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)
•Viruses
Most 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.
How Cells Grow:
ΣS
+
X
=
nutrients
+ cells
=
ΣP
+
extra-cellular products
n. X
+ 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 = dX
net spec. growth rate
dt
 1 dX 


 X dt 
 1 dX 
 gross
X
dt


net = 
- 
1 dX 

 X dt 
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
1 dN
N dt
Microbial Growth and its Control:
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.
Measurement of growth
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.
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 Zterm
• 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.
B.
C.
D.
Heat: boiling, autoclaving, dry heat. pasteurization.
Membrane Filters.
Chemicals – name of chemical ?
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 ßlactam 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