Fundamentals of Microbiology

What are microbes?

Small organisms, generally smaller than what human eye can detect.

Examples:Bacteria (in Kingdom Monera) • Archaea, protists (in Kingdom Protista).• Algae (in Kingdom Protista or Plantae, depending on

taxonomy) • Fungi (in Kingdom Fungi) • VirusesMost microbes live as unicellular cells or cell clusters; some multi-cellular (e.g. filamentous multi-cells), but not as complex as animals or plants

The Structure of Microbes

Two basic cell architectures: prokaryotes & eukaryotes

Prokaryotes:• "pro" = before, + "karyos" =

nucleus • Includes bacteria and

cyanobacteria and archaea• Bacteria have a single circular

DNA molecule ("chromosome")• Divide by binary fission, not by

mitosis or meiosis. • Size: 0.5-5 µm (micrometer) diameter

Eukaryotes• "eu" = true, + "karyos" = nucleus• Contain membrane-bounded

organelles (e.g. mitochondria, lysosomes, etc.

• Size: 5 micrometers (yeast cells) - 100 micrometers.

• Includes protists, fungi, animals & plants.

• Divide by mitosis (asexual reproduction), or meiosis

The variety of Bacteria 1. Rods = bacilli (sing. bacillus).

2. Spheres = cocci (sing. coccus).

3. Spiral forms = spirilla (sing. spirillum).

4. Filamentous forms.

5. Pleiomorphic shapes.

6. Mycoplasmas (Acholeplasmas) lack cell walls, so they have no well defined shape.

7. Square bacteria.

Growth curve:

Lag phase: Previous cells ran out of food, shut down many metabolic pathways needed for active growth, made adaptations necessary for dormancy and protection. Need to regenerate pools of essential nutrients before growth can.

Exponential phase: Cells in optimum growth state, divide repeatedly by binary fission at maximal rate.

Stationary phase Can be due to exhaustion of some critical nutrient, or to accumulation of waste products that slow down growth

Death phase Continued accumulation of wastes, exposure to oxygen, loss of cell's ability to detoxify toxins, etc.

Σ S + X = Σ P + n. X

nutrients + cells = extra-cellular products + more cells

Microbial growth is an autocatalytic rxn, i.e. the rate of growth is directly related to cell concentration. Cellular production is the outcome of this rxn. Rate of microbial growth:µ net = net spec. growth rate

net = gross - death

µ net = µs – kd (6.2b)where, kd = 1st order rate const. or “spec. death rate” describing the loss of cell mass due to endogenous metabolism or cell lysis during stationary phase.Endogenous metabolism – catablolism of cellular reserves needed for ne building blocks and energy.Microbial growth in terms of cell # conc (N)µR ≡ net spec. replication rate

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How Cells Grow:

Batch vs. Continuous culture methods

Batch method: put small inoculum of pure culture into sterile medium, let grow. Common lab procedure, but not typical of many real environments.

Continuous culture : Use chemostat or turbidostat. Trickle fresh medium into culture at slow but steady rate, displace = volume of culture as overflow. Cells remain in exponential (but suboptimal) state, growing at known rate. Good simulation for study of many natural environments.

Microbial Growth and its Control:

1. Total Cell count Petroff-Hausser chamber slide -- needs large conc. (107 cells/ml minimum) Coulter Counter: For larger microbes; fungi, yeasts, protozoa, etc.)

2. Viable count:

This is typically carried out by CFU (colony forming units) assay: 1. Carry out dilution series. 2. Plate known volumes on plates 3. Count only plates with 30-300 colonies (best statistical accuracy).

Spread plate: bacteria are spread on the surface of agar using some sterile spreading device. Pour plate: bacteria are mixed with melted agar and cooled; colonies grow throughout the agar.

Measurement of growth

3. Optical techniques: Estimate cell numbers accurately by measuring visible turbidity. Light scattered is proportional to number of cells.

Eyeball method. This is not a precise measurement, but should allow estimation within an order of magnitude.

4. Absorbance method: Use a spectrophotometer to accurately measure absorbance, usually at wavelengths around 400-600 nm.

Effects of temperature on growth

Higher temperatures speed up chemical reactions, ~ double rate for every 10 deg. C in temperature.

Expect cells to grow more rapidly as temp. rises, up to a point. But too high temperatures denaturation of proteins and nucleic acids, loss of critical enzymes and loss of metabolism.

Cardinal temperatures: every organism can be characterized by thee temperatures:

Minimum temperature, below which no growth occurs

Optimum temperature, at which fastest growth occurs

Maximum temperature, above which no growth occurs

Thermal Destruction of Microorganisms

• Heat treatment is a practical method for destruction of harmful microorganisms

• Death is the inability of organisms to form a viable colony

• Death rate is the reduction in number of viable cells per minute

Decimal Reduction Time, D-value

• D-value is time (minutes) required to destroy 90% of the population, (D65c=5)

• Plot of logarithmic value of surviving cells against time of exposure to heat is a straight line

• The rate of death at any given temperature is constant and independent of initial number of cells

Rate Of Destruction Curve

Thermal Death Time, TDT

• D-value shows the resistance of bacteria at a given temperature

• Plot TDT vs exposure temperature (log D vs T)

• The slope of the curve is expressed by Z-term

• Points on TDT curve indicate the relative resistance at different tempetatures

Thermal Death-Time Curve

Effects of oxygen on growth

Obligate aerobes -- grow only when oxygen is present.

Facultative anaerobes -- grow with or without oxygen, grow better in oxygen (respire).

Aerotolerant anaerobes -- ignore oxygen, grow equally well with or without.

Obligate anaerobes -- die in presence of oxygen.

Micro-aerophiles -- won't grow at normal atmospheric oxygen (20%), but require some oxygen for growth (2-10%)

Effects of pH on growth

Acidophiles = acid pH optimal (1 to 5.5). Neutrophiles = pH 5.5 to 8 optimal

Alkaliphiles = pH 8.5 to 11.5

Extreme alkaliphiles = optimum pH 10 or greater

Most bacteria are neutrophiles (Exceptions: some bacteria in hot springs have optimum of 1-3)

Most fungi prefer slight acid (pH 4 to 6)

Control of Microbial Growth

Overall Effectiveness (from least to most specific)

1. Sterilizing Agents-- kill everything (e.g. heat, radiation) 2. Disinfectants-- kill most things. Too strong for living tissues (e.g. lysol, NH3) 3. Antiseptics-- milder in action. Can be used topically, but not ingested. (e.g.

alcohol, iodine) 4. Chemotherapeutics-- can be ingested (e.g. penicillin, sulfa drugs)

1: Sterilizing Agents:

A. Heat: boiling, autoclaving, dry heat. pasteurization.B. Membrane Filters. C. Chemicals – name of chemical ?D. Radiation – kind of radiation??

2. & 3: Disinfectants & Antiseptics (not mutually exclusive; depends on concentration)

Heavy metals: (Mercury, Silver, Arsenic)- cause protein denaturation

Halogens: (Chlorine, Iodine, Hypochlorite)- oxidizing agents. Good for swimming pools, etc.

Phenols & Cresols: Dissolve membranes, denature proteins

Alcohols: Denature proteins, dissolve membranes.

Detergents: Dissolve membranes

4: Antimicrobial Inhibitors

Antibiotics: a. Cell Wall antibiotics:

Example: Penicillins. First widely available drug, introduced in 1945. Contains ß-lactam ring.Benzylpenicillin (Penicillin G) was first natural isolate.

b. Inhibitors of protein synthesis:

•Aminoglycosides : streptomycin, gentamicin, kanamycin.

•Tetracyclines.

•Glycopeptide antibiotics. E.g. Vancomycin

•Macrolide antibiotics. E.g. Erythromycin

Drug Resistance Testing for Drug Resistance

Ways for bacteria to develop drug resistance:

Mutations affecting cell surface can affect entry of drug

Receptor normally used by drug altered- no binding to altered receptor

Bacteria or plasmids can produce enzymes which inactivate drug; e.g. pencillinases hydrolyze ß-lactam ring

Some plasmids carry genes for antibiotic resistance (enzymes that degrade antibiotic). Called R-plasmids

Plasmid encoded drug pump

Production of protein "pumps" to pump drug out of cell

Ways to deal with antibiotic resistance:Higher dose, different antibiotic, more than one drug simultaneously