MICROBIAL GROWTH REQUIREMENTS Microorganisms must have the proper

MICROBIAL GROWTH REQUIREMENTS; Microorganisms must have the proper PHYSICAL and CHEMICAL conditions for growth.

PHYSICAL REQUIREMENTS Temperature II. p. H III. Osmosis I.

I. Temperature; (3 groups of microorganisms based on temp) 1. Psychorophiles; usually grow at temp between 0 and 20 degrees Celsius. 2. Mesophiles; usually grow at temperatures between 20 and 40 degrees Celsius. p Psychrotrophs are mesophilic organisms that can grow in the psychrophilic range. p These organisms are responsible for food spoilage. p

p Mesophiles include most of the common spoilage and disease organisms. Ex. S. aureus grows at 5 degrees C. in refrigerated food and produce toxins. p Proteus vulgairis causes eggs to spoil. p Human pathogens are mesophiles 3. Thermophiles: capable of growth at high temperatures, 40 to 90 degrees C.

p p p Extreme thermophiles have optimal growth at 80 degrees or above. The known record for bacterial growth at high temperatures is about 110 degrees C near deepocean hydrothermal vents. Optimal growth temperature; temperature at which the species grows best. Most mesophilic organisms have an optimal growth temperature of approx. 37 degrees C. This means that most human pathogens are mesophilic organisms.

II. PH: refers to the acidity or alkalinity of a solution. ' p Most bacteria grow best at a p. H range near neutrality, between p. H 6. 5 and 7. 5 ( neutrophilic). p Few bacteria grow at a p. H below 4. p Acidophiles grow at the low p. H and have been found in acid having a p. H of 1. p Alkalinity also inhibits microbial growth but is rarely used to preserve foods. p Acid tolerant bacteria are valuable for the food and dairy industries.

Genera of Lactobacillus and Streptococcus produce acid to convert p milk to buttermilk, p cream to sour cream and milk curds to cheese. p Acid in stomach deters disease in organisms. p Acid foods are rarely contaminated p

III. Osmotic pressure; p Osmosis; the movement of water from an area of greater concentration of water to an area of lower concentration of water across a semi permeable membrane. p Hypertonic solution; solution containing more solutes and less water than the cells suspended in it. Hypotonic solution; solution containing less solutes and more water than the cells suspended in it. Isotonic solution; solution containing the same concentration of water as the cells suspended in it. p p

p When microbial cells are in a hypertonic solution, the water passes out of the cell through the cell membrane, and the membrane pulls away from the cell wall. p This inhibits the growth of the microorganism. Therefore the addition of salts (or other solutes) to a solution, and the resulting increase in osmotic pressure, can be used to reserve foods. Salted fish, honey, and sweetened condensed milk are preserved largely by this mechanism: the high salt or sugar cone. Draws water out of any microbial cells that are present and thus prevent their growth. p p p

p Extreme halophiles; bacteria that have adapted to high salt cone, and require them for growth. Ex. Bacteria from dead sea require nearly 30% salt. p p Facultative halophiles: do not require high salt cone. But are able to grow at salt cone, up to 2%, a cone. That inhibits the growth of many other bacteria. Most media used to grow bacteria is nearly all water. Ex agar is usually only 1. 5% salt.

CHEMICAL REQUIREMENTS; 1. p 2. p 3. p 4. p oxygen carbon nitrogen nutrients

1. Oxygen: a. aerobic; organisms that use oxygen to produce energy. b. Obligate aerobes require oxygen to live. Facultative anaerobes; use oxygen if present, but can growing its absence. c. d. Anaerobic; organism that do not use oxygen for energy. e. Obligate anaerobes; are unable to use molecular oxygen for growth and are usually harmed by it. They are unable to produce the enzyme superoxide dismutases (SOD) to neutralize superoxide free radicals

a. b. c. aerotolerant; organisms that cannot use oxygen for growth but they tolerate it fairly well. They produce the enzyme superoxide dismutase. Microaerophilic; organism that require oxygen concentrations at lower levels than those found in air. Capnophilic; organisms that have environment rich in carbon dioxide.

2. Carbon; Virtually all chemical substances in microorganisms contain carbon in some form. p About 50% bacteria dry weight is carbon. p Both chemoautotrophic and photoautotropic microorganisms obtain their energy and produce their nutrients from simple inorganic compounds. Ex carbon dioxide p

3. Nitrogen Used in synthesis of proteins, amino acids, DNA, RNA. p Bacteria that obtain nitrogen directly form atmosphere are called nitrogen fixing bacteria. Ex Rhizobium, Azotobacteria, both found in soil. p

4. p p p Nutrients: Organisms need: a. water: solvent b. vitamins and minerals: coenzymes c. carbohydrates: energy source d. proteins: building blocks e. lipids: building blocks p Heterotrophes: (other feeders) organisms that obtain food from preformed organic carbon compounds. Ex. All animals, most bacteria, and all pathogenic bacteria. p Autotrophes; (self feeder) organisms that synthesize food from inorganic compounds.

Organisms synthesize food through 2 processes: p Photosynthesis: organism takes carbon dioxide from the air and water from the environment and in the presence of energy from the sun converts the carbon dioxide and water into glucose and oxygen. EX green plants and preen and purple sulfur bacteria. p Chemosythesis: makes energy for ATP production through chemical reactions taking place within the organism. p

Culture media p p p p p a nutrient material prepared for the growth of microorganisms in the laboratory. Criteria a culture media must meet. must contain the appropriate nutrients. must contain sufficient moisture. must contain a properly adjusted p. H. must contain suitable levels of oxygen. media must initially be sterile. Culture media may be a liquid (broth) or a solid (agar). The agar is the solidifying agent and is added to the nutrient broth. Complex media - (natural) not certain of exact components of media. Media whose exact chemical composition varies slightly from batch to batch. (Cheaper)

Chemically Defined Media - nature and quantity of each, component is known. p The exact chemical composition is known. Fastidious organisms require many growth factors and do not grow well without enriched media. p Ex. Streptococcus - whole blood is added to the agar. p

Selective media - inhibits the growth of unwanted bacteria, and encourages the growth of desired bacteria. p Ex EMB : Eosin Methylene Blue agar contains carbohydrates fermented by gram negative bacteria most contain eosin and methylene blue that inhibits gram positive organisms. p

Differential media p p Differential media - media in which colonies of different bacteria can be distinguished. Pure cultures of microorganisms have identifiable reactions with differential media. Ex. a. Blood agar is used to identify bacterial species that destroy red blood cells. Manitol salt agar - high salt concentration inhibits most bacteria" and. The manitol may be fermented to select for the growth

CULTURE MEDIA: p Pure culture: if only one type of organism is present. p p p Mixed culture : if 2 or more types of organisms are present. Many media for microorganisms are complex reflecting the growth requirements of the microorganism: p Ex Rickettsia and Virus; must use living cells as their media p Anaerobic microorganism: atmosphere must be free from oxygen ( placed w/in container where CO 2 and H 2 are present) To preserve microbial cultures: place them in a refrigerator which will slow down the metabolic reactions of the culture. p

GROWTH OF BACTERIAL CULTURES p During their growth cycles, microorganisms undergo reproduction many times, causing the number in the population to increase.

REASON FOR BACTERIAL DEATH: 1. 2. 3. Unfavorable environmental conditions. Accumulation of waste. Lack of nutrients.

Stages of bacteria growth ( in numbers) 1. 2. p p p lag phase - population remains temporarily. unchanged at the beginning. . Lag phase usually last for a few hours and would appear as a straight horizontal line on a graph. logarithmic (exponential) phase - Organism. . is expressing maximum reproductive potential. There is a logarithmic (exponential) progression of number is the population. During this time growth is the highest and death is the lowest. at this time. Symptoms develop in humans and colonies appear on the agar medium during the log phase. This phase may last for hours or days.

3. 4. stationary phase - living count remains constant at the maximum value. Living count levels off and would appear as a straight horizontal line on a graph. The count remains constant because the same number of cells are dying as dividing. This is due to the depletion of nutrients and the accumulation of waste products. Spore-forming bacteria will form spores during the stationary phase. Death phase - The living count decreases. Death exceeds reproduction. Cells weaken, change into odd shapes, undergo lysis and die.

The environment exerts LIMITING FACTORS p p It has been calculated that if E. coli grew unchecked for 36 hours, there would be enough bacteria to cover the face of the earth. This can not happen due to limiting environmental factors. In the lab a population of bacteria can be kept in the log phase of growth indefinitely by using an instrument called a chemostat. This instrument continuously drains off spend media and adds fresh media.

Plate count method; Sample bacteria diluted, placed in petri dishand set aside to incubate. p Generally, plates with 30 to 300 colonies are selected for determining final count, which is expressed as number of bacteria per original ml of sample. p Most probable number; used to get number of bacteria in contaminated water. p

ISOLATION OF BACTERIA; • "To obtain separated colonies from mixed culture

WAYS TO ISOLATE BACTERIA 1. Streak plate; sample of mixed bacteria is streaked several times along one edge of petri dish containing medium. 2. Pour plate method: a sample of bacteria is diluted in several tubes of melted medium. After dilution, the melted agar is poured into separate petri dishes and allowed to harden, since bacteria have been diluted in various tubes, the plates contain various dilution of bacteria and where the bacteria are most diluted, they form isolated colonies. p

3. Dry weight; liquid culture is dried out and the amount of microbial mass is weighed. 4. Turbidity method; assess bacteria growth. As bacteria multiply in liquid media, they make media cloudy. p Placing culture tube in a beam of light and noting the amount of light transmitted gives idea of Turbidity of culture.

CONTROL OF MICROBIAL GROWTH; Sterilization; destruction of all forms of microbial life. p Disaffection; destruction of pathogenic organism on inanimate objects. p Antisepsis; destruction of living object such as skin surface. p Sanitization; reduction in number of pathogens to level deemed safe for public health guidelines. p

p Degerming; physical removal of microorganisms by using soap ect. . . p Germicide; any chemical agent that kills microorganism ( condition affecting germicide include: 1. Type of microorganism 2. Environment) p Bactericide; kills bacterial p Viricide: kill virus

PHYSICAL METHOD TO CONTROL MICROBIAL GROWTH; Divided into : heat method and nonheat method p Thermal death point; lowest temp at which microorganism are killed in 10 min, . p Thermal death time; The minimal amount of time needed to kill microorganism. p Decimal reduction; time for the destruction of 90% of microbe population. p

HEAT METHODS p p Dry heat; kill microorganisms by reacting with oxidizing their protein. Ex Bunsen burner Moist heat; ex boiling water. Usually denature microbe proteins ex autoclave Pasteurization; used to lower bacterial content of milk and dairy products, it doesn't achieve sterilization. It is set up to eliminate Tuberculosis bacillus and rickettsia. . . pasteurize for 30 min at 62 C for 15 to 27 sections at 72 C

NON HEAT METHODS; p p filtration drying; when water is removed from cell it shrivels and dies cold temperature; decrease microbial metabolism. Radiation; gamma rays, x-rays

CHEMICAL METHOD: p p p p Most reduce microbial population to safe level or remove pathogen from object. Among important criteria for selecting antiseptic/disinfectant: Concentration of disinfectant whether agent is bactericidal or bacteriostatic nature of material to be treated (organic or not) temperature and p. H that chemical will be used at. time chemical agent will be left in contact with surface

p Evaluation method; various dilutions of chemical agent are prepared and tested against equivalent dilution of phenol with such bacteria as Staphylococcus aures and Salmonella typhi

Phenol; one of first chemicals used for disinfecting (1860 Lister) It damages cell membrane and inactivates enzymes of microorganisms. Ex Lysol ( against Staphylococcus) 2. Halogens: ex Iodine ( betadine), cholorine (bleach), ammonia 3. Alcohol: No effect on bacterial spores. Ex Isopropyl (rubbing) alcohol evaporates quickly and leaves no residue, degerms skin injections. 4. Heavy metals; ex silver nitrate: eyes of newborns to guard against Neisseria gonorrhea and to cauterize wounds, Copper sulfate: retard algae growth in pools, Zinc Choride: mouth wash, Zinc oxide: antifungal agent in paint 1.

5. 6. 7. Soap and detergents; degerming Aldehydes: ex glutaraldehyde: used to sterilize hospital equip. Oxidizing agents; Ex hydrogen peroxide: kills microorganism by releasing large amount of oxygen which contributes to alteration of microbial enzyme.

ANTIBIOTICS; The products of microorganisms that react with and inhibit growth of other microorganisms.

1. p 2. 3. Penicillin; prevents gram pos bacteria from forming peptidoglycan (main component of cell wall) If person is allergic the rxn may be localized or whole body rxn ( anaphylaxis) Cephalosporin; first produced by the mold Cephalosprium and is useful against gram pos bacteria and penicillin resistant bacteria. Aminoglycoside; inhibit protein synthesis in gram neg bacteria ex Streptomycin (fights tuberculosis) many have debilitating side effects.

4. p Tetracycline; broad spectrum of drugs that inhibit gram neg bacteria growth, rickettsia, chlamydia, some gram pos by inhibiting protein synthesis. One side effect is to inhibit calcium deposit in body so shouldn't be used in young children. 5. Other antibacterial antibiotics include; a. Ervthromvcin; sub in for penicillin when allergy exists. Useful against gram pos bacteria by inhibiting protein synthesis. Bacitracin; treat skin infection caused by gram pos bacteria Vancomycin; Effective against bacteria displaying resistance to penicillin, cephalosporin and other antibiotics, is very expensive and has may side effects, it interferes with cell wall formation in b b. c.

d. p e. Chloroamphericoil: against bacteria, rickettsia, chlamydia. Has serious side effects such as aplastic anemia (blood cells without hemoglobin), gray syndrome (cardiovascular collapse)in babies, is used for most serious bacterial infections ex typhoid fever and meningitis sulfa drugs: interfere with folic acid production by bacteria. Ex sufamethoxazole, sulfisoxazole against gram pos bacteria.

6. 7. 8. Antifungal drugs: ex griseofulvin ( ringworms, athletes foot) Antiviral drugs; not widely used, virus have few functions or structures with which drug can interfere. Ex Azidothymidine (AZT), Acyclovir (herpes), interferon (cancer) Antiprotozoal drugs: many of the same sued against bacteria work here ex tetracycline flagyl ( against trichomonas vaginalis), quinine (malaria)

p p p COMMON PRESCRIPTION ABBREVIATIONS Ever wonder what those abbreviations on your prescription mean? They tell the phar macist how often or when during the day you need to take a drug. Here a few of the most common ones: bid—twice a day qd—once a day hs—at bedtime qid—four times a day pe—after meals fid—three times a day prn—as needed