Microbial Growth and ControlMicrobial Growth and ControlI. Environmental Factors Influencing Microbial Growth A. The Omnipresence of Microorganisms 1. Microorganisms live in all parts of the biosphere where there is liquid water, including soil, hot springs, on the ocean floor, high in the atmosphere and deep inside rocks within the Earth's crust. 2. One teaspoon of topsoil contains about 1 billion bacteria, 120,000 fungal cells and about 25,000 algal cells 3. The human body is composed of about 10 trillion cells and populated with about 100 trillion microorganisms

II. Role of Microorganisms A. Decomposers of Nutrients 1. Microorganisms return nutrients back to the soil

B. Decomposers in Landfills 1. Microorganisms breakdown garbage and return nutrients back to the soil

C. Decomposers in Sewage Treatment 1. Microorganisms breakdown raw sewage to make it safe to be released

into streams and other waterways

2. The Warwick water treatment plant sewage mixed with the local river water after it had overflowed its banks after the great flood

D. Decomposers in Bioremediation of Oil 1. Microorganisms helped breakdown the oil after the BP oil catastrophe

E. Oxygen Production 1. Microorganisms produce oxygen as a by-product of photosynthesis

F. Nitrogen Cycle 1. Microorganisms fix nitrogen by changing nitrogen gas into nitrogen compounds that green plant can use

G. Role in Biotechnology and Genetic Engineering 1. Through genetic engineering with microorganisms crop yields have increased

2. Some foods that have been genetically engineered have changed the face

of farming

H. Role in Producing Food and Beverages 1. Microorganisms are used to produce many foods and beverages

I. Role in Fuel Production 1. Microorganisms change corn into biofuels

J. Biological Insecticides 1. Microbes that are pathogenic to insects are alternatives to chemical pesticides to prevent insect damage to agricultural crops and disease transmission 2. Bacillus thuringiensis infections are fatal in many insects but harmless to other animals including humans and to plants

K. Role in Disease 1. Some microorganisms cause disease in human populations

III. Some Newly Emerging Infectious Diseases A. West Nile Encephalitis 1. West Nile Virus

2. First diagnosed in the West Nile region of Uganda in 1937 3. Appeared in New York City in 1999

B. Bovine Spongiform Encephalopathy

1. Prion

2. Also causes Creutzfeldt-Jakob disease (CJD) C. Escherichia coli O57:H7

1. Toxin-producing strain of E. coli 2. First seen in 1982 3. Leading cause of diarrhea worldwide D. Invasive group A Streptococcus 1. Rapidly growing bacteria cause extensive tissue damage. 2. Increased incidence since 1995

E. Hantavirus pulmonary syndrome 1. Hantavirus 2. First identified in 1951 in Korea as cause of hemorrhagic fever and named for Hantaan River 3. A new disease involving respiratory symptoms was seen in the U.S.

in 1993 4. The U.S. virus, called Hantavirus Sin Nombre virus, probably came to the U.S. with rats around 1900 F. Ebola hemorrhagic fever 1. Ebola virus 2. Causes fever, hemorrhaging, and blood clottingG. Acquired immunodeficiency syndrome (AIDS) 1. Human immunodeficiency virus (HIV) 2. First identified in 1981. 3. Sexually transmitted disease affecting males and females

IV. Bacteria Cell Division A. Binary Fission

1. The most common method of bacterial reproduction is by binary fission 2. Prior to binary fission a filamentous temperature sensitive protein (Fts) form a division apparatus called the divisome which ushers in binary fission

3. Binary fission

B. Budding 1. Some bacteria reproduce by budding 2. Below Hyphomicrobium sulfonivorans demonstrates budding

V. The Growth Cycle A. Exponential Growth in Bacteria 1. The generation time for E. coli cultured under ideal condition is 20 minutes 2. This growth pattern is called exponential growth 3. Phases of growth: lag, log, stationary, death phases

VI. Direct and Indirect Measurement of Microbial Growth A. Direct Methods of Assessing Microbial Growth 1. Petroff-Hausser method allows microorganisms to be counted

directly

2. Serial dilution and plate count technique

3. Pour plate and spread plate technique

4. Membrane filter technique

5. Coulter Counter - counts cells as they disrupt an electric current flowing across a detector

B. Indirect Method of Assessing Growth 1. Spectrophotometry -based on transmittance or absorption of light

VII. Environmental Effects on Microbial GrowthA. pH adaptations 1. The pH Scale is based on the concentration of hydrogen ions (H+)in solution

2. The pH range of most microorganisms is 6.0 to 8.0 pH 3. Most bacteria have a pH range between 6.5 to 7.5 4. Acidophiles are acid loving microbes that live in a acidic environment 5. Euglena mirabilis is an acidophile that can live in water with a pH = 1.0

6. Thermoplasma (Archaea) lives in an acidic environment of pH = 1.2-1.8

7. Alkalinophiles can live in alkaline soda lakes often at a pH = 10-11

B. Temperature Adaptations

Extreme hyperthermophiles live in hot springs and around deep sea hydrothermal vents

The danger of microbial growth and food poisoning

C. Osmotic Pressure in a Hypertonic Solution

1. Halophiles- can live in a high osmotic environment

Halococcus (bacteria) Halobacterium salinarum (Archaea)

2. Facultative halophiles- can live in an elevated osmotic environment

Staphylococcus aureus

D. Adaptations to High Pressure 1. Barophiles can survive in a high pressure environment

VIII. The Role of Oxygen A. Oxygen, Free Radicals and Enzymes

1. Toxic forms of oxygen form free radicals 2. Free radicals are very dangerous and can have profound effects on living things -singlet oxygen is one of the reactive oxygen species, its electrons are energized -superoxide radical (O2

-) is negatively charge oxygen that readily forms

free radicals 3. Enzymes can nullify the effects of free radical oxygen -superoxide dismutase can change superoxide radical into hydrogen peroxide and oxygen- anaerobes lack this enzyme and die in its presence 4. The peroxide ion (O2

2-) can cause free radicals to form

5. Catalase neutralizes the peroxide ion

Free Radicals and Enzymes

Superoxide free radicals: O2

B. Oxygen Requirements of Microorganisms (Fluid Thioglycollate Media)

Obligate aerobes

Facultative anaerobes

Obligate anaerobes

Aerotolerant anaerobes Microaerophiles

IX. Control of Microbial Growth

A. Factors Influencing the Effectiveness of Antimicrobial Treatments 1. Microbial population size 2. Environmental factors 3. Exposure time to antimicrobial agent 4. Microbial characteristics

X. Physical Methods of Microbial Control

A. Heat and Control of Microbial Growth 1. Thermal death point (TDP) -lowest temperature at which all microorganisms are killed in 10 min 2. Thermal death time (TDT) -minimal length of time for all bacteria in a solution to be killed in a particular temperature 3. Decimal reduction time (DRT) -time, in minutes, in which 90% of all microorganisms are killed at a given temperature

B. Control of Microbial Growth

1. Boiling

-denatures proteins -lethal to bacteria, fungi and viruses after 10 min but some spores may take up to 20 hours to kill

2. Autoclave -denatures proteins -15 psi at 121 degrees C for 15 min -sterilizes media, glassware, solutions, utensils, equipment -an industrial autoclave is called a retort

Autoclave

3. Pasteurization -denatures proteins -classical method: heat to 63 degrees C for 30 min -HTST- high temperature-short time: 72 degrees C for 15 sec -UHT- ultra high temperature: involves heating milk or cream to 138°to 150° C for one or two seconds

4. Dry heat -direct flame -incineration -hot-air sterilization- heat in oven at 170 degrees C for 2 hours 5. Low temperatures - refrigeration -deep freeze

6. Lyophilization (cryodesiccation) -freeze-drying process of a material and then reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to

gas

7. Filter sterilization

8. High pressure -use in a vacuum to preserve the colors and flavors of juices 9. Desiccation -removes water from microorganisms 10. Osmotic pressure -causes plasmolysis in microorganisms

11. Radiation -gamma and x-ray radiation ionizing radiation that causes breaks in the DNA molecule -UV radiation non ionizing radiation that causes pyrimidine dimers

Xeroderma pigmentosum

XI. Chemical Methods of Microbial Control A. Disk-Diffusion Method

B. Chemicals that Control Microbial Growth

1. Phenol -destroys membranes

2. Phenolics -destroys membranes -used on environmental surfaces, skin, mucous membranes 3. Bisphenols -destroys membranes -used in soaps, toothpaste, hand lotions -lysol -triclosan

4. Halogens -oxidizing agent that destroys proteins -iodine- combined with alcohol to form tincture of iodine -chlorine- forms hypochlorous acid -used in pools, on utensils, glassware

5. Alcohols -destroys proteins and disrupts cell membranes -isopropyl alcohol -ethanol -commonly used as a swab to degerm

6. Heavy metals -destroys proteins -silver, mercury, copper

7. Surfactants- surface active agents -soaps and detergents -mechanical removal of microorganisms -used to degerm

8. Quaternary ammonium compounds (QUATS) -protein denaturation, destroys membranes -used on skin instruments, utensils, rubber goods

9. Chemical food preservers a. organic acids -metabolic inhibitors usually against mold -sorbic acid, benzoic acid, calcium propionate b. nitrates and nitrites -used to inhibit enzyme action of anaerobes -forms nitrosamines when cooked which may be carcinogenic

10. Aldehydes -destroys proteins -gluteralderhyde and formaldehyde

11. Gaseous chemosterilizers -protein denaturation -ethylene oxide

XII. Antibiotics and Related Drugs

A. Alexander Fleming 1. Isolated the first antibiotic, called penicillin 2. Awarded the Nobel Prize for his accomplishment

B. Paul Erhlich

1. Did a systematic search for a chemical to cure syphilis 2. Came up with arsenic 606 3. Highly effective in treating patients who contracted syphilis 4. Awarded the Nobel Prize for his accomplishment

C. Gerhard Domagk

1. Discovered sulfanilamide 2. Was effective in treating Streptoococcal infections in animals 3. Awarded the Nobel Prize for his accomplishment

XIII. The Action of Antimicrobial Drugs A. Inhibition of Cell Wall Synthesis 1. Cell walls can be targeted by antibiotics that interfere with

peptide cross bridges of peptidoglycan causing lysis of bacteria

2. Penicillin -effective against gram-positive bacteria -penicillin and other antibiotics have the beta-lactam ring

3. Cephalosporins

-over two dozen in current use -most are semi-synthetics derived from the secretion of the mold

Cephalosporium -similar to penicillins but are more b-lactamase resistant

B. Inhibition of Protein Synthesis 1. Chloramphenicol -binds to 50s portion of ribosomes and inhibits formation of peptide bonds 2. Erythromycin -binds to 50s and prevents movement of ribosome along mRNA -not a broad spectrum drug 3. Tetracyclines -interferes with attachment of tRNA to mRNA-ribosome complex 4. Streptomycin -changes shape of 30s causing code to be read wrong

C. Injury to Plasma Membrane

1. Polymyxin B -primarily used for resistant gram negative infections -bind to the cell membrane and alter its structure making it more permeable resulting in water uptake which leads to cell death. 2. Ketoconazole -used on fungal diseases -prescribed for topical infections such as athlete’s foot, ringworm, candidiasis (thrush) and jock itch -the over-the-counter shampoo version can also be used as a body wash for the treatment of tinea versicolor -interferes with the fungal synthesis of ergosterol, a constituent of fungal cell membranes

D. The Inhibition of Nucleic Acid Synthesis 1. Quinolones -inhibits DNA synthesis -prevents bacterial DNA from unwinding and duplicating 2. Rifampin -inhibits mRNA synthesis

E. Inhibition of Metabolite Synthesis 1. Gerhard Domagk and Jacques and Therese Trefouel (1935) are generally credited with the discovery of sulfanilamide as a chemotherapeutic agent 2. As an antibiotic, it functions by competitively inhibiting (ie, by acting as a substrate analogue) enzymatic reactions involving para-aminobenzoic acid (PABA) 3. PABA is needed in enzymatic reactions that produce folic acid in the synthesis of purine, pyrimidine and other amino acids

How Sulfanilamides Mimic PABA an Intermediate in the Making of Folic Acid