop of 1% AgNO₃ solution is placed in eyes of newborn to protect against infection by the gonococcus, Neisseria gonorrhoeae. Silver nitrate can also be combined with an antimicrobial drug for use in treatment of burns.

4. Alcohol. It is an effective antiseptic, applied to skin. The most common is ethyl alcohol, though propyl, butyl and pentyl alcohols have a greater germicidal ability. But they are more expensive and do not easily mix with water. Methanol is toxic to tissues. Ethanol acts particularly on vegetative bacterial cells. It is strong dehydrating agent. Ethyl alcohol (70%) is mostly used.

5. Alkylating agents. Formaldehyde is most common. This compound is a gas at high temperature but a solid-paraformaldehyde at room temperature. Formalin is prepared by suspending 40g of this solid in water. This is used in anatomical specimens. It is also used in inactivation of viruses in vaccine preparations and in production of toxoids from toxins. Ethylene oxide (Eto) is used in sterilisation of plastic materials used in laboratory. It is used in combination with freon in a ratio of 12 : 88, available as cryoxide or steroxide. Carboxide contains Eto and carbondioxide. The gas is released into a tightly sealed chamber. Other alkalyting agents are, beta-propiolactone, and glutaraldehyde.

6. Hydrogen peroxide. It is used as a rinse in wounds, scrapes or abrasions. New forms of stable H₂O₂, like Super D hydrogen peroxide have also appeared.

7. Soaps and detergents. Since the pH of soaps is about 8.0, it destructs microorganisms to some extent due to alkalinity. Soap is used for mechanical washing of the skin surface. Soaps are wetting agents, emulsifying and solubilising particles that cling to a surface. Detergents are synthetic chemicals developed for their ability to be strong wetting agents and surface tension reducers.(Control of Microorganisms Notes Study Material)

8. Dyes. A group of dyes-triphenyl methane dyes is useful as antiseptics for Bacillus spp. and Staphylococcus spp., and in higher concentration to typhoid bacilli. This group includes malachite green and crystal violet (traditionally used as gentian violet for trench mouth and Candida infections). A second group of dyes, the acridine dyes are good antiseptics for gonococcal and staphylococcal infections. Generally dyes are more valuable for Gram-positive than for Gram-negative forms. They act by combining directly with DNA and halt RNA synthesis.

9. Acids. Some acids are good disinfectants and antiseptics. The common ones are benzoic, salicylic and undecylnic acids for tinea infections of skin. Organic acid as lactic acid and acetic acid are good food preservatives.

Chemotherapeutic Agents

It could become possible only since 1940’s that a successful treatment of fatal disease was achieved. Early efforts of microbiologists towards control of diseases were mainly centered at enhancing the role of immune system. Vaccines for rabies, diphtheria and tetanus were developed. Among the pioneers in this field were Emil von Behring, Elic Metchnikoff, and Paul Ehrlich, all Nobel Prize winners in Physiology or medicine. Ehrlich, is created with the development of the first chemotherapeutic agent, who conceived the antibody molecules as “magic bullets”. In the early 1900’s his attention turned to magic bullets of a purely chemical nature, the chemotherapeutic agents. Ehrlich and his associates had synthesised hundreds of arsenic-phenol derivatives. These were tested by one of his associates, Sahachiro Hata, against the syphilis organism. Later, however, their attention focused on a single chemical, compound 606. This chemical, after trials in animals was made available to physicians under the trade name Salvarsan that soon became the first useful chemotherapeutic durg. The technical or generic name was arsphenamine. Ehrlich’s death in 1915 and the emerging World War eroded the enthusiasm for chemotherapy. Further progress in this area was made only after about 20 years. German chemists developed some industrial dyes, that exhibited antimicrobial qualities also. A red dye, prontosil was synthesised in 1932, that showed activity against some Gram-positive bacteria such as streptococci and staphyococci. In 1935, a French group headed by Jacques and Thevese Trefouel announced that sulfanilamide was the active component of prontosil. Gerhard Domagk was awarded Nobel Prize in 1939 for successful treatment of war-related infections with sulfanilamide.

Following are the common groups of chemotherapeutic agents used in control of infectious microorganisms:

[I] Sulfanilamide and other sulfonamides

Sulfanilamide was the first of a group of chemotherapeutic agents known as sulfonamides. They interfere with metabolism of bacteria through the mechanism known as competitive inhibition. Modern sulfonamides are typified by sulfamethoxazole, prescribed for urinary tract infections due to Gram-negative rods. The drug is combined with trimethoprim, available commercially under the trade name Bactrim. There are two kinds of name for each chemical, generic name and trade name.(Control of Microorganisms Notes Study Material)

Other common sulfonamides are, sulfacetamide, sulfabenzamide and sulfathiazole, commercially available as Triple Sulfa. This is used for vaginal infections due to Haemophilus sp. Another sulfonamide, sulfisoxazole is marketed a cream for vaginal infections.(Control of Microorganisms Notes Study Material)

[II] Other chemotherapeutic agents

There are many other agents, which became common these days. They are not related to sulfonamides. Some common ones include isonicotinic acid hydrazide, effective for tuberculosis: trimethoprim used in urinary tract infections; nalidixic acid for some Gram-negative bacteria of urinary tract infections and nitrofurantoin, also used for such infections.(Control of Microorganisms Notes Study Material)

Metronidazole (Flagyl) is effective against Trichomonas vaginalis infections and amoebiasis. However, there is evidence that it causes tumors in mice Primaquine destroys the malarial parasite. Dapsone is used in treatment of leprosy.

[III] Antibiotics

Alexander Fleming was a student of Almroth Wright, the British investigator who described opsonins. Fleming described the nonspecific enzyme. lysozyme. In 1928, he observed that a Petri dish culture of staphylococci had become contaminated with a green mold, and that the bacteria were disappearing as the mold grew over the plate. The mold isolated, was identified as Penicillium, and found that the broth contained an active principle with antibacterial characteristics. Although, he failed to isolate the substance, he called it penicillin. He recognised the mold’s potential for the treatment of human diseases, trying filtrates on infected wounds. Rene Dubos in 1939 indicated that soil bacteria could produce antibacterial chemicals like Fleming’s penicillin. A group at Oxford University, led by the British pathologist, Howard Florey and the German biochemist, Ernst Boris Chain, reisolated penicillin and carried out careful trials with highly purified samples. In 1940, their successful attempts were published. American pharmaceutical companies developed technology for large-scale production of penicillin. Fleming, Florey and Chain received Nobel prize in 1945 for the discovery and development of penicillin. Since this was a naturally occurring product, a term antibiotic was introduced in medicine.

Following are some of the most commonly used antibiotics:

1. Penicillin. A large group of penicillin derivatives are available. Penicillin G, or benzylpenicillin is the most popular of the penicillins. Other types are penicillin F or penicillin V. All have the basic same structure with a beta-lactam nucleus and several attached groups as shown in Figure.

Penicillins are active against a variety of Gram-positive bacteria, including staphylococci and streptococci. Penicillin functions during the synthesis bacterial cell wall. The molecule blocks the cross-linking of hexoses in the peptidogylycan layer during wall formation, causing bursting of cell. There are, however, two major drawbacks to the use of penicillin. One, the anaphylactic reaction in those who are allergic to penicillin. It causes swelling about the eyes or wrists, itchy skin etc. Second, the evolution of some penicillin-resistant bacteria that produce an enzyme, penicillinase. This converts penicillin into harmless penicilloic acid. Modern penicillins are mostly produced from Penicillium notatum and P. chrysogenum.(Control of Microorganisms Notes Study Material)

2. Semisynthetic penicillins. In the late 1950s, the betalactum nucleus of the penicillin molecule was identified and synthesised. Various groups then could be attached to this nucleus, creating a number of new penicillins. At present thousands of penicillins are prepared by this semi-synthetic process. Ampicillin is less effective than Penicillin G against Gram-positive cocci, but valuable against some Gram-negative rods. It can be taken orally and absorbed from the intestine. Amoxicillin is a similar penicillin. Both are useful to treat urinary tract infections. Another semisynthetic penicillin, carbenicillin is used for Pseudomonas and Proteus infections of urinary tract. Others are, methicillin, nafcillin and oxacillin, which are resistant to penicillinase.

3. Cephalosporins. They were developed in 1960s. Cephalosporin C was isolated from the blue mold. Cephalosporium. A number of related semisynthetic drugs developed from it are, cephalexin, cephalothin, cefazolin and cephaloridine. Cephalosporins are alternatives to penicillin, and are effective for staphylococcal boils or wounds, streptococci, and bacterial pneumonia, and urinary tract infections by Gram-negative bacteria.

4. Streptomycin (an aminoglycoside). This was discovered by Selman A. Waksman. Several moldlike soil bacteria-actinomycetes produced antimicrobial chemicals. Of these, Streptomyces griseus was most effective producer. The substance produced by it was named Streptomycin by Waksman, Elizabeth Bugie and Albert Shatz. This drug was made available in 1947 and Waksman received Nobel Prize in 1952 in Physiology or medicine. Streptomycin in combination with isoniazid is important for treatment of tuberculosis. Gram-negative infections as plague, brucellosis are also treated.

5. Other aminoglycoside antibiotics. Gentamicin is the first drug to be given for infections by Gram-negative bacteria. It is combined with carbenicillin for Pseudomonas infections, with ampicillin for strepytococcal infections of intestine, and with cephalosporin for staphlococcal disorders. Neomycin is now used as topical antibiotic for eye bacterial conjunctivitis or other Gram-negative infections. Commercially, it is available as neosporin when combined with polymyxin, and as cortisporin when combined with cortisone, bacitracin and polymyxin. The mixtures are useful for a variety of mild skin infections due to Gram-negative or Gram-positive bacteria. All aminoglycosides are derived from the species of Streptomyces.(Control of Microorganisms Notes Study Material)

6. Chloramphenicol. This is the first broad-spectrum antibiotic discovered. It was isolated in 1947 by Ehrlich, Burkholder and Gotlieb. It inhibits a wide variety of Gram-positive and Gram-negative bacteria, as well as several rickettsiae and fungi. Chloramphenicol was originally isolated from the metabolites of Streptomyces venezuelae. However, there are two main disadvantages during its use. In bone marrow it prevents hemaglobin incorporation into the red blood cells—aplastic anemia. Moreover, due to its accumulation in blood of newborn child, it causes a toxic reaction and sudden breakdown of cardiovascular system-gray syndrome.(Control of Microorganisms Notes Study Material)

7. Tetracyclines. They are also a broad-spectrum antibiotics with range of activity similar to chloramphenicol. They include naturally occurring chlorotetracycline and oxytetracycline, isolated from species of Streptomyces. They may be taken orally. Though they have side effects problems, they remain the drugs of choice for most rickettsial and chlamydial diseases. They are used in Gram-negative infections as brucellosis, plague, cholera, for primary atypical pneumonia, as substitutes for penicillin in syphilis, anthrax, gonorrhea, and pneumonia; and therapy of some protozoan infections as amoebiasis.

8. Other antibiotics. Erythromycin, obtained from Streptomyces is useful for primary atypical pneumonia, staphylococcal and streptococcal infections and syphilis. Vancomycin, also a product of Streptomyces is given intravenously against Gram-positive infections. Other antibiotics are rifampin for leprosy and tuberculosis, clindamycin and lincomycin, active against streptococo staphylococci and other Gram-positive organisms.

Bacitracin and polymyxin, obtained from Bacillus species are used topically. The former is used as ointment for staphylococci, and latter for Gram-negative bacilli. Spectinomycin, a product of Streptomyces became popular in late 1970s as substitute for penicillin in case of gonorrhea caused by PPNG. It is given intramuscularly.(Control of Microorganisms Notes Study Material)

9. Antifungal antibiotics. Nystatin, a product of Streptomyces is used as cream or ointment or in suppository form, for infection of oral cavity, vagina or intestine due to Candida albicans. Griseofulvin is used for fungal infections of skin, hair and nails. It is effective against ringworm and eczema. This is a product of Penicillium. For serious systemic fungal infections, amphotericin B is used. This is effective for organisms of histoplasmosis, blastomycosis, cryptococcosis etc.

Mode of Action of Antibacterial Agents

A knowledge of the mode of action of antibiotics should help designing new and better chemotherapeutic agents.(Control of Microorganisms Notes Study Material)

It may be seen that antibacterials act at different sites of the pathogenic bacterium to which they are applied. They may act accordingly as follows:

1. Cell-wall inhibitors. These include two widely used classes of antibiotics, the penicillins and cephalosporins. Both contain a ß- lactam ring. They act on various Gram-positive and Gram-negative rods and cocci, responsible for various diseases. They inhibit the formation of peptide cross-linkages within the peptidoglycan backbone of the cell wall. Other cell-wall inhibitors are vancomycin, bacitracin and cycloserine.(Control of Microorganisms Notes Study Material)

2. Protein synthesis inhibitors. Such antibiotics are streptomycin, gentamicin, neomycin, kanamycin, tobramycin and amikacin. These are called aminoglycoside antibiotics, and used for Gram-negative bacteria. They bind to the 30s ribosomal subunit of the 70s prokaryotic ribosome. In addition to these, a number of other antibiotics inhibit protein synthesis. These are tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin and spectinomycin. Chloramphenicol, unlike others acts primarily by binding to the 50s ribosomal subunit, preventing the binding of tRNA molecules to both the amino-acyl and peptidyl binding sites of the ribosome. Erythromycin also binds to 50s ribosomal subunits.(Control of Microorganisms Notes Study Material)

3. Membrane transport inhibitors. The polymyxins, such as polymyxin B changes structure of cell membrane and causes leakage of cell contents of Gram-negative bacteria. Pseudomonas spp and other Gram-negative bacteria, resistant to penicillins and aminoglycoside antibiotics are controlled by polymyxins B and E.(Control of Microorganisms Notes Study Material)

4. DNA inhibitors. They block DNA replication. In particular quinolones interfere with DNA gyrase, preventing the establishment of replication fork. The quinolones include nalidixic acid, ciprofloxicin, norfloxicin, amifloxicin and enoxicin, effective against a range of Gram-positive and Gram-negative bacteria.

5. Other sites of inhibition. Sulphonamides, sulphones and para-aminosalicylic acid are structural analogues of the vitamin para-aminobenzoic acid. These analogues are effective competitors with the natural substrate for the enzymes involved in synthesis of folic acid, as such, and thus inhibit the formation of this required co-enzyme (folic acid) causing a bacteriostatic effect. Trimethoprim is an inhibitor of dihydropholate reductase. DihyTrofolic acid is a co-enzyme required for 1-carbon transfers, as those required in synthesis of thymidine and purines. This antibiotic is broad-spectrum one used to treat bacterial infections of urinary and intestinal tracts.(Control of Microorganisms Notes Study Material)

Antibiotic Sensitivity Assays

These assays are used to study the inhibition of a test organism by one or more antibiotics or other chemotherapeutic agents. Two general methods are commonly used:

[I] Tube dilution method

This is often used to determine the smallest amount of antibiotic necessary inhibit a test organism. This amount is known as the minimum inhibitory oncentration (MIC). A set of tubes with different concentrations of a particular antibiotic are prepared. The tubes are inoculated with the test organism, incuhated, and examined for growth of bacteria. Growth is seen to diminish as the concentration of antibiotic increases, and eventually an antibiotic concentration may be observed at which growth fails to occur. This is the MIC.

[II] Agar diffusion method

The principle used here is that antibiotic will diffuse from a paper disc or small cylinder into an agar medium that contains test organisms. Inhibition is observed as a failure of the organism to grow in the region of the antibiotic. A common application of this method is the Kirby-Bauer test, developed in the 1960s. The procedure is used to determine the sensitivity of an organism isolated from a patient to a series of antibiotics. The results serve a guide to physician to prescribe a drug.(Control of Microorganisms Notes Study Material)

An agar medium such as Mueller-Hinton medium is inoculated with the organism and poured to the plate. Paper discs containing known concentrations of antibiotics are applied to the surface, and the plate is incubated. The appearance of a zone of inhibition surrounding the disc is indicative of sensitivity. By comparing the diameter of the zones to a standard table, one may determine if the test organism is susceptible, or resistant to the antibiotic. If the organism is susceptible, it is likely to be killed in the blood stream of the patient if that concentration of the drug is reached. Resistance indicates that the antibiotic will u be effective at that concentration in the blood stream.

BSc Microbiology Control of Microorganisms Notes Study Material

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