EXAM THREE NOTES

 

 

 

Bacterial Growth Oxygen Effects

 

Toxic Oxygen - Forms

Singlet oxygen

Superoxide free radicals

Peroxides

Hydroxyl free radicals

 

Toxic Oxygen - Neutralization

Catalase:

2 H2O2 2 H2O + O2

Superoxide dismutase:

2 O2 - + 2 H+ O2 + H2O2

Peroxidases:

H2O2 H+ + H2O

 

Oxygen Classification

aerobic - grows in presence of oxygen

obligate aerobe - requires oxygen

anaerobic - grows in absence of oxygen

obligate anaerobe - will only grow in absence of oxygen

facultative anaerobe - will grow both anaerobically or aerobically

 

Oxygen Classification

Aerotolerant - only grow anaerobically but will tolerate oxygen

Microaerophillic - growth occurs only in presence of low concentrations of oxygen

 

Bacterial Growth Culture & Media

 

Media Components

Carbon Source

Nitrogen Source

Minor Nutrients

Solid Support

If not liquid media

 

Carbon Sources

Usually sugars

Alternatives include:

Fats or lipids

Proteins or amino acids

Fix CO2

 

Nitrogen Sources

Usually ammonium ion

Alternatives include:

Nitrate ion

Nitrite ion

Amino acids

Nucleic acids

Other compounds containing nitrogen

 

Minor Nutrients

Sulfur in cystiene and methionine

Phosphate DNA RNA energy intermediates

Iron electron transport

Magnesium kinases

Other metals

Some organisms very particular

 

Solid Support

Gelatin

Melts near room temperature

Digested by proteases which are produced by many bacteria

Agar derived from algal polysaccharide

 

Bacterial Growth

 

Introduction

Population vs. Cellular Growth

Exponential vs. Arithmetic Growth

Bacterial Growth - Binary Fission

 

Bacterial Growth Curve

Lag Phase

Log Phase

Stationary Phase

Death Phase

 

Calculations - Number of cells

Bacterial Growth

Calculations - Number of Generations

Bacterial Growth

Calculations - Generation Time

Time for single cell to double

Bacterial Growth

Measurement of Growth

Biomass

Viable Counts

Total Counts

Most Probable Number (MPN)

 

Biomass

Dry weight (increase in mass)

Turbidity (using a spectrophotometer)

Metabolic activity (O2 used or CO2 evolved)

 

 

Viable Counts

Plate counts

Membrane filters

 

Total Counts

Direct Microscopic Count

Counting Chamber

Electronic Counter

 

Most Probable Number

5 tubes at 10 mL, 1 mL and 0.1 mL sized inoculum

Use combinations with table to calculate the most probable number

Confidence limits set range of likely number of cells per 100 mL

 

 

 

Control of Microbial Growth

 

Definitions

Sterilization - Processes that kill kill living organisms including spores

Disinfection - Destruction of vegetative pathogens (not spores)

Antisepsis -Destruction of vegetative pathogens on living tissue

Degerming - Removal of microbes from a limited area (alcohol swab prior to injection)

Sanitization - Lowers microbial counts

 

Physical Methods of Control

Temperature Effects

Filtration

Desiccation

Osmotic Pressure

Radiation

 

Effects of Temperature

Moist Heat

Dry Heat

Pasteurization

Refrigeration

 

Temperature - Moist Heat

Autoclave:  120-130C (15-20 lbs/in2)

Tyndalization

Effect on instruments

Foil or cloth wrap

Must use on liquids

 

Temperature - Dry Heat

Glassware and instruments

160-170C for at least 90 min

Usually foil wrap

Incineration (loops & needles)

 

Temperature - Pasteurization

Milk:  63C for 30 min (old)

Milk:  72C for 15 sec (new)

Juices

 

Temperature - Refrigeration

Does not usually kill

Slows metabolism although psychrophillic organisms can still grow

Best between 0 and 7 C

Freeze thaw cycles can kill some organisms

 

Filtration

Used for heat sensitive liquids like serum or urea containing media

0.45 or 0.22 pores in membrane

Often used commercially with beer, wine and fruit juices

 

Desiccation

Inhibits growth more than kills

Dried meats and vegetables

Freeze dry processes in foods

 

Osmotic Pressure

High concentrations of salts or sugars inhibit growth of bacteria;  examples includes jams and jellies, sugar and salt cured meats

 

Radiation

Short wave (X-rays, gamma rays) high penetration power; breaks DNA

Non-ionizing (UV) longer wave; no penetrating power; forms thymine dimers

Organisms contain multiple repair systems

 

Chemical Methods

Phenols

Halogens

Alcohols

Surface active agents

Aldehydes

Heavy metals

Acids & bases

Gases

Peroxygens

 

Phenols & Phenolics

Lister & carbolic acid

penetrate plasma membrane & precipitate proteins

phenol coefficient - chemical agents evaluated relative to phenol

 

Halogens

Chlorine & drinking water

Ca(OCl)2  bleach

Iodine

Betadine (Iodine and organic solvent)

Chloramines

 

Alcohols

70% ethanol

isopropanol

require some water to be effective

used in conjunction with other agents

 

Surface Active Agents

soaps - emulsification of oils increases bacterial removal - deodorant soaps contain triclocarban or triclosan

acid-anionic agents used in dairy industry

quaternary ammonium compounds - work best against G+ bacteria, less effective against G-; also kill fungi protozoa and viruses; Psuedomonas resistant

 

Aldehydes

react with a variety of organic functional groups:  -NH2, -OH, -SH 

Formaldehyde

Glutaraldehyde

used for embalming

 

Heavy Metals

usually toxic

Silver nitrate used on infants eyes (replaced by antibiotics)

Copper sulfate used as algicide

Mercury toxicity and resistance

Zinc chloride and oxide in toothpaste

 

Acids and Bases

pH extremes inhibit growth of bacteria

acid (pickles and tomatoes)

trisodium phosphate in detergent

Propionic acid in bread

Benzoic acid in soft drinks

 

Gases

ethylene oxide - strong oxidizer and alkylating agent

very penetrating

possible carcinogen

does not damage instruments but expensive to use

 

Peroxygens - Oxidizing Agents

Ozone - being used in water treatment

Hydrogen peroxide -surface disinfecting

Benzoyl peroxide - skin treatment

Peracetic acid - used in food processing because residue is non-toxic

 

 

 

 

Antibiotics / Chemotherapy

History

Properties

Testing

Spectrum of Antimicrobial Action

Modes of Action

Survey of Drugs

 

History

Quinine for malaria

Willow bark for treating fever

Paul Ehrlich - staining of bacteria led to ideas for chemotherapy

Fleming (1928) observed the effect of Penicillium of on Staphylococcus

Flory & Chain (1940) developed penicillin and clinically tested it

 

Properties

Selective toxicity (e.g. sulfanilamide mimics PABA in folic acid synthesis)

Sources

Microorganisms

Synthetic agents

Plants

 

Testing

Broth dilution

Agar dilution

Disc diffusion

 

Broth dilution

MIC - minimal inhibitory concentration; smallest concentration that stops growth

Successive dilutions inoculated with same number bacteria

Turbidity measure when compared to control (could also do dilutions & plate counts)

MBC - minimal bactericidal concentration

 

Agar dilution

Dilute drug into agar at varying concentrations

Can test multiple species of bacteria

Not very quantitative

 

Disc-Diffusion

Discs with known concentrations of antibiotics seeded onto lawn of bacteria

Zone of clearing around disk a measure of effectiveness of antibiotic

 

Spectrum of Antimicrobial Activity

Selectively toxic drugs; uses differences between prokaryotic and eukaryotic cells

Broad spectrum - both G+ and G-

Antibiotic effect, e.g. penicillin and Candida albicans

 

Modes of Action

Bactericidal vs. Bacteriostatic

Cell Wall

Protein Synthesis

Plasma membrane

Nucleic Acid Synthesis

Essential Metabolites

 

Bactericidal vs. Bacteriostatic

Bactericidal - kills

Bacteriostatic - inhibits growth but once remove growth can resume

 

Inhibition of Cell Wall Synthesis

Uniqueness of bacterial cell wall

Prevent peptidoglycan synthesis or peptide cross-linking from forming

Penicillins & cephalosporins

 

Inhibition of Protein Synthesis

Bacterial protein synthesis significantly different than eukaryotic e.g. 70S vs. 80S ribosome or elongation & termination factors

Amyloglycosides (streptomycin and gentamicin)

 

Injury of Plasma Membrane

Alteration in permeability

Interference with required consituents, e.g. sterols in fungal lipid membranes

Polymixin B (bacteria)

Amphotericin B or miconazole (fungal)

 

Essential Metabolites

Para-aminobonzoic acid is an essential cofactor used by bacteria to synthesize folic acid (a vitamin that functions as a coenzyme in the synthesis of nucleic acid precursors)

animals ingest folic acid

Sulfanilamide is an analog of PABA

 

Inhibition of Nucleic Acid Synthesis

Nucleic acid synthesis especially mRNA and DNA

Rifampin and quinolones

Limited utility because of RNAs and DNAs essential role in both prokaryotic and eukaryotic cells

 

Survey of Drugs - Cell Wall Synthesis

Penicillins (G, V)

Semisynthetic penicillins (Ampicillin)

Monobactams

Vancomycin - Glycopeptide topical

Cephalosporins

Bacitracin - bacterial origin; topical use

Isoniazid - tuberculosis

Ethambutol - tuberculosis

 

Survey of Drugs - Protein Synthesis

Amyloglycosides (Streptomycin, neomycin Gentamicin)

Tetracyclines - Bacteriostatic

Chloramphenicol

Macrolides - Erythromycin - Bacteriostatic

 

Survey of Drugs - Plasma Membrane

Polymyxin B - topical; works against G-

 

Survey of Drugs - Nucleic Acids

Rifampin

Quinolones

Fluorquinolones

 

Survey of Drugs - Essential Metabolites

Sulfonamides