Environmental Microbiology -Laboratory Manual- prepared for Environmental Microbiology III...

Environmental Microbiology -Laboratory Manual- prepared for Environmental Microbiology III Microorganism Growth Factors Controlling Growth [email protected] ENVIRONMENTAL MANAGEMENT TECHNOLOGY FACULTY OF CIVIL AND ENVIRONMENTAL ENGINEERING ITB, 2010

The capacity to grow, and ultimately to multiply, is one of the most fundamental characteristics of living cells (Posten & Coney).

GROWTH In biophysics cells are open systems far from a thermodynamic equilibrium, exchange material and energy with their environment, and especially exhibit a large outflow of entropy. In chemical engineering growth is referred to as an increasing amount of biocatalyst Mathematical growth are restricted to a couple of equations employing hyperbolic and exponential terms. Growth is usually considered as an increase of cell material expressed in terms of mass or cell number. Ref: Posten & Coney

Microbial Nutrition Why is nutrition important? The hundreds of chemical compounds present inside a living cell are formed from nutrients. This slide is taken from : MICR 300 : Microbiology, California State of University Macronutrients : elements required in fairly large amounts Micronutrients : metals and organic compounds needed in very small amounts

Main Macronutrients Carbon (C, 50% of dry weight) and nitrogen (N, 12% of dry weight) Autotrophs are able to build all of their cellular organic molecules from carbon dioxide Nitrogen mainly incorporated in proteins, nucleic acids Most Bacteria can use NH 3 and many can also use NO 3 - Nitrogen fixers can utilize atmospheric nitrogen (N 2 ) This slide is taken from : MICR 300 : Microbiology, California State of University

Other Macronutrients Phosphate (P), sulfur (S), potassium (K), magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe) Iron plays a major role in cellular respiration, being a key component of cytochromes and iron-sulfur proteins involved in electron transport. Siderophores : Iron-binding agents that cells produce to obtain iron from various insoluble minerals. This slide is taken from : MICR 300 : Microbiology, California State of University

Representative Siderophore Ferric enterobactin Aquachelin This slide is taken from : MICR 300 : Microbiology, California State of University

Micronutrients Need very little amount but critical to cell function. Often used as enzyme cofactors This slide is taken from : MICR 300 : Microbiology, California State of University

Growth factors This slide is taken from : MICR 300 : Microbiology, California State of University

Culture Media: Composition Culture media supply the nutritional needs of microorganisms defined medium : precise amounts of highly purified chemicals complex medium(or undefined) : highly nutritious substances. Inclinical microbilogy, Selective : contains compunds that selectively inhibit Differential: contains indicator terms that describe media used for the isolation of particular species or for comparative studies of microorganisms. This slide is taken from : MICR 300 : Microbiology, California State of University

Media Composition Natural Semi- synthetic Synthetic Use GeneralSelectiveDifferentialEnriched

Culture Media: Physical Properties Liquid Bouillon or broth Solid Addition of a gelling agent (typically 1% agar) to liquid media Immobilize cells, allowing them to grow and form visible, isolated masses called colonies (Figure 5.2). Semisolid Reduced amount of agar added Allows motile microorganism to spread This slide is taken from : MICR 300 : Microbiology, California State of University

Bacterial Colonies on Solid Media S. marcescens (Mac) P. aeruginosa (TSA) S. flexneri (Mac) This slide is taken from : MICR 300 : Microbiology, California State of University

Laboratory Culture of Microorganisms Microorganisms can be grown in the laboratory in culture media containing the nutrients they require. Successful cultivation and maintenance of pure cultures of microorganisms can be done only if aseptic technique is practiced to prevent contamination by other microorganisms. This slide is taken from : MICR 300 : Microbiology, California State of University

Microbial Growth Binary fission This slide is taken from : MICR 300 : Microbiology, California State of University

Microbial growth involves an increase in the number of cells. Growth of most microorganisms occurs by the process of binary fission Cell Growth and Binary Fission This slide is taken from : MICR 300 : Microbiology, California State of University

Microbial Growth Peptidoglycan layer This slide is taken from : MICR 300 : Microbiology, California State of University

Microbial populations show a characteristic type of growth pattern called exponential growth, which is best seen by plotting the number of cells over time on a semi- logarithmic graph. Microbial Growth Pattern This slide is taken from : MICR 300 : Microbiology, California State of University

Growth Curve Microorganisms show a characteristic growth pattern (Figure 6.8) when inoculated into a fresh culture medium. This slide is taken from : MICR 300 : Microbiology, California State of University

Measuring Microbial Growth Growth is measured by the change in the number of cells over time. Cell counts done microscopically measure the total number of cells in a population whereas viable cell counts (plate counts) measure only the living, reproducing population. This slide is taken from : MICR 300 : Microbiology, California State of University

Total Cell Count

Viable Cell Count : Determination of Colony Forming Units

Serial Dilution of Cells This slide is taken from : MICR 300 : Microbiology, California State of University

Indirect Cell Number Measurement : Turbidity Turbidity measurements are an indirect but very rapid and useful method of measuring microbial growth (Figure 6.12). However, to relate a direct cell count to a turbidity value, a standard curve must first be established. This slide is taken from : MICR 300 : Microbiology, California State of University

Turbidity Measurements of Microbial Growth This slide is taken from : MICR 300 : Microbiology, California State of University

Environmental Effects on Bacterial Growth Temperature pH Osmotic pressure Oxygen classes This slide is taken from : MICR 300 : Microbiology, California State of University

Temperature and Microbial Growth Cardinal temperatures minimum optimum maximum Temperature is a major environmental factor controlling microbial growth. This slide is taken from : MICR 300 : Microbiology, California State of University

Classification of Microorganisms by Temperature Requirements This slide is taken from : MICR 300 : Microbiology, California State of University

Temperature Classes of Organisms Mesophiles Midrange temperature optima Found in warm-blooded animals and in terrestrial and aquatic environments in temperate and tropical latitudes Psychrophiles Cold temperature optima Most extreme representatives inhabit permanently cold environments Thermophiles Growth temperature optima between 45C and 80C Hyperthermophiles Optima greater than 80C These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100C This slide is taken from : MICR 300 : Microbiology, California State of University

Heat-Stable Macromolecules Thermophiles and hyperthermophiles produce heat-stable macromolecules, such as Taq polymerase, which is used to automate the repetitive steps in the polymerase chain reaction (PCR) technique. This slide is taken from : MICR 300 : Microbiology, California State of University

pH and Microbial Growth The acidity or alkalinity of an environment can greatly affect microbial growth. Most organisms grow best between pH 6 and 8, but some organisms have evolved to grow best at low or high pH. The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic. Acidophiles : organisms that grow best at low pH Alkaliphiles : organismsa that grow best at high pH This slide is taken from : MICR 300 : Microbiology, California State of University

Osmotic Effects on Microbial Growth Osmotic pressure depends on the surrounding solute concentration and water availability Water availability is generally expressed in physical terms such as water activity Water activity is the ratio of the vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure of pure water. This slide is taken from : MICR 300 : Microbiology, California State of University

Halophiles and Related Organisms In nature, osmotic effects are of interest mainly in habitats with high salt environments that have reduced water availability Halophiles : have evolved to grow best at reduced water potential, and some (extreme halophiles) even require high levels of salts for growth. Halotolerant : can tolerate some reduction in the water activity of their environment but generally grow best in the absence of the added solute Xerophiles : are able to grow in very dry environments This slide is taken from : MICR 300 : Microbiology, California State of University

Oxygen and Microbial Growth Aerobes : Obligate : require oxygen to grow Facultative : can live with or without oxygen but grow better with oxygen Microaerphiles : require reduced level of oxygen Anaerobes : Aerotolerant anaerobes : can tolerate oxygen but grow better without oxygen. Obligate : do not require oxygen. Obligate anaerobes are killed by oxygen

This slide is taken from : MICR 300 : Microbiology, California State of University

Test for Oxygen Requirements of Microorganisms Thioglycolate broth : contains a reducing agent and provides aerobic and anaerobic conditions a)Aerobic b)Anaerobic c)Facultative d)Microaerophil e)Aerotolerant This slide is taken from : MICR 300 : Microbiology, California State of University

Toxic Forms of Oxygen and Detoxifying EnzymesHydrogenperoxide Superoxide This slide is taken from : MICR 300 : Microbiology, California State of University

Control MethodsPhysical Methods Temperature (heat, cold) RadiationFiltrationGasChemical AgentsAntisepticsDisinfectants Chemotherapeutics (in vivo)

46 Heat 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. Nabeel Al-Mawajdeh RN.MCS

47 Heat (2) 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. - 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) - 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. Nabeel Al-Mawajdeh RN.MCS

48 Cold 2.Cold: Most microbes are not killed by cold temperatures and freezing, but their metabolic activities are slowed, greatly inhibiting their growth. Nabeel Al-Mawajdeh RN.MCS

49 Radiation Radiation: a. Nonionizing radiation: - ultraviolet (UV) rays (has low degree of penetration): A ultraviolet lamp (germicidal lamp) is useful for reducing the number of microorganisms in the air and on surfaces. 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. Many biologic materials, such as toxins, and vaccines, are sterilized with UV rays. 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. Nabeel Al-Mawajdeh RN.MCS

50 Filtration 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. Nabeel Al-Mawajdeh RN.MCS

51 Gases 5.Gases (ethylene oxide, propylene oxideetc): Suitable for plastics, hormones, surgical dressing, all antibiotics and thermo labile powder. Nabeel Al-Mawajdeh RN.MCS

53 Antiseptics 1.Antiseptics: chemicals inhibit the growth or kill microbes on living tissues like human skin and mucus membranes. Nabeel Al-Mawajdeh RN.MCS

54 Disinfectants 1.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. Nabeel Al-Mawajdeh RN.MCS

55 Disinfectants (2) Characteristics of good disinfectant: Rapid action, easy to use. Wide range of action. Good penetration. Capability of mixing with water. Activity in organic matter (like blood, feces, vomit) Resistance to decomposition. Nonstaining and noncorrosive. Odorless. Stable in various temperature and light. Cheep. Nabeel Al-Mawajdeh RN.MCS

56 Disinfectants (3) - 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. 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. Nabeel Al-Mawajdeh RN.MCS

USING ANTI MICROBIAL AGENTS TO CONTROL MICROBIAL GROWTH IN VIVO Nabeel Al-Mawajdeh RN.MCS 57

58 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. d. Antibiotics Nabeel Al-Mawajdeh RN.MCS

59 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. Nabeel Al-Mawajdeh RN.MCS

60 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. Nabeel Al-Mawajdeh RN.MCS

61 Ideal Qualities Of Antimicrobial Agents (Contd) 2.Should Kill or inhibit the growth of pathogens. 3.Cause no damage to the host. 4.Be stable when stored in solid or liquid form. 5.Remain in specific tissues in the body long enough to be effective. 6.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. Nabeel Al-Mawajdeh RN.MCS

62 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. 2.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. Nabeel Al-Mawajdeh RN.MCS

63 3.Inhibition of nucleic acid synthesis or replication (DNA or RNA). e.g., rifampicin, metronidazole, nalidixic. 4.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. Nabeel Al-Mawajdeh RN.MCS Mechanisms Of Action Of Antimicrobial Agents (Contd)

64 Mechanisms of Resistance to Antimicrobial Agents 1.The organism produces enzymes that destroy the drug. E.g., production of beta lactamases that destroys penicillin. 2.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. 3.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

65 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) Nabeel Al-Mawajdeh RN.MCS

66 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. 2.Idoxuridine: it inhibits the replication of DNA viruses. It is severely cytotoxic, used topically for treatment of herpes simplex infection. 3.Methisazone: it blocks replication of poxviruses. 4.Interferon: used systemic and topical as a ''broad spectrum'' antiviral agent. Nabeel Al-Mawajdeh RN.MCS

67 Clinical Use of Antibiotics 1.Antibiotics should not be given for trivial infections. 2.Treatment should be based on a clear clinical and bacteriological diagnosis. According to results of antibiotic sensitivity testing in vitro. 3.Antibiotics for systemic treatment should be given in full therapeutic doses for adequate period. 4.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. Nabeel Al-Mawajdeh RN.MCS

68 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 Nabeel Al-Mawajdeh RN.MCS

a.Effect of temperature on bacteria and yeast cells b.Effect of temperature on vegetative and spore cells of bacteria, yeast, and fungi

8.A. Effect of temperature on bacteria and yeast cells m.o. culture agar plate Streak plate Incubate in 4 C Incubate in 20/25 C Incubate in 37 C Incubate in 44 C broth tube + Durham tube Inoculatio n Incubate in 4 C Incubate in 20/25 C Incubate in 37 C Incubate in 44 C

Durham Tube Durham tubes are used in microbiology to detect production of gas by microorganisms. This small tube is initially filled with the solution in which the microorganism is to be grown. If gas is produced after inoculation and incubation, a visible gas bubble will be trapped inside the small tube.

8.B. Effect of temperature on vegetative and spore cells of bacteria, yeast, and fungi m.o. culture 4 Broth tube Hot water 80 C Cold water Streak in 4 glucose broth Room tempera ture 4 days Gram & spore staining

Exp.9 - pH m.o. culture Nutrition broth pH 3 Inoculate E. coli 24-48 jam incubate 37 C Nutrition broth pH 5 Inoculate Alcaligenes faecalis 24-48 jam incubate 37 C Nutrition broth pH 7 Inoculate S.aureus 24-48 jam incubate 37 C Nutrition broth pH 9 Inoculate S.cereviceae room temp. 48- 72 hours

spectrophotometer

Exp.10 - Oxygen Requirements m.o. culture divided agar plate Streak plate 24-48 hours incubate 37 C divided agar plate Streak plateanaerob jar 24-48 hours incubate 37 C

m.o. culture slant agar NaCl 0,5 % Inoculate 24-48 hours incubate 37 C slant agar NaCl 5 % Inoculate 24-48 hours incubate 37 C slant agar NaCl 10 % Inoculate 24-48 hours incubate 37 C slant agar NaCl 15 % inoculate 24-48 hours incubate 37 C Exp.11 Osmotic Pressure

Exp.12 Chemotherapeutic Agent m.o. culture agar plate, incubate to drain swab Press immersed bloaters in plate 24-48 hours incubate 37 C Using tweezers, immerse bloaters in penicillin Using tweezers, immerse bloaters in tetracycline Using tweezers, immerse bloaters in streptomycin A B C D

a.Alcohol effectivity evaluation b.Antiseptic evaluation using blotter c.Effectivity of handwashing using soap

13.A. Alcohol effectivity evaluation A = left thumb without alcohol B = left thumb without alcohol C = right thumb without alcohol D = right thumb with alcohol Incubate the dish A B C D

13.B. Antiseptic evaluation using blotter m.o. culture agar plate, incubate to drain swab Press immersed bloaters in plate 24-48 hours incubate 37 C Using tweezers, immerse bloaters in alcohol

13.C. Effectivity of hand washing using soap Washed HandUnwashed Hand

Environmental Microbiology -Laboratory Manual- prepared for Environmental Microbiology III...

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