GROWTH, CONTROL, CHEMOTHERAPY

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GROWTH, CONTROL, CHEMOTHERAPYGROWTH -

Binary Fission, Growth Rate, Generation Time, N = No2 n

Growth Phases of Culture - Lag, Exponential, Stationary, DeathPhysical Factors- Temperature: psychrophile, mesophile,

thermophile, hyperthermophilepH, Buffer, acidophile,

alkalophileOsmotic Pressure,

halophileOxygen, Aerobic,

Anaerobic

CONTROL OF MICROBIAL GROWTHSterilization by Heat, PasteurizationSterilization by FiltrationAntimicrobial Agents- bacteriocidal, bacteriostatic

Disinfectants, Antiseptics

CHEMOTHERAPEUTIC AGENTSEhrlich - Salvarsan - Selective Toxicity

Domagk - Sulfonamides - Sulfa DrugsSulfanailamide - para-Aminobenzoic Acid -

Folic Acid Synthesis Inhibitor

Fleming- Penicillin - Peptidoglycan Synthesis InhibitorPenicillium notatum

Waksman- Streptomycin, Streptomyces griseusMycobacterium tuberculosisProtein synthesis inhibitor (70S ribosomes)

Antiviral AgentsAzidothymidine (AZT) - antiretroviral agentInterferon - species-specific antiviral protein

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AEROBIC - O2 TERMINAL ELECTRON ACCEPTORANAEROBIC - 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

OXYGEN 3

GR

OW

TH

RA

TE

ºC

TEMPERATURE

PS

YC

HR

OP

HIL

ES

ME

SO

PH

ILE

STH

ER

MO

PH

ILE

SH

YP

ER

-TH

ER

MO

PH

ILE

S

4

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

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TYPICAL BACTERIAL CELL - BINARY FISSIONGRAM

NEGATIVE BACILLUS OUTER MEMBRANE

PEPTIDOGLYCANCYTOPLASMIC

MEMBRANENUCLEOIDNEW SYNTHESIS OF

OUTER MEMBRANEPEPTIDOGLYCAN

CYTOPLASMIC MEMBRANE

DNA (NUCLEOID)

~1x3 µm

0 MIN

20 MIN

40 MIN

60 MIN

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DEALING WITH NUMBERS OF BACTERIA

N = N02n

N = NUMBER OF BACTERIA AFTER nGENERATIONS

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

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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 HOURS0.5 HOURS/GENERATION

g == 0.5

HOURS/GENERATION

10 HOURS20 GENERATIONS

g = t n

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HOURS CELLS/ML LOG10CELLS/ML

0 1 0

1 4 0.60

2 16 1.20

3 64 1.80

4 256 2.40

5 1024 3.01

6 4096 3.61

10 1,050,000 6.02

10

HOURS

CE

LL

S/M

L

11

HOURS

LOG10

CELLS/ML

12

~ 12 HRS

TIME

LOG10

LIVING CELLS/

MLLAG

EXPONENTIAL

STATIONARY

DEATH

GROWTH IN LIQUID

INOCULUM = STARTING CELLS

ABSORBANCETURBIDITY

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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

BACTERIA IN LIQUID CULTURE ABSORB ANDSCATTER 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

PHYSICAL MEANS OF MICROBIAL CONTROL

STERILIZATION- COMPLETE KILLING OR REMOVALAUTOCLAVE - 121o C, 15 PSI, 15-30 MIN

PASTEURIZATION - KILLS MOST PATHOGENS 63-66oC 30 MINUTESHTST- HIGH TEMPERATURE SHORT TIME

FLASH PASTEURIZATION

74oC 15 SECONDSUHT- ULTRA HIGH TEMPERATURE

140oC < 1 SECONDSTABLE, ROOM

TEMPERATURE 30 DAYS

DESICCATION- DRYINGKILLS: HIV, NEISSERIA GONORRHOEAESURVIVE: 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

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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 - LISTERPHENOL DERIVATIVES:

HEXACHLOROPHENE, LYSOL

HALOGENS- CHLORINE-BLEACHCl2 + H2O H+ + Cl- + HOCl

(HYPOCHLOROUS ACID)

IODINE- TINCTURE OF IODINE = 2% SOLUTION IN ALCOHOL,

ANTISEPTIC IODINE TABLETS FOR WATER PURIFICATIONISODINE = ORGANIC IODINE

DERIVATIVE

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HEAVY METALS- ENZYME POISONSILVER NITRATE- AgNO3

ANTISEPTIC AGAINST NEISSERIA GONORRHOEAE

MERCURY- Hg, ORGANIC DERIVATIVES:

MERTHIOLATE, MERCUROCHROME

HYDROGEN PEROXIDE- H2O2 WOUND CLEANING

ETHYLENE OXIDE- POISONOUS GAS

FORMALDEHYDE- TISSUE PRESERVATIVE

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EHRLICH- 1906 SYPHILIS CURESALVARSAN, ARSENIC DERIVATIVETREPONEMA PALLIDUMCOINED TERM: CHEMOTHERAPY

1930s- SULFA DRUGS, SULFONAMIDESANTIBACTERIAL, STRUCTURAL ANALOGS OF PARA AMINO BENZOIC ACID

CHEMOTHERAPY-DRUG TREATMENT, SELECTIVE TOXICITY

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PARA AMINO BENZOIC ACID

SULFANILAMIDE

ENZYMES AND OTHER

PRECURSORS

FOLIC ACID

VITAMINNUCLEIC ACID SYNTHESIS

SOME AMINO ACIDS

REACTS WITH AND INHIBITS ONE

ENZYME OF FOLIC ACID SYNTHESIS

WHY SELECTIVE?

NOBEL

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PENICILLIN - FLEMING - 1929PENICILLIUM NOTATUM

FLOREY - PURIFIED PENICILLIN

R =NATURAL

PENICILLIN G

b LACTAM RING

PENICILLIN - A b LACTAM

NOBEL

b LACTAM RINGS ARE STRUCTURAL ANALOGS OF D-ALANINE-D-ALANINE

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D-ALA D-ALA

TRANSPEPTIDATION

D-ALANINEPENICILLIN INHIBITS - IT IS A STRUCTURAL ANALOG

OF D-ALA-D-ALA

CROSS LINK BOND WILL BE FORMED

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STREPTOMYCIN - WAKSMAN - 1945STREPTOMYCES 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

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SIDE EFFECTS

TOXICITY - STREPTOMYCIN - OTIC NERVE DAMAGE

- AZIDOTHYMIDINE (AZT) - LYMPHOCYTE

DEVELOPMENT

ALLERGY - ANAPHYLAXISPENICILLIN

HYPERSENSITIVITY

NORMAL FLORA DESTRUCTIONCANDIDA

ALBICANS – MUCOUS

MEMBRANE INFLAMMATIONCLOSTRIDIUM

DIFICILE - DIARRHEA

SELECTION OF DRUG-RESISTANT PATHOGEN MUTANTS- PENICILLIN RESISTANT NEISSERIA

GONORRHOEAE- CEFTRIAXONE [CEPHALOSPORIN;

PEPTIDOGLYCAN SYNTHESIS INHIBITION]- AZT-RESISTANT HIV

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SINGLE-STRAND RNA

REVERSE TRANSCRIPTASE AND LOW MOLECULAR WEIGHT DNA PRECURSORS

RNADNA

RNA DEGRADATIONSECOND DNA STRAND SYNTHESIS

HIV DNA SYNTHESIS FROM RNA A RETROVIRUS

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SINGLE-STRAND RNA

HIV DNA SYNTHESIS INHIBITION BY AZT

REVERSE TRANSCRIPTASE

+ AZT [STRUCTURAL ANALOG OF THYMIDINE]

+ LOW MOLECULAR WEIGHT PRECURSORS

AZT

GROWING DNA STRAND CONTAINS AZT - IT CANNOT BE EXTENDED FURTHER

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TAKE AWAY:PHYSICAL/CHEMICAL FACTORS IN MICROBIALGROWTHBINARY FISSION, N=No2 TO THE n POWER

CONTROL OF MICROBES ENVIRONMENT – PHYSICAL/CHEMICAL

HUMANS/ANIMALS – SELECTIVE TOXICITYMECHANISMS OF ACTION