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GROWTH, CONTROL, CHEMOTHERAPY

1. GROWTH, CONTROL, CHEMOTHERAPY. 2. GROWTH, CONTROL, CHEMOTHERAPY GROWTH - Binary Fission, Growth Rate, Generation Time, N = N o 2 n Growth Phases of Culture - Lag, Exponential, Stationary, Death Physical Factors- Temperature: psychrophile, mesophile, thermophile, hyperthermophile

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GROWTH, CONTROL, CHEMOTHERAPY

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  1. 1 GROWTH, CONTROL, CHEMOTHERAPY

  2. 2 GROWTH, CONTROL, CHEMOTHERAPY GROWTH - Binary Fission, Growth Rate, Generation Time, N = No2n Growth Phases of Culture - Lag, Exponential, Stationary, Death Physical Factors- Temperature: psychrophile, mesophile, thermophile, hyperthermophile pH, Buffer, acidophile, alkalophile Osmotic Pressure, halophile Oxygen, Aerobic, Anaerobic CONTROL OF MICROBIAL GROWTH Sterilization by Heat, Pasteurization Sterilization by Filtration Antimicrobial Agents- bacteriocidal, bacteriostatic Disinfectants, Antiseptics CHEMOTHERAPEUTIC AGENTS Ehrlich - Salvarsan - Selective Toxicity Domagk - Sulfonamides - Sulfa Drugs Sulfanailamide - para-Aminobenzoic Acid - Folic Acid Synthesis Inhibitor Fleming- Penicillin - Peptidoglycan Synthesis Inhibitor Penicillium notatum Waksman- Streptomycin, Streptomyces griseus Mycobacterium tuberculosis Protein synthesis inhibitor (70S ribosomes) Antiviral Agents Azidothymidine (AZT) - antiretroviral agent Interferon - species-specific antiviral protein

  3. OXYGEN 3 AEROBIC - O2 TERMINAL ELECTRON ACCEPTOR ANAEROBIC - SOMETHING OTHER THAN O2 OBLIGATE AEROBIC - REQUIRE O2 ex, PSEUDOMONAS AERUGINOSA MICROAEROPHILIC - REQUIRE O2; BUT IN LOW CONCENTRATION ex, NEISSERIA GONORRHOEAE FACULTATIVE ANAEROBIC - GROW WITH OR WITHOUT O2; BETTER WITH O2 ex, ESCHERICHIA COLI AEROTOLERANT ANAEROBIC - GROW EQUALLY WELL WITH OR WITHOUT O2 ex, ENTEROCOCCUS FECALIS OBLIGATE ANAEROBIC - CANNOT GROW IN O2 ex, CLOSTRIDIUM TETANI

  4. 4 TEMPERATURE HYPER- THERMOPHILES PSYCHROPHILES THERMOPHILES MESOPHILES GROWTH RATE ºC

  5. 5 pH = -log10[H+] CONCENTRATION IN MOLES/LITER H2O H+ + OH- [H+] = 1 x 10-7 MOLAR [OH-] = 1 x 10-7 MOLAR [H+] x [OH-] [H2O] Keq = [H2O] = 55M; VERY BIG; ~ CONSTANT Kw = [H+] x [OH-] = [1 x10-7] x [1 x10-7] = 1 x10-14 pH = -log10 1 x10-7 = -(-7) = 7

  6. 6

  7. 7 TYPICAL BACTERIAL CELL - BINARY FISSION GRAM NEGATIVE BACILLUS 0 MIN OUTER MEMBRANE PEPTIDOGLYCAN CYTOPLASMIC MEMBRANE NUCLEOID 20 MIN NEW SYNTHESIS OF OUTER MEMBRANE PEPTIDOGLYCAN CYTOPLASMIC MEMBRANE DNA (NUCLEOID) 40 MIN 60 MIN ~1x3 µm

  8. 8 DEALING WITH NUMBERS OF BACTERIA N = N02n N = NUMBER OF BACTERIA AFTER n GENERATIONS N0 = INITIAL NUMBER OF BACTERIA n = TOTAL NUMBER OF GENERATIONS EXAMPLE: CALCULATE N, IF N0 = 1,000 AND n = 5 N(after 5 generations) = 1,000 x 25 = 1,000 x 32 = 32,000 USUALLY MEASURE CELLS/UNIT VOLUME N(CELLS/ML) = 1,000(CELLS/ML) x 25 = 32,000 CELLS/ML CELLS USUALLY DIVIDE EVERY 30 - 60 MINUTES IN LAB CONDITIONS

  9. 9 10 HOURS 0.5 HOURS/GENERATION g = t n NUMBER OF GENERATIONS AND GENERATION TIME n = t/g n = NUMBER OF GENERATIONS t = TIME IN HOURS g = GENERATION TIME IN HOURS/GENERATION n = = 20 GENERATIONS 10 HOURS 20 GENERATIONS g = = 0.5 HOURS/GENERATION

  10. 10

  11. 11 CELLS/ML HOURS

  12. 12 LOG10 CELLS/ML HOURS

  13. 13 GROWTH IN LIQUID STATIONARY LOG10 LIVING CELLS/ML EXPONENTIAL DEATH LAG ~ 12 HRS TIME INOCULUM = STARTING CELLS ABSORBANCE TURBIDITY

  14. ABSORBANCE (A) AND CONCENTRATION A = log10 (INCIDENT LIGHT INTENSITY/INTENSITY OF LIGHT WHICH GOES THRU A SOLUTION) e.g., if chemical absorbs 90% of light, A = log10 (100/10) = log10(10) = 1.0 if chemical absorbs 99% of light, A = log10 (100/1) = log10 (100) = 2.0 A IS PROPORTIONAL TO CONCENTRATION A = εlc ε= CONSTANT FOR EACH CHEMICAL; l = DISTANCE THRU SOLUTION; c = CONCENTRATION

  15. BACTERIA IN LIQUID CULTURE ABSORB AND SCATTER LIGHT OPTICAL DENSITY = LOG10 (IO/I) ALSO CALLED ABSORBANCE IO - LIGHT INTENSITY GOING IN (INCIDENT) I = INTENSITY OF LIGHT WHICH GOES THRU OPTICAL DENSITY – PROPORTIONAL TO BACTERIAL CONCENTRATION

  16. 14 PHYSICAL MEANS OF MICROBIAL CONTROL STERILIZATION- COMPLETE KILLING OR REMOVAL AUTOCLAVE - 121oC, 15 PSI, 15-30 MIN PASTEURIZATION - KILLS MOST PATHOGENS 63-66oC 30 MINUTES HTST- HIGH TEMPERATURE SHORT TIME FLASH PASTEURIZATION 74oC 15 SECONDS UHT- ULTRA HIGH TEMPERATURE 140oC < 1 SECOND STABLE, ROOM TEMPERATURE 30 DAYS DESICCATION- DRYING KILLS: HIV, NEISSERIA GONORRHOEAE SURVIVE: MYCOBACTERIUM, ENDOSPORES, MOLD SPORES HIGH OSMOTIC PRESSURE - SALT, SUGAR RADIATION- UV, X-RAYS, GAMMA RAYS, MUTATIONS, CARCINOGENS FILTRATION- STERILE MEMBRANE 0.45 MICRON (MICROMETER) DIAMETER PORE

  17. 15 CHEMICAL CONTROL DISINFECTION- REMOVE OR KILL MOST PATHOGENS ON INANIMATE OBJECTS, DISINFECTANT ANTISEPTIC- CHEMICAL APPLIED TO BODY TO KILL MOST PATHOGENS BACTERICIDAL- KILLING BACTERIA BACTERIOSTATIC- PREVENTING BACTERIAL GROWTH ACIDS- FOOD ADDITIVES, PROPIONIC ACID, CALCIUM PROPIONATE, GLUTAMIC ACID, MONOSODIUM GLUTAMATE ALCOHOLS- ISOPROPYL ALCOHOL, ETHYL ALCOHOL, ANTISEPTICS, ALCOHOLS DENATURE PROTEINS PHENOLICS- PHENOL - LISTER PHENOL DERIVATIVES: HEXACHLOROPHENE, LYSOL HALOGENS- CHLORINE-BLEACH Cl2 + H2O H+ + Cl- + HOCl (HYPOCHLOROUS ACID) IODINE- TINCTURE OF IODINE = 2% SOLUTION IN ALCOHOL, ANTISEPTIC IODINE TABLETS FOR WATER PURIFICATION ISODINE = ORGANIC IODINE DERIVATIVE

  18. 16 HEAVY METALS- ENZYME POISON SILVER NITRATE- AgNO3 ANTISEPTIC AGAINST NEISSERIA GONORRHOEAE MERCURY- Hg, ORGANIC DERIVATIVES: MERTHIOLATE, MERCUROCHROME HYDROGEN PEROXIDE- H2O2 WOUND CLEANING ETHYLENE OXIDE- POISONOUS GAS FORMALDEHYDE- TISSUE PRESERVATIVE

  19. 17 CHEMOTHERAPY-DRUG TREATMENT, SELECTIVE TOXICITY EHRLICH- 1906 SYPHILIS CURE SALVARSAN, ARSENIC DERIVATIVE TREPONEMA PALLIDUM COINED TERM: CHEMOTHERAPY 1930s- SULFA DRUGS, SULFONAMIDES ANTIBACTERIAL, STRUCTURAL ANALOGS OF PARA AMINO BENZOIC ACID

  20. 18 FOLIC ACID VITAMIN NUCLEIC ACID SYNTHESIS SOME AMINO ACIDS ENZYMES AND OTHER PRECURSORS PARA AMINO BENZOIC ACID NOBEL REACTS WITH AND INHIBITS ONE ENZYME OF FOLIC ACID SYNTHESIS WHY SELECTIVE? SULFANILAMIDE

  21. PENICILLIN - FLEMING - 1929 PENICILLIUM NOTATUM FLOREY - PURIFIED PENICILLIN 19 NOBEL b LACTAM RING PENICILLIN - A b LACTAM NATURAL PENICILLIN G R = b LACTAM RINGS ARE STRUCTURAL ANALOGS OF D-ALANINE-D-ALANINE

  22. 20 D-ALA D-ALA CROSS LINK BOND WILL BE FORMED TRANSPEPTIDATION PENICILLIN INHIBITS - IT IS A STRUCTURAL ANALOG OF D-ALA-D-ALA D-ALANINE

  23. 21 STREPTOMYCIN- WAKSMAN - 1945 STREPTOMYCES GRISEUS WAKSMAN COINED THE TERM ANTIBIOTIC: SOMETHING PRODUCED BY ONE ORGANISM THAT IS TOXIC FOR ANOTHER ORGANISM AMINO GLYCOSIDE – AMINO SUGARS LINKED TO CYCLO – HEXANE RING BINDS PROKARYOTIC RIBOSOMES (70S) 30S SUBUNITS, CAUSES tRNAs TO FAIL TO PAIR CORRECTLY WITH CODONS; PROTEINS HAVE INCORRECT AMINO ACID SEQUENCE – STRESS – OXYGEN RADICAL FORMATION - DEATH FIRST CURE FOR MYCOBACTERIUM TUBERCULOSIS

  24. 22 SIDE EFFECTS TOXICITY - STREPTOMYCIN - OTIC NERVE DAMAGE - AZIDOTHYMIDINE (AZT) - LYMPHOCYTE DEVELOPMENT ALLERGY - ANAPHYLAXIS PENICILLIN HYPERSENSITIVITY NORMAL FLORA DESTRUCTION CANDIDA ALBICANS – MUCOUS MEMBRANE INFLAMMATION CLOSTRIDIUM DIFICILE - DIARRHEA SELECTION OF DRUG-RESISTANT PATHOGEN MUTANTS - PENICILLIN RESISTANT NEISSERIA GONORRHOEAE - CEFTRIAXONE [CEPHALOSPORIN; PEPTIDOGLYCAN SYNTHESIS INHIBITION] - AZT-RESISTANT HIV

  25. 23 HIV DNA SYNTHESIS FROM RNA A RETROVIRUS SINGLE-STRAND RNA REVERSE TRANSCRIPTASE AND LOW MOLECULAR WEIGHT DNA PRECURSORS RNA DNA RNA DEGRADATION SECOND DNA STRAND SYNTHESIS

  26. 24 HIV DNA SYNTHESIS INHIBITION BY AZT SINGLE-STRAND RNA REVERSE TRANSCRIPTASE + AZT [STRUCTURAL ANALOG OF THYMIDINE] + LOW MOLECULAR WEIGHT PRECURSORS AZT GROWING DNA STRAND CONTAINS AZT - IT CANNOT BE EXTENDED FURTHER

  27. TAKE AWAY: PHYSICAL/CHEMICAL FACTORS IN MICROBIAL GROWTH BINARY FISSION, N=No2 TO THE nPOWER CONTROL OF MICROBES ENVIRONMENT – PHYSICAL/CHEMICAL HUMANS/ANIMALS – SELECTIVE TOXICITY MECHANISMS OF ACTION

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