Download Microbial physiology. Microbial metabolism. Enzymes. Nutrition

Microbial physiology.
Microbial metabolism.
Enzymes. Nutrition.
Bioenergetics. Bacterial
growth and multiplication.
Growth and multiplication
mode: Binary fission
Bacterial Cell Division
1. Replication of chromosome
2. Cell wall extension
3. Septum formation
4. Membrane attachment of
DNA pulls into a new cell.
Growth
 It is an increase in all the cell components,
which ends in multiplication of cell leading
to an increase in population.
 It involves - an increase in the size of the
cell & an increase in the number of
individual cells.
 Bacteria divide by binary fission.
Generation time
 Interval of time between two cell divisions
OR
 The time required for a bacterium to give
rise to 2 daughter cells under optimum
conditions
 Also called population doubling time.
Generation time
 Coliform bacilli like E.coli & other medically
important bacteria – 20 mins
 Staphylococcus aureus- 27-30 mins
 Mycobacterium tuberculosis - 792-932 mins
 Treponema pallidum -1980 mins
Growth form in Laboratory
 Colony – formed by bacteria growing on
solid media. (20-30 cell divisions)
 Each bacterial colony represents a clone of
cells derived from a single parent cell.
 Turbidity – liquid media
- 107-109 cells/ml
 Biofilm formation – thin spread over an
inert surface.
Solid medium
Colony
Liquid medium
Bacterial counts
 Cell Counts ... many ways
 2 methods – Total cell count
- Viable cell count
Total Count

Total number of cells in the sample = living
+ dead.
Can be obtained by :
 Direct counting under microscope using
counting chambers.

Counting in an electronic device – Coulter
counter.
Counting chambers
Over method

Direct counting using stained smears - by
spreading a known volume of culture over a
measured area of slide.

Opacity measurements using an
absorptiometer/ nephalometer.

Chemical assays of cell components.
Turbidity- a spectrophotometer
measures how much light gets through
Viable Cell Count

Measures the number of living cells.

Methods – Surface colony count



Dilution method
Plating method
Number of colonies that develop after
incubation gives an estimate of the viable
count.
Plate counts
Bacterial Growth Curve
 When a bacterium is added to a suitable liquid
medium and incubated, its growth follows a
definite course.
 If bacteria counts are made at intervals after
inoculation & plotted in relation to time, a growth
curve is obtained.
 Shows 4 phases :
 Lag,
 Log or Exponential,
 Stationary
 Decline.
Phases of Growth Curve
 1. Lag phase – No increase in number
but there may be an increase in the size
of the cell.
 2. Log OR Exponential phase – cells
start dividing and their
increases exponentially.
number
Phases of Growth Curve
 3. Stationary phase – cell division stops
due to depletion of nutrients &
accumulation of toxic products.
- equilibrium exists between dying cells and
the newly formed cells, so viable count
remains stationary
 4. Phase of Decline – population decreases
due to the death of cells – autolytic
enzymes.
Morphological & Physiological
alterations during growth
 Lag phase – maximum cell size towards the end of
lag phase.
 Log phase – smaller cells, stain uniformly
 Stationary phase – irregular staining, sporulation
and production of exotoxins & antibiotics
 Phase of Decline –involution forms(with ageing)
Factors Affecting Bacterial
Growth
 Availability of Nutrients & H2O
 Temperature
 Atmosphere – O2 & CO2
 H-ion concentration
 Moisture & drying
 Osmotic effects
 Radiation
 Mechanical & sonic stress.
Bacterial Nutrition
 Water constitutes 80% of the total weight of
bacterial cells.
 Proteins, polysaccharides, lipids, nucleic acids,
mucopeptides & low molecular weight compounds
make up the remaining 20%.
Moisture & Drying
 Water – essential ingredient of bacterial
protoplasm. Hence drying is lethal to cells.
 Effect of drying varies :


T. pallidum – highly sensitive
Staphylococci sp– stand for months
 Spores – resistant to desiccation, may
survive for several decades.
Nutrients
Functions
–
–
–
Generation of energy
Synthesis of cellular materials
Essential nutrients (basic bioelements needed for bacterial
cell growth)
–
–
–
–
–
–
H2O: universal solvent; hydrolyzing agent
Carbon: food & E* source; in form of prot., sugar, lipid
Nitrogen: for prot. syn; nucleic acid syn (purines &
pyrimidines)
Sulfur (sulfate): AA syn (i.e., Cystine)
Phosphate: key component of DNA & RNA, ATP, and
inner & outer membrane phospholipids
Minerals: assoc’d w/ PRO (i.e., Fe:PRO); common
component of enzymes.
Nutrients
2 types
1.
Macronutrients – needed in large quantities
for cellular metabolism & basic cell structure

2.
Micronutrients – needed in small quantities;
more specialized (enzyme & pigment
structure & function)

–
C, H, O, N
Mn, Zn
Fastidious Bacteria: microbes that
require other complex - nutrients/growth
factors ( i.e., Vitamins or AAs)
Temperature
 Vary in the temperature requirements.
 Temperature range – growth does not
occur above the maximum or below the
minimum.
 Optimum Temperature – growth occurs
best, 37ºC for most pathogenic bacteria.
Uptake of nutrients by
bacteria
o
Passive diffusion
o
o
o
simple diffusion
facilitated diffusion
Active transport
 Psychrophiles: -10 to 20C
 Psychrotrophs: 0 to 30 C
 Mesophiles: 10 to 48C
e.g. most bacterial pathogens
 Thermophiles: 40 to 72C
 Hyperthermophile: 65 to 110C
29
 Some pathogens can multiply in the
refrigerator: Listeria monocytogenes
30
H-ion Concentration
 Neutral or slightly alkaline pH (7.2 – 7.6) –
majority of pathogenic bacteria grow best.
 acidic pH – Lactobacilli
 alkaline pH -Vibrio cholerae
Osmotic Pressure or Osmolarity

Most bacteria require an isotonic environment or
a hypotonic environment for optimum growth.

Osmotolerant - organisms that can grow at
relatively high salt concentration (up tp 10%).

Halophiles - bacteria that require relatively high
salt concentrations for growth, like some of the
Archea that require sodium chloride
concentrations of 20 % or higher.
Similar effect with sugars
34
Radiation, stress
 Radiation
 X rays & gamma rays exposure – lethal
 Mechanical & Sonic Stress
 May be ruptured by mechanical stress.
Growth Factors
Some bacteria require certain organic
compounds in minute quantities – Growth
Factors OR Bacterial Vitamins.
It can be :
 Essential – when growth does not occur in
their absence.
 Accessory – when they enhance growth,
without being absolutely necessary for it.

Growth Factors
Identical with eukaryotic nutrition







Vitamin B complex –
thiamine
riboflavine
nicotinic acid
pyridoxine
folic acid &
Vit.B 12
Presence or Absence of Gases
 Primary gases = O2, N2, & CO2

O2 - greatest impact on microbial growth
(even if the microorganism does not require it)
 Aerobic respiration – terminal electron
acceptor is oxygen.
 Anaerobic respiration – terminal electron
acceptor is an inorganic molecule other
than oxygen (e.g. nitrogen).
Depending on the O2
requirement
Strict (Obligate) Aerobes – O2 present, require O2 for growth

e.g. Pseudomonas aeruginosa


Obligate aerobe – 20% O2: only grows with O2
Microaerophile – 4% O2: best growth with small amount O2

e.g. Campylobacter spp, Helicobacter spp
Strict (Obligate) Anaerobes – O2 depleted, grow in the absence
of O2 & may even die on exposure to O2
e.g. Bacteroides
fragilis



Obligate anaerobe: only grows in absence of O2
Aerotolerant anaerobe: anaerobes that “tolerate” +/or survive in O2, but
do NOT utilize O2 during E* metabolism


e.g. Clostridium perfringens
Facultative Anaerobe – grows both in presence & absence of O2;
but grows BEST under Aerobic conditions; considered to be
aerobic organism; O2 present – aerobic respiration for E*; O2 absent –
anaerobic pathways (fermentation)


e.g. Staphylococcus spps
Capnophilic organism – requires high CO2 levels eg Neisseria
spps
Oxygen-related growth zones in
a standing test tube
 Oxygen is readily converted into radicals
(singlet oxygen, superoxide, hydrogen
peroxide, hydroxyl radical)
 Most important detoxifying enzymes are
superoxide dismutase and catalase
 Cells differ in their content of detoxifying
enzymes and hence, ability to grow in the
presence of oxygen
42
 Classification of gram-positive cocci
 Staphylococci are catalase +
 Streptococci are catalase -
Staphylococci
Streptococci
43
pH
Majority of bacteria grow BEST at neutral or
slightly alkaline pH

pH 7.0 – 7.4 => this is near most normal body
fluids
•

Acidophiles: grow BEST at low pH (acid: pH 0
– 1.0)


T.B. - pH 6.5-6.8
Alkalophiles: grow BEST at high pH (alkaline:
pH 10.0)

V. cholerae - pH 8.4-9.2