MEDICAL MICROBIOLOGY (MMIC 201)

Prepared by

Nabeel Al-Mawajdeh RN,MSC.

King Saud University-Aflaj College

CHAPTER 6 Controlling the Growth of Microorganisms

Nabeel Al-Mawajdeh RN.MCS

CONTROLLING MICROBIAL GROWTH IN VITRO

Nabeel Al-Mawajdeh RN.MCS

Factors Affecting Microbial Growth

1. Availability of Nutrients: Many nutrients are energy sources; organisms will obtain energy from these chemicals by breaking chemical bonds. Nutrients also serve as sources of carbon, oxygen, hydrogen, nitrogen, phosphorus, and sulfur as well as other elements (e.g., Na+, Cl-, Ca++, Mg++etc.) that are required in lesser amount. About 25 of the 92 naturally occurring elements are essential to life.

Nabeel Al-Mawajdeh RN.MCS

Factors Affecting Microbial Growth (Contd)

2. Moisture: All living organisms require water to carry out their normal metabolic processes, and most will die in environments containing too little moisture. Temperature: Every microorganism has an optimum growth temperature the temperature at which the organism grows best.

3.

Nabeel Al-Mawajdeh RN.MCS

Factors Affecting Microbial Growth (Contd)

a. b. c. d. Thermophiles: are microorganisms that prefer to live in hot temperatures. Mesophiles: prefer moderate temperature. Psychrophiles: prefer cold temperature. Psychrotrophs: are a group of Psychrophiles that prefer to live at refrigerator temperature (4C).

Nabeel Al-Mawajdeh RN.MCS

Factors Affecting Microbial Growth (Contd)

4. PH: The term ''PH'' refers to the acidity or alkalinity of a solution. Most microorganisms prefer a neutral or slightly alkaline growth medium (PH 7.0 to 7.4) a. Acidophiles: are microbes preferred to live in acidic environments. b. Alkaliphiles: live in alkaline environments. - Fungi prefer acidic environments.

Nabeel Al-Mawajdeh RN.MCS

Factors Affecting Microbial Growth (Contd)

5. 6. 7. Osmotic Pressure: Is the pressure that is exerted on a cell membrane by solutions both inside and outside the cell. Barometric Pressure: Barophiles live where there is high barometric pressure. Gaseous Atmosphere: Some microbes (obligate aerobes) prefer the same atmosphere that humans do (e.g., about 20% to 21% O2 and 78% N2) Obligate anaerobic are killed by the presence of oxygen. Some microbes require increased concentration of CO2.
Nabeel Al-Mawajdeh RN.MCS

Encouraging The Growth Of Microorganisms In Vitro

The growth of microorganisms is encouraged in microbiology laboratories, research laboratories, and in various industries (e.g., antibiotic industry and certain food, beverage, and chemical industries). To culture microorganisms in the laboratory, they must be provided with appropriate nutrients, moisture, and the temperature, PH, and atmosphere that they require.

Nabeel Al-Mawajdeh RN.MCS

INHIBITING TH GROWTH OF MICROORGANISMS IN VITRO

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A. Using Physical Methods

The physical methods commonly used in hospitals, clinics, and laboratories to destroy or control pathogens. 1. Heat: Is the most practical, efficient, and inexpensive method of sterilization of those inanimate objects and materials that can withstand high temperatures. Two factors, temperature and time, determine the effectiveness of heat for sterilization. Sterilization is the process whereby all viable microbes including spores are removed or killed.

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A. Using Physical Methods (contd)

a. Dry: At 160 to 165 C for 2 hours or at 170 to 180C for 1 hour. - Red heat and incineration (burning): Direct exposure of material to flame till it becomes red. E.g., culture loops. - Direct flaming: Passing material over flame many times without reaching redness. E.g., flaming mouths of bottles, slides, flasks and culture tubes.

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A. Using Physical Methods (Contd)

- Hot air oven: Supplied with a fan from inside to distribute hot air in all chamber. It has temperature thermostat. It is used widely in hospitals, clinics, and laboratories. E.g., test tubes, glass pipettes, scissors, blades. b. Moist: Heat applied in the presence of moisture, as in boiling or steaming, is safer and more effective than dry heat, and can be accomplished at a lower temperature for 30 min.; thus, it is less destructive to many materials. Moist heat causes proteins to coagulate (as occurs when eggs are hard boiled)
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A. Using Physical Methods (Contd)

- Boiling and steaming: At 100 C. clean articles made of metal and glass, such as syringes, needles may be disinfected by boiling for 30 minutes. Boiling is not always effective because heat- resistance bacterial endospores, mycobacteria and viruses may be present. - Autoclaving (steam under pressure): An autoclave is like a large metal pressure cooker that uses steam under pressure to completely destroy all microbial life.

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A. Using Physical Methods (Contd)

E.g., surgical instruments and dressing, towels, peptone water broth and agar, aqueous injection...etc. Is the best and effective sterilization technique, at a pressure of 15 pounds/square inch, at temperature of 121 C for 20 minutes kills vegetative microorganisms, bacterial endospores, and viruses.

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A. Using Physical Methods (Contd)

2. Cold: Most microbes are not killed by cold temperatures and freezing, but their metabolic activities are slowed, greatly inhibiting their growth. 3. Radiation: a. Nonionizing radiation: - ultraviolet (UV) rays (has low degree of penetration): A ultraviolet lamp (often called a germicidal lamp) is useful for reducing the number of microorganisms in the air and on surfaces.

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A. Using Physical Methods (Contd)

They do, however, penetrate cell and, thus, can cause damage to DNA. When this occurs, genes may be so severely damaged that the cell dies. Its main component is a low- pressure mercury vapor tube. Such lamps are found in newborn nurseries, operating roomsetc. Many biologic materials, such as toxins, and vaccines, are sterilized with UV rays.

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A. Using Physical Methods (Contd)

b. Ionizing radiation: - X-rays and gamma rays (has high degree of penetration): Are used in industry for sterilization of plastic catheters, syringes, surgical equipments, preparation of vaccinesetc. 4. Filtration: Is the passage of a liquid or gas through a filter with pores small enough to allow microbes to pass. This method used for sterilization of serum, hormones, antibiotic solution.

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A. Using Physical Methods (Contd)

Gases (ethylene oxide, propylene oxideetc): Suitable for plastics, hormones, surgical dressing, all antibiotics and thermo labile powder.

5.

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B. Using Chemical Agents

1. Antiseptics: chemicals inhibit the growth or kill microbes on living tissues like human skin and mucus membranes. 2. Disinfectants: chemicals inhibit the growth or kill microbes. - Factors that determine the effectiveness of any disinfectant: a. Time. b. Temperature. c. Concentration. d. Type and number of microbes. e. Presence of spores. f. Presence of proteins in feces, blood, vomitus, pus.
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B. Using Chemical Agents (Contd)

- Characteristics of good disinfectant: a. Rapid action, easy to use. b. Wide range of action. c. Good penetration. d. Capability of mixing with water. e. Activity in organic matter (like blood, feces, vomit)

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B. Using Chemical Agents (Contd)

f. Resistance to decomposition. g. Nonstaining and noncorrosive. h. Odorless. i. Stable in various temperature and light. l. Cheep.

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B. Using Chemical Agents (Contd)

- Kinds of Disinfectants: a. Alcohol: 70% to disinfect skin and thermometer. b. Phenolics: 5% phenol is useful for disinfection of stool, sputum. 0.25% - 0.5% phenol for preservation of sera and vaccines. c. Chlorine: for sterilization of water supplies (1% in million) after the treatment of water by precipitation or filtration for removal of organic matters since they can not act efficiency in the presence of organic matter.

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B. Using Chemical Agents (Contd)

d. Iodine (povidone): for skin disinfection (available as a tincture ,2% iodine with 70% alcohol). e. H2O2 (hydrogene peroxide) 3 6 % for wounds, ulcers, and mouth wash. f. Formaldehyde: for rubber, leather, shoes, books, and blankets.

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USING ANTI MICROBIAL AGENTS TO CONTROL MICROBIAL GROWTH IN VIVO

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Using Chemotherapeutic agents

- Chemotherapeutic agent is any chemical (drug) used to treat an infectious disease, either by inhibiting or killing pathogens in vivo. a. Antifungal agents are used to treat fungal diseases. b. Antiprotozoal agents are used to treat protozoal diseases. c. Antiviral agents are used to treat viral diseases.

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Using Chemotherapeutic agents (Contd)

d. Antibiotics are substances produced by microorganisms (usually a soil organism) that effective in killing or inhibiting the growth of other microorganisms. Some antibiotics (e.g., penicillin and cephalosporin) are produced by molds, whereas others (e.g., tetracycline, erythromycin, and chloramphenicol) are produced by bacteria. Many antibiotics have been chemically modified to kill a wider variety of pathogens or reduce side effects; these modified antibiotics are called semisynthetic antibiotics.
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Ideal Qualities Of Antimicrobial Agents

1. Antimicrobial agents should have selective toxicity for microorganisms. E.g., it can kill or inhibit the growth of a microorganism in concentrations that are not harmful to the cells of the host.
Therapeutic index= large amount of antimicrobial can be given without harm Lowest dose that can kill microorganism

The higher the therapeutic index the better the antimicrobial.

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Ideal Qualities Of Antimicrobial Agents (Contd)

2. 3. 4. 5. 6. Should Kill or inhibit the growth of pathogens. Cause no damage to the host. Be stable when stored in solid or liquid form. Remain in specific tissues in the body long enough to be effective. Kill the pathogens before the mutate and become resistant to it. Unfortunately, most antimicrobial agents have some side effects, produce allergic reactions, or permit development of resistant mutant pathogens.

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Mechanisms Of Action Of Antimicrobial Agents

1. Inhibition of cell wall synthesis. Due to its unique structure and function, the bacterial cell wall is an ideal point of attack by selective toxic agents. Some antibiotics e.g., penicillin, cephalosporins, interfere with cell wall synthesis and cause bacteriolysis. Inhibition of cytoplasmic membrane function. Some antibiotics cause disruption of the membrane and leakage of cellular proteins and nucleotides leading to cell death. E.g., nystatin, polymyxins.

2.

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Mechanisms Of Action Of Antimicrobial Agents (Contd)

3. Inhibition of nucleic acid synthesis or replication (DNA or RNA). e.g., rifampicin, metronidazole, nalidixic. Inhibition of protein synthesis. Many antimicrobial agents block protein synthesis by acting on the 30s or 50s subunits of the bacterial ribosome. E.g., chloramphenicol, tetracycline, erythromycin, gentamycin, streptomycin, lincomycin.

4.

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Mechanisms of Resistance to Antimicrobial Agents

1. The organism produces enzymes that destroy the drug. E.g., production of beta lactamases that destroys penicillin. The organism changes its permeability to the drug, by modification of protein in the outer cell membranes, thus impairing its active transport into the cell e.g., resistance to polymyxins. The organism develops an altered receptor site for the drug e.g., resistance to aminoglycosides is associated with alteration of a specific protein in the 30s subunit of the bacterial ribosome that serves as a binding site in susceptible organisms.
Nabeel Al-Mawajdeh RN.MCS

2.

3.

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Mechanisms of Resistance to Antimicrobial Agents (Contd)

4. The organism develops an altered metabolic pathway that bypasses the reaction inhibited by the drug e.g., sulphonamide- resistant bacteria acquire the ability to use performed folic acid with no need for extracellular PABA (p- aminobenzoic acid)

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Antiviral Drugs
1. Amantadine hydrochloride: it inhibits the penetration of the virus into the susceptible cells. It is used usually in treatment of influenza A virus infection. Idoxuridine: it inhibits the replication of DNA viruses. It is severely cytotoxic, used topically for treatment of herpes simplex infection. Methisazone: it blocks replication of poxviruses. Interferon: used systemic and topical as a ''broad spectrum'' antiviral agent.

2.

3. 4.

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Clinical Use of Antibiotics

1. 2.
3. 4.

Antibiotics should not be given for trivial infections. Treatment should be based on a clear clinical and bacteriological diagnosis. According to results of antibiotic sensitivity testing in vitro. Antibiotics for systemic treatment should be given in full therapeutic doses for adequate period. Combined therapy with two or more antibiotics is required in some conditions. E.g,
a. Serious resistant infections e.g., infective endocarditis or meningitis. b. In treatment of tuberculosis 2 or 3 drugs are given by lowering the dose of each to decrease toxic effects of drugs. c. Severe mixed infections e.g., peritonitis following perforation of the colon.

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Complications of Antibacterial Chemotherapy

1. Development of drug resistance. This can be due to inadequate dosage, prolonged treatment and the abuse of antibiotics without in vitro susceptibility testing. 2. Drug toxicity. This can be due to overdosage, or prolonged. E.g., tetracyclines inhibit growth and development of bones and teeth in the developing fetus and infant, chloramphenicol may cause depression of the bone marrow, aspirin lead to deafness. Voltarin may cause renal failure. 3. Allergy. May occur if sensitivity test was not done before administering an antibiotic. 4. Superinfection. This may lead destruction of human indigenous microflora of the mouth, vagina, and intestine
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The End

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