BIOL 2421 Microbiology
Lecture Notes
Dr. Weis

Bacterial Reproduction

• Metabolic activity designed to increase the number of cells
• Asexual process known as binary fission. 
• Other methods for prokaryotes include budding and fragmenting.

            Binary Fission steps

1)     DNA is duplicated by continuous synthesis (vs. semi conservative)

2)     Cells elongate

3)     Cell wall and Plasma membrane pinch to divide into two parts

4)     Cross walls form around DNA, nuclear material is distributed

5)     Cells separate

Genetic Recombination can take place through three mechanisms that transfer viable amounts of DNA

a)     Transformation: external DNA is incorporated by bacterial cells

b)     Conjugation: direct transfer of genes from one bacteria to the next

c)     Transduction: transfer of genes between bacteria via viruses

End Results: growth to increase the population of a discrete bacterial colony.

Generation Time is the time from one binary fission to the next, usually 1-3 hours but can extend to 24 hours. Short generation times allow prokaryotes to adapt to rapidly changing conditions, such as temperature and pH. Their population doubles every generation time and this is known as geometric progression.

            Mutations and genomes from recombination have created

·       diversity

·       success

·       variety of nutritional and metabolic mechanisms

Chemical nutrients required to build organic molecules and cell structures include

• Macro Elements: C, H, N, O2, Na P, K, S, CL, Ca, Mg which make up 99.5% of the cell
• Trace Elements: Fe, Co, Cu, Mn, Ni, Se, Zn, Si which usually functions as cofactors for enzymes

All nutrients for the cell must be transported via passive or active processes. Any disruption in the amount of available nutrients to derive needed elements or problem with transport processess will affect growth and thus the generation time.

Generation times are expressed as a power of 2

                        2º = 1 cell

                        2¹ = 2 cells

                        2² = 4 cells

                        2³ = 8 cells

Therefore logarithmic representations are needed

                        5 generations (2 to the 5^th power) = 32 cells

                        10 generations (2 to the 10^th power) = 1024 cells

Growth curves are established that are based on generation times. 

The curve consists of four phases:

1)     Lag phase – adaptation to new environment, store nutrients, prepare for binary fission, increased metabolism.  Can last for 1 hour to days.

2)     Log phase – active growth stage, exponential as generation time doubles.

     Most metabolically active phase, sensitive to any environmental conditions

      Disease conditions and immunological responses occur during this phase.

3)     Stationary phase – growth = death due to decreased nutrients, increased waste production, changes in pH, and the response of the immune system..  Slowing of metabolic activity

4)     Decline phase – exponential death phase, as death > growth.  Possible spore formation

Measurement of Microbial Growth

·       Cell numbers = plate count of visible colony forming units (CFU)

·       Population’s total mass

·       Serial Dilutions

            1 ml sample with 9 ml sterile H20

a)     Pour Plate: sample with heated agar, mixed, re-plated, colonies grow in media

b)     Spread Plate: inoculate spread over agar, colonies grow on surface

·       Filtration: water through filter, filter to dish with medium for growth

·       Most probable number (MPN): statistical estimate

·       Direct Microscopic Count: sample on slide and stained

            Calculate number in each field of view on special counter

Indirect Measurement of Growth

         * Turbidity: spectrophotometer to measure % transmission/absorbance

         * Metabolic activity: CO2 production

         * Dry Weight – specimen removed, filtered, dried

Bacterial Groupings Based on Growth Requirements

                        ~ physical: pH, temperature, osmotic pressure

                        ~ chemical: O2, element source/availability for C, S, N, P, etc.

A>  Temperature requirements (minimum, maximum, optimal). Temperature is important in maintaining hydrogen bonds that help form the 3D structure of a molecule.

a.      Psychropiles  0ºC to 20ºC; 5ºC

b.     Pshycrotrophic : variant of psychrophile, bridges psychrophile/mesophile range 0 C to 30 C, prefers room temperature 15-25 C

c.      Mesophiles 20ºC to 40ºC; 37ºC

d.     Thermophiles 40ºC to 90ºC; 60ºC

e.      [Extreme] Hyperthermophiles above 100ºC

Temperature Group Summary Chart

Group	Minimum Temp	Optimum Temperature	Maximum Temperature	Comments
Psychrophil	Below 0	10-15	Below 20	best at low temp
Psychrotroph	0	15-30	Above 25	prefer moderate temp refrigeration
Mesophile	10-15	30-40	Below 45	most bacteria live in association with warm blooded animals
Thermophile	45	50-85	above 100 [boiling]	wide variation of optimum and maximum temp

B>  Oxygen requirements (aerobic vs anaerobic): Atmospheric [ATM} O2 is at 21% of air at sea level

a.      Obligate Aerobes – require the presence of oxygen, as O2 is the final electron acceptor in aerobic respiration

b.     Microareophiles – limited amounts of oxygen, normally below 0.2 ATM (2% to 10% at maximum)

c.      Obligate Anaerobes [aka Aerophobes] – absence of O2, if O2 is present, will create a toxic free radical O2-

d.     Aerotolerante anaerobes – tolerate O2, do not use O2, but also, these organisms do not create toxic O2- free radicals

e.      Faculative anaerobes – can grow in the presence or absence of O2. Anaerobic growth by fermentation; Aerobic growth by aerobic respiration

f.       Capnophilic – decreased O2, increased CO2

                      
 Normal form of oxygen: O2

                       Toxic forms of O2 occur because O2 is highly reactive and can steal electrons (oxidize) other compounds. Thes reactive oxygen species (ROS) can accumulate and further damage cells beyond repair.

Exampes of Toxic forms of O2

• Singlet Oxygen [1,O2]: electrons in higher energy state
• Superoxide Radical [O2-]: incomplete reduction of O2 during ETC process. Must be detoxified with enzyme: Superoxide dismutases [SOD] to convert the oxygen radical to hydrogen peroxide and molecular oxygen (O2).
• Peroxide Anion [O2 =] : contained within the hydrogen peroxide that is generated by other reactions. Enzyme needed to detoxify peroxide anion can be
  1. Catalase: converts Hydrogen peroxide to water and oxygen
  2. Peroxidase: breaks down hydrogen peroxide to water and a hydrogen ion carrier (NAD+)
• Hydroxyl Radical [OH*]: incomplete reduction of hydrogen peroxide usually due to ionizing radiation resulting in water and the hydroxyl radical. OH* is the most reactive toxic form of oxygen.

Other antioxidants such as Vitamin C and Vitamin E help protect against ROS.

                 
Summary of Enzyme presence with Different O2 tolerances

C>  pH requirements (range 2-9, most prefer pH around 7.0) Range : minimum, optimal, maximum

a.      acidophiles – acid tolerant (pH 4-6), usually seen with fungi, yeast, molds, Archeae

b.     neutrophils – pH 6.5-7.5; most bacterial groups

c.      alkalinity inhibits most microbe growth, but alkinophiles can be found in the environment (soil and water) at a pH range of 8.5 - 12.0; Nitrobacter sp.

D> Osmotic Pressure (Water movement due to solutes such as salts)

                  a. Extreme Halophiles – 30% salt; Archaea

                  b. Obligate [moderate] Halophiles – 15% salt

                  c. Faculative [mild] Halophiles - ~ 2% salt

Tonicity

• Hyptertonic solutions (10%) creates plasmolysis, shriveling of the cell
• Isotonic solutions (0.85%) allows for normal organism structure and function
• Hypotonic (distilled water) causes lysis

Bacteria that can grow in moderate salt conditions, but that do not need NaCl are halotolerant

The osmotic pressure of water can exert hydrostatic pressures in proportion to its depth. Barophiles are organisms that can withstand a great amount of hydrostatic pressure and therefore are found at the depths in the ocean. The water activity [Aw] of pure H20 is 1.0 [100% water]. Water activity is affected by the presence of solutes such as salts or sugars, that are dissolved in the water. The higher the solute concentration of a substance, the lower is the the water activity and vice-versa. Most microorganisms can live in an Aw can range from 1.0 to 0.7. Examples are listed below

Spirillum require 1.0 Aw
E. coli require 0.91 Aw
Lactobacillus require 0.90 Aw
Staphylococcus require 0.85 Aw

• The Aw of human blood is 0.99
• Aw of seawater = 0.98
• Aw of maple syrup = 0.90
• Aw of Great Salt Lake = 0.75
  

E> Chemical Requirements: 95% of the bacteria is made up of C, H, N, O

                Carbon makes up ½ [50%] of the bacteria,

                                    From proteins, CH2O, lipids à Chemoheterotroph

                                    From CO2 à Chemoautotroph, Photoautotroph

                N, S, P

                        Needed for synthesis of cellular material

                                       1) Protein synthesis requires N, S

                                       2) NA synthesis requires N, P

Nitrogen requirements for proteins and NA production as well as metabolism are also important as it makes up 14% of the cell.
  Certain bacteria can convert one nitrogen product to the other

                        NH3 to NO2-

                        NO2- to NO3- + N2

                        N2 to NH3

                         Nitrogen makes up 14% of the bacterial cell

                                     Use:  NH3 of AA, NH4+ for other cell material

                                              NO3- for metabolism

                                              N2 as byproduct

     Sulfur –

                  Use: Sulfur containing AA

                          Vitamins (Thiamine, Niacin)

                  Source: H2S, SO4=

   Phosporus-

                 Use: NA synthesis

                          Phospholipids of cell membrane

                          ATP

                 Source: PO4=

Others macro elements: : K+, Mg++, Ca++ are used as cofactors for enzymes

 Other important trace elements: Fe, Cu, Mb, Zn are also used as cofactors for enzymes

Summary of Nutritional Elements

F> Nutrional Requirements Overview

a. Autotrophy = synthesize own food from carbon source, Energy from sun

                                                    i.     Phototrophs – Energy from light

b. Heterotrophy = obtain Carbon from organic sources in environment

                                                  ii.     Chemotrophs (from environment)

         Various combinations of the above give us four possible groupings

1)     Photoautotrophs : Use light energy to synthesize organic compounds from CO2 (photosynthetic eukaryotes)

2)     Chemoautotrophs: require CO2 as carbon source and obtain energy from inorganic compounds such as H2S, NH3, Fe++ (Archaea bacteria)

3)     Photoheterotrophs: Light energy to synthesize ATP from organic sources

4)     Chemoheterotrophs: must obtain organic molecules for energy and carbon source.  (Many bacteria, most all eukaryotes)

Additional classifications can also be made regarding the aquisition of hydrogen, which is usually always available (H2O).

• Organotrophs: acquire electrons from same organism that provided C and ATP
• Lithotrops: acquire electrons or H+ from inorganic compounds such as NO=, H2S, Fe++
  

Since most bacteria are chemoheterotrophs, they can be classified into two subgroups:

(a)   Saprobes – decomposers  that absorb nutrients form dead organic matter

(b)  Parasites – absorb nutrients from living host

D>  Relationships – Symbiosis (the interaction of two populations)

a.      Mutualism: benefits both

b.     Commensalism: benefits one, the other neutral

c.      Synergism : both interact to increase benefit of either separately

d.     Parasitism: one benefits, the other is harmed.

Biofilms also consist of complex relationships using cell receptors and signaling molecules to allow growth and response to environmental changes and allow the expression of suppressed genes that enable the group to have alternate functions not normally seen in an individual colony. About 65% of bacterial diseases are the result of biofilm formation such as the dental plaque found on teeth.

Most Bacteria also require a small amount of certain organic compounds for growth, as they may not be able to synthesize them from available nutrients. Such compounds are called growth factors.

• Purine and pyrimadines: Synthesize NA [DNA, RNA]
• Amino Acids: Synthesize proteins
• Vitamins: Need as coenzymes and functional groups of enzymes

Some bacteria may not need growth factors and can synthesize them during intracellular metabolism. Other require growth factors that must be provided exogenously.

For any bacterium to be propagated for any purpose it is necessary to provide the appropriate biochemical and biophysical environment.

Bacterial Cultivation

            Nutrient material prepared for microorganism growth

                        a) Inoculum (introduce) a sample of microorganisms to a selected medium for culture. Sources can include environmental and clinical.

                        b) Culture (growth) microorganisms in broth or for isolation of pure cultures on solid media

            Chemicals used

                        a) Provide Energy source (C, N, S, P)

                        b) Organic growth factors

            Complex Media: contain mixture for Energy, C / N / S source, growth factors, AA

Agar is used to form the basis of solid media and is formed from an undigestable hydrocolloid polysaccharide derived from red algea that can be liquified and poured into petri dishes for plates, or tubes to create slants or deeps. Additional components are added to form various types of media for culture:

1) Chemically Defined Media : known chemical composition to study the minimal nutritional requirements for a physiological process

2)     General Use Complex Undefined Media– Nutrient Broth, Nutrient Agar (petri dish, slants, deeps)

3)     Enriched Complex Media – special nutrients (Blood agar, Nitrogen Enriched)

4)     Selective – Ingredients removed or added such as dyes or salts that inhibit or stimulate growth (MSA, SS)

5)     Differential – Distinguish between colonies based on media or colony changes; can stimulate or inhibit growth (MAC, Blood Agar, EMB, TSI)

6)     Combinations : Selective and Differential Media (MSA, MAC)

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            Anaerobic Growth Media

                        1) Reducing media (deplete O2) [Thioglycollate]

                        2) Container that produces H2 & CO2 by chemical reactions and special disc [Gas Pack]

            Special Techniques

                        1) Living specimens and cells for culturing leprosy, Rickettsia, Chlamydia

                        2) CO2 incubators for culturing Campylobacter

            Isolation of pure colonies [CFU] from streak plate or other growth meida using aseptic techniques

                              Gram Stain to verify pure colony

                              Select specific tests for final identification of Genus and species

            Preservation of colonies

* Refrigeration for short periods

                            * Deep freezing (-50°  C to – 90°  C)

                            * Freeze drying (-54º  C to -72 ° C) with water removal. Process called lyophilization