Microbial Growth and its Control
Last revised: Monday, February 3, 2003
Reading: Ch. 6, 7 in text

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 (see text pp. 121-122): 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.

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Mechanics of batch growth.

• Prokaryotes grow by binary fission: 1 cell grows, divides into 2 identical daughters, etc.
  
• View movie of dividing E. coli

Growth curve (see Fig. 6.1 in text)

4 phases: lag, exponential, stationary, and death

1. Why 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 resume, requires new enzyme synthesis and time for pathways to function.
   • Can reduce or eliminate lag phase by using cells that are not in stationary phase.
2. Why exponential phase?
   • Cells in optimum growth state, divide repeatedly by binary fission at maximal rate.
   • Note: useful to calculate doubling time; can vary from 20 min to several days
3. Why stationary phase?
   • Can be due to exhaustion of some critical nutrient, or to accumulation of waste products that slow down growth (e.g. acid buildup from fermentation).
4. Why death phase?
   • Continued accumulation of wastes, exposure to oxygen, loss of cell's ability to detoxify toxins, etc. Note that death is exponential; 90% of cells die in certain time, another 90% in same time period, etc.
   • Note: in practice, try to prolong stationary phase, reduce death phase. Don't store cultures at room temperature on plates (ready exposure to oxygen, dessication, high temperature speeds oxidation reactions). Better, transfer to slants (tubes), store capped in refrigerator once grown. Still better, transfer to stab tube (soft nutrient agar), stopper and seal with airtight seal. Can recover viable culture even after a year of sitting on shelf!

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Mathematics of growth; generation time

Growth equation

# of generations	# of bacteria	log2N	log10N
0	1 or 20	0	0
1	2 or 21	1	.301
2	4 or 22	2	.602
3	8 or 23	3	.903
4	16 or 24	4	...
5	32 or 25	5	...
n	2n	n	.301n

• where n = number of generations
• N_f = final conc. of cells (e.g. 10⁹/ml)
• N_o = initial conc. of cells (e.g. 10³/ml)
• and .301 is factor to convert log₂ to log₁₀

Example:

measure culture at 9 a.m.:	No = 10,000 cells/ml
measure culture at 3 p.m.:	Nf = 100,000 cells/ml
calculate n	= (5 - 4)/0.301 = 1/0.3 = 3.33 generations
total time	= 6 hours = 360 minutes
360 minutes/3.33 generations	= 108 minutes/generation
Conclude:	generation time = 108 minutes

Note: be able to calculate g.t. Pay attention to units, ask if your answer is reasonable.

Graphical measurement of growth

See text fig. 6.1

Plotting # of cells vs. time gives a curved line.
Plotting log # of cells vs. time gives a straight line -- easier to interpolate, use.
Plotting # of cells vs time on semilog paper also gives a straight line -- easiest way in practice to work with growth measurements.
Note: often what is plotted on the Y-axis of semilog paper is not # of cells, but something more easily measurable, such as Absorbance (see below).

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Measurement of growth

1. Total Cell count
   • Petroff-Hausser chamber slide -- needs large conc. (10⁷ cells/ml minimum) -- see Fig. 6.3
   • Coulter Counter (for larger microbes; fungi, yeasts, protozoa, etc.) -- uses electrical charge difference in passing through small hole. Not so useful with bacteria, get errors due to clumping, debris, 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)
     4. extrapolate to undiluted cell conc.
   • CFU may or may not be same as number of cells --
   • Method is accurate, but requires time for incubation.
   • Two ways to carry out viable count:
     1. Spread plate: bacteria are spread on the surface of agar using some sterile spreading device.
        • Advantages: if properly carried out, all colonies should be easily counted.
        • Disadvantages: takes some time, not always reliable in inexperienced hands, cells with low tolerance to oxygen won't grow. If "spreaders" are present, may overgrow plate surface.
     2. Pour plate: bacteria are mixed with melted agar and cooled; colonies grow throughout the agar.
        • Advantages: almost fail-proof technique, colonies well separated. Can allow growth of organisms with lower oxygen tolerance in agar.
        • Disadvantages: colonies variable size, harder to see similarity in colony morphology between those on surface and in agar. Counting may be more difficult. Heat may kill some cells before agar cools and gels.
3. Optical techniques
   • Often, can estimate cell numbers accurately by measuring visible turbidity. Light scattered is proportional to number of cells.
   • This only works above cell densities of 10⁷ in pure cultures. With less than 10⁷ cells/ml, cannot detect bacteria.
     1. Eyeball method. This is not a precise measurement, but shoud allow estimation within an order of magnitude
        • no turbidity means less than 10⁷ cells/ml
        • slight turbidity = 10⁷-10⁸ cells/ml
        • high turbidity = 10⁸-10⁹ cells/ml
        • Very high turbidity = greater than 10⁹ cells/ml (cultures rarely get as high as 10¹⁰ cells/ml)
     2. Absorbance method
        • Use a spectrophotometer to accurately measure absorbance, usually at wavelengths around 400-600 nm.
        • Accurate measure of cells when concentration not too high. Easy and quick to measure (can sample in less than a minute)

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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: (see Table 6.5 for examples)
  1. minimum temperature, below which no growth occurs
  2. optimum temperature, at which fastest growth occurs
  3. maximum temperature, above which no growth occurs

Different microbes adapted to different temperature ranges

• Typical bacterium can grow over ~ 30 deg. C temp. range (stenothermal); some can grow over wider range (eurythermal).
  1. Psychrophiles -- optimum temp. typically 15 deg C or lower. Note: some organisms are psychrotolerant -- optimum temperature is 20-40 deg, but can grow as low as 0 deg. These are not considered psychrophiles.
  2. Mesophiles -- optima from 20-45 deg, minimum around 15-20 deg.
  3. Thermophiles -- optima 55 deg or higher. Some (hyperthermophiles) have optima of 80 deg or higher (mostly Archaea in this group). Found in hot springs, deep-sea hydrothermal vents, other locations.
  
  
  
• Physiological and structural adaptations are related to temperature:
  1. psychrophiles produce enzymes with lower temperature optima. They often denature at room temperatures.
  2. psychrophiles have higher unsaturated fatty acids in membrane lipids, keeps membranes fluid at lower temperatures.
  3. thermophiles have enzymes that are heat stable, also ribosomes work at higher temps. Only a few amino acid changes from mesophile proteins seem necessary in some cases to allow high temperature stability. Also more salt bridges in proteins.
  4. thermophile membranes have many long-chain fatty acids, lots of saturated fatty acids. membrane lipids "freeze" into solid form at what we would consider warm temperatures, thus inhibiting transport. But at very high temperatures, membranes function well.
  
  

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Effects of oxygen on growth

Note: higher organisms all require oxygen; we are not used to thinking about other alternatives. But many microbes grow anaerobically some or all of the time.

Terminology

1. Obligate aerobes -- grow only when oxygen is present
2. Facultative anaerobes -- grow with or without oxygen, grow better in oxygen (respire)
3. Aerotolerant anaerobes -- ignore oxygen, grow equally well with or without
4. Obligate anaerobes -- die in presence of oxygen
5. Microaerophiles -- won't grow at normal atmospheric oxygen (20%), but require some oxygen for growth (2-10%)

Anaerobic habitats more common than expected. Ex: in human mouth, plaque contains bacterial zoo. Facultative anaerobes consume oxygen, create anaerobic microenvironment fit for obligate anaerobes.In general, wherever organic matter accumulates, microbes will use up oxygen faster than it can be replaced, create anaerobic environment. Esp. true under water, since oxygen is poorly soluble in water. Lakes and ponds stratify into aerobic (upper) and anaerobic (lower) zones in summer due to vigorous microbial growth on sediments.

Why obligate anaerobes?

Oxygen itself is reactive (oxidizing agent), capable of degrading organic molecules. But oxygen can easily generate very toxic byproducts, strong oxidizing agents that react indiscriminately with any organic molecules, including DNA, proteins, etc.:

• superoxide (O₂^-)
  
• peroxide (H₂O₂
• hydroxyl radical (OH^.)

Aerobes (and all cells able to tolerate oxygen) must have enzymes to get rid of these radicals. Superoxide dismutase and catalase are two crucial enzymes.

superoxide (O₂^-)+ H⁺ ---(superoxide dismutase) O₂ + H₂O₂
peroxide (H₂O₂) ---(catalase) O₂ + H₂O

Note: If E. coli (a facultative anaerobe) is mutated so it loses these two enzymes, resulting mutant behaves like an obligate anaerobe -- good confirmation of idea.

Culture techniques

• for aerobes: shake or rotate culture to add more oxygen, or bubble filtered air through culture
• for anaerobes: use media with reducing agent (combines with oxygen chemically)
  pump out air, flush with pure nitrogen gas GasPak jar, seal plates in jar, use catalyst + hydrogen gas to remove oxygen

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Effects of pH on growth

• pH measures acidity. pH = log ₁₀ of H⁺ concentration.
• Pure water has pH of 7; 1 molar acid pH = 0. See figure 6.13 in text

Diff microbes have diff pH optima:

• 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

• Note: most bacteria are neutrophiles (Exceptions: some bact in hot springs have optimum of 1-3)
• But most fungi prefer slight acid (pH 4 to 6)
• Saboraud's Medium (used in lab first week) -- uses low pH to stop bacterial growth, selective for fungal growth.

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Effects of solutes and water activity on growth

• Cells require certain amount of free water to be able to carry out metabolism. When placed in hypertonic environments, many cells stop growing.
• Some can compensate, synthesize compatible solutes (= molecules whose function is to balance osmotic strength). Examples: choline, proline, betaine, glutamic acid, etc.
• Staphylococci are good examples; grow on skin, where salts are common. Staph can tolerate up to 10% salt; can design culture media with 7.5% salt, suppress growth of most other bacteria, select for Staph (will do this in lab later on this semester)
• Some bacteria require very high osmotic strengths for growth = Halophiles; Ex. Halobacterium halobium grows in Dead Sea, Great Salt Lake, evaporating salt flats. Won't grow if salt concentration much less than 3M!
• Note: these are members of Archaea; have very modified cell walls and membranes. Accumulate enormous amounts of potassium as compatible solute.

Effects of radiation on growth

• Light and UV are parts of EM spectrum; extends to very strong radiation (gamma rays), very weak radiation (heat, radio)
• Visible light (esp. more energetic violet and blue) are quite strong, can kill bacteria. Many bacteria that are spread by air are pigmented; pigments adsorb radiation, prevent damage to cell.
• Note: pigment-less mutant shows much more sensitivity to light than pigmented form.

Mechanisms of damage:

• light adsorbed by some pigment (e.g. cytochrome, flavin, chlorophyll), energy transferred to oxygen to generate singlet oxygen = very strong oxidizing agent, causes lots of damage
• UV light causes specific damage to DNA, max. effect at 260 nm --> thymine dimers
• Ionizing radiation causes many types of damage: breaks H-bonds, oxidizes many groups, can break DNA strands (most vulnerable target).

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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)

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1: Sterilizing Agents

A. Heat

1. Boiling. OK for most food, but not sterilizing. Endospore formers, hepatitis virus can resist.
2. Autoclaving. Most common sterilizing procedure. 15 min @ 121 deg. Celsius. Adequate for l liter volumes. Longer times for larger volume.
3. Dry Heat. Used for dry products. Typically 170-200 deg. C. overnite.
4. Pasteurization. Not a sterilizing treatment, but kills pathogens in milk. 63-67 deg. C. for 30', Now 71 deg. C. for 15 sec.

B. Membrane Filters.

1. 0.45 um filters retard bacteria. Good for heat-labile materials. Rapid. But expensive, and filters will clog.
2. View examples of membrane filters

C. Chemicals

• Ethylene Oxide = alkylating agent

  

• chemically adds on to proteins, nucleic acids, etc.
• Widely used (in special cabinets) to sterilize heat-sensitive items, e.g. syringes, disposable plastics, rubber items, surgical supplies, etc.
• usually carried out for 1-10 hours at 60 deg. C. Very flammable, must use special precautions. Products retain residues of gases. (e.g. petri dishes have enough to cause mutations in bacteria).

D. Radiation

1. UV light. Reacts with DNA, causes DNA damage -- death. Thymine dimers!. But much damage can be repaired, esp. if light available (PHOTOREACTIVATION). NOTE: cannot penetrate glass.
2. Ionizing radiation. Gamma rays produce free radicals, destroy all kinds of chemicals. E.g. OH^-

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

1. heavy metals (Mercury, Silver, Arsenic)- cause protein denaturation
2. halogens (Chlorine, Iodine, Hypochlorite)- oxidizing agents. Not usually used as antiseptics, but good for swimming pools, hot tubs, water supplies. Household bleach = 5% soln of hypochlorite- good for all-purpose disinfectant. But don't use with ammonium compounds or acids, can produce explosive gases (nitrogen tricholoride or chlorine gas).
3. phenols & cresols- dissolve membranes, denature proteins
4. alcohols- denature proteins, dissolve membranes.
5. detergents- dissolve membranes

Note: disinfectants are classified into 3 groups:

1. High level: effective against all life, incl. endospores. E.g. ethylene oxide, 2% glutaraldehyde. May require 10 hours to kill all pop of endospore-forming bacteria
2. Intermediate level: defined as tuberculocidal (kill Mycobacterium tuberculosis ), as well as more resistant viruses (hepatitis, rhinovirus). Not effective against endospores.
3. Low level: not effective against tuberculosis or endospores, or viruses without membranes. But do kill vegetative bacteria and fungi, used extensively. Economical, not overly toxic to humans. E.g. Lysol, detergents, mercurials.

4: Antimicrobial Chemotherapy

A. History:

1. Antibiotics known for long time= chemicals produced by certain organisms that killed other organism. E.g. mushroom poisons. Early searches for antibiotics (ca. 1900) had bad side-effects. People decided that therapeutic applications were probably too dangerous.
2. Ehrlich's "magic bullet": 1909, discovered "Salvarsan", chemical used to treat syphillis. Ehrlich stressed Selective toxicity as key factor in success.

B. Structural analogues as drugs

• 1935. Domagk discovered sulfa drugs.

  

• this drug prevented Staph aureus infections in vivo, but not on petri plates. Domagk discovered that compound is split in liver into two parts, including:

  

• Bacteria normally synthesize folic acide (cofactor for synthesis of bases for DNA and RNA) from PABA:

  

• But sulfanilamide is competitive inhibitor, blocks folic acid synthesis, blocks NA production.
• Humans can't make folic acid (vitamin requirement), hence are not affected.
• optimal use: E. coli urinary tract infections, certain protozoal infections, Nocardia infections. Not good if pus or dead tissue involved.
• Sulfa drugs had enormous impact on WWII. First battles in which more men died in battle than afterwards as result of infection.
• Note: later modified: sulfanilamide is insoluble in acidid urine, causes kidney problems. Chemical modification can produce drug that has same activity, but is more soluble in urine:

  

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C. Antibiotics:

• After Sulfa drug success, more search for drugs.
• Penicillin was discovered in 1929, not commercially developed until WWII.
• Since then major search for antibiotics. Found in 3 major groups of microorganisms:
  1. Certain molds (Penicillium, Cephalosporium). e.g. penicillin,cephalosporin
  2. Certain strains of Bacillus e.g. bacitracin
     
  3. Many strains of Actinomycetes (soil bacteria that grow in long filamentous masses). Especially from Genus Streptomycetes. e.g. streptomycin. Majority of antibiotics come from these organisms.

1. Cell Wall antibiotics

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

  

• Activity: binds to enzymes that carry out transpeptidation linkage in bacterial cells. Unique target in procaryotes.
• Note beta-lactam ring -- this is critical for activity.
• INITIAL PRODUCTION: 1-10 ug/ ml. Gradual strain improvement over years, today 85,000 ug/ml!!!
• BenzylPenicillin G -- low activity vs Gram-, ß-lactamase sensitive
• Modifications: side chain can be chemically modified. e.g.
  1. Methicillin, Oxacillin -- acid stable, ß-lactamase resistant
     Methicillin
     
     Oxacillin
     
     
  2. Ampicillin -- broader spectrum (esp. vs Gram-), acid stable, ß-lactamase sensitive.
     
     
  3. Carbenicillin -- broader spectrum (esp. vs Pseudomonads), acid stable but not effective orally, ß-lactamase sensitive
     
     
• Other antibiotics active against growth of cell wall: Cephalosporins, Cycloserine, Bacitracin.
  1. Cephalosporins (e.g. cefoxitin, cephalothin) are ß-lactam antibiotics, dihydrothiazine ring instead of thiazolidine ring. Broader spectrum than penicillins, low toxicity. Relatively resistant to pencillinase.
  2. Bacitracin does not block transpeptidation, but previous step in wall synthesis. Limited to topical application, because of severe toxic reactions.

2. Inhibitors of protein synthesis

• Aminoglycosides: (have amino sugars linked by glycosidic bonds). streptomycin, gentamicin, kanamycin, tobramycin, amikacin. Used mainly for Gram- infections. Not very effective against anaerobes or Gram+. Bind to 30s ribosomal unit, block protein synthesis. Also cause misreading of mRNA. Useful for a number of diseases. Different degrees of toxicity (e.g. Gentamicin is very toxic, only used for severe infections) .
  Amikacin is best antibiotic for Gram- rod hospital infections, because such infections (nosocomial) are often caused by strains with R-factors, resistant to many common antibiotics. Generally group is used as reserve antibiotics, when others fail.
• Tetracyclines. (4 ring system) Also bind to 30S subunit,block protein synthesis. Effective against variety of pathogenic bacteria- broad spectrum. Together with ß-lactam antibiotics, the most important group commercially.
  
• Glycopeptide antibiotics. E.g. Vancomycin.
  • Two antibiotics commercially available: vancomycin & teicoplanin.
  • Vancomycin (Vancocin) -- worldwide use. Discovered 1956, produced by fungus Amycolatopsis orientalis, from Indonesia. Drug has relatively high toxicity and requires IV administration. Currently the drug of last resort for treating methicillin-resistant staph infections.
  • Teicoplanin (Targocid) -- another glycopeptide, approved for use in some European countries in 1988, under clinical trials in U.S. and Canada. Has longer half-life than vancomycin, can be administered only once a day by intramuscular or IV. Is effective against some vancomycin-resistant strains.
• Macrolide antibiotics. E.g. Erythromycin. Large lactone rings connected to sugar groups. Binds to 50S ribosome, blocks protein synthesis. Most active vs. Gram+ organisms, eg. Strep. pyogenes. Now routinely applied to eyes of newborns to prevent gonnorhaea and chlamydia from infecting eye.
  

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D. Drug resistance

Testing for Drug Resistance

• Plate sensitivity test: (will be done in MCB 229 lab)
  1. add test bacteria to small amount of melted agar
  2. pour over surface of nutrient agar plate, let gel
  3. add paper disks with known dose of antibiotic to surface
  4. incubate: antibiotic will diffuse into medium as cells grow
  5. examine plate: look for clear zones around disk where growth is inhibited
  6. measure diameter of clear zones: consult table to find if this is clinically useful
  
  
  

History:

• Drug resistance first noted in Japanese hospitals; serious increase in bacterial strains resistant to variety of standard antibiotics.
• Since then, many examples of drug resistance developing. Ex: gonorrhea initially treated by pencillin. But pencillin-resistant strains now account for more than 25% of isolates, must use different antibiotic.
• Note: antibiotic resistance has always been present; frozen bacterial cultures from before WW II have been shown to include drug resistant individuals even though antibiotics weren't yet used by humans. Conclude that antibiotics are natural part of biological activity, not surprising that some resistance should have developed in course of evolution.
• What is new, and different, is rate of development of resistance. Some disesase, like TB, never easy to treat even with the few antibiotics that were effective. Now drug resistant strains appearing, TB becoming much harder to treat.

Different ways for bacteria to develop drug resistance

1. mutations affecting cell surface can affect entry of drug
   • prevents entry of drug into cell
2. receptor normally used by drug altered- no binding.
   • example: mutations can affect drug target in cell (e.g. slight change in ribosomal RNA can change affinity of ribosomes for erythromycin)
3. bacteria or plasmids can produce enzymes which inactivate drug; e.g. pencillinases hydrolyze ß-lactam ring.
   • Plasmids = small, circular DNA elements that reside in bacterial cells, duplicate separately from bacterial chromosome.
   • Some plasmids carry genes for antibiotic resistance (enzymes that degrade antibiotic). Called R-plasmids. Have been found for most classes of antibiotics.
   • When antibiotics are in use, most bacteria are killed. If R-plasmid exists, can be transferred to other cells, resistance spreads through population. Result: new population is resistant to drug.
   • Note: possible for a single plasmid to carry multiple drug resistance genes, spread all of these as a single unit!
4. plasmid encoded drug pump
   • production of protein "pumps" to pump drug out of cell

Ways to deal with antibiotic resistance

1. higher dose, different antibiotic, more than one drug simultaneously
2. also restraint by physicians and control (no over the counter use)
3. CORRECT use of drug. Most people take drugs improperly, miss doses, allow conditions that favor selection of drug resistant mutants.

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