Contains slides describing essential elements for bacterial growth, bacterial growth curve, mechanism of energy production and metabolism, principle of in-vitro bacterial culture

1. BACTERIAL GROWTH AND
PHYSIOLOGY
Dr. Tarek Mahbub Khan
MBBS, M.Phil
Assistant Professor
Department of Virology
tarekviro@yahoo.com
SSMC2017

2. TLO
Students will be able to:
• explain bacterial growth and generation time
• describe bacterial growth cycle
• describe aerobic and anaerobic growth
• describe nutrient requirements and metabolism by
bacteria
• define the terminologies related to in-vitro culture
• outline steps of bacterial culture in the laboratory
• outline of bacterial classification
tarekviro@yahoo.com
SSMC2017

3. GROWTH
• Growth is an orderly increase of sum of a total
of an organism.
• Bacterial growth is measurable:
– Cell concentration: Number of cell increases per
unit volume of culture
– Biomass density: Dry weight of bacterial cell
increases per unit volume of culture
tarekviro@yahoo.com
SSMC2017

4. BACTERIAL GROWTH
• Bacteria reproduced by binary fission a process by
which one parent cell divides to form two progeny
cell
• Bacterial growth is exponential (logarithmic): 2n
Number of cells 1 2 4 8 16
Exponential 20 21 22 23 24
tarekviro@yahoo.com
SSMC2017

5. DOUBLING TIME or GENERATION
TIME
• Time required by a bacterium to become double in
number or double in biomass concentration
• Varies according to the species
– EXAMPLE: 20 minutes for Escherichia coli, 24 hours for
Mycobacterium tuberculosis
• Bacteria with long generation time may have TWO
important significance:
– May require prolong duration of Antibiotic treatment
– Chance of development of antibiotic resistance
tarekviro@yahoo.com
SSMC2017

6. CALCULATION OF GENERATION TIME
Generation time (G) = (t1- t2) = 2.3 log10 (N2/N1) /Ƙ
t1- t2 = time interval in hours or minutes
N1= number of bacteria at the beginning of time interval
N2= number of bacterial at the end of the time interval
k= Growth rate constant
Example: What is the generation time of a bacterial
population that forms turbidity in the liquid culture medium
at the final cell concentration of 10 7 cells per ml (growth rate
constant is 0.02 /h?
tarekviro@yahoo.com
SSMC2017

7. FACTORS THAT MODIFY GENERATION
TIME
• Nutrients of the bacterium
• Required temperature
• PH
• Bacterial species
• Other environmental factors
tarekviro@yahoo.com
SSMC2017

8. BACTERIAL GROWTH CURVE
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SSMC2017

9. BACTERIAL GROWTH CYCLE
• Lag phase
– Last few minutes to few hours
– Cell adaptation starts in a new environment
– Can be a continuation from the previous death phase
– Accumulations of nutrients initiate this phase
– Metabolic activities occurs
– Cells do not divide
– Surface acting antimicrobials can inhibit the cell growth at
this stage
tarekviro@yahoo.com
SSMC2017

10. • Log phase
– Rapid cell division occurs
– Steady state growth though number of viable cell increases
– Phase continue until nutrients become exhausted
– Cell wall acting antibiotic (e.g., β Lactam drug) acts in this
phase, as cell divides
tarekviro@yahoo.com
SSMC2017
BACTERIAL GROWTH CYCLE

11. • Stationary phase
– Depletion of nutrient
– Toxic product accumulates
– Bacterial growth become slow
– Number of viable and death bacteria counts equal
– Production of spore, release of toxin
BACTERIAL GROWTH CYCLE
tarekviro@yahoo.com
SSMC2017

12. • Death or declining phase
– Nutrient exhausted, toxin accumulates more
– Marked decline in number of viable bacteria
– Death rate decreases after majority of the cells have died
– Small amount of viable cells may enter into lag phase to
continue with the cycle
tarekviro@yahoo.com
SSMC2017
BACTERIAL GROWTH CYCLE

13. GROWTH REQUIREMENT
• ESSENTIAL NUTRIENT
– Carbon, Nitrogen
• TRACE ELEMENT
– Ca, Mg, Iron, sulphur, phosphate, Mn
• ENVIRONMENTAL FACTORS
– Oxygen: Aerobic, anaerobic, microaerophillic
– Carbon dioxide: Capnophillic
– PH
tarekviro@yahoo.com
SSMC2017

14. ESSENTIAL NUTRIENTS
• Carbon source:
– Autotrophs:
• Do not require organic carbon
• Use photosynthetic energy to reduce CO2 to H1O
– Heterotrophs:
• Needs organic carbon for growth
tarekviro@yahoo.com
SSMC2017

15. • Nitrogen source:
– Major componant of protein and nucleic acid
– Comprises 5% of the dry weight of bacterial cell
– Inorganic nitrogen is very prevalent
– Bacteria assimilate nitrogen from NH3 ( N2 fixation)
– NH3 can be use in organic matter
•
tarekviro@yahoo.com
SSMC2017
ESSENTIAL NUTRIENTS

16. • Sulfur:
– Component of many organic cell substances
– Can be used to for co-enzymes
– Many organism use sulphate sulfur source and reduce
sulphate to H2S
tarekviro@yahoo.com
SSMC2017
TRACE ELEMENT

17. • Iron:
– Require as co-enzymes of cytochrome and peroxidase.
– Irons are capture and transported by siderophores, a
molecule produced by bacteria
• Mg2+ and K+ are essential for function and integrity of
ribosome
tarekviro@yahoo.com
SSMC2017
TRACE ELEMENT

18. GROWTH FACTORS
• Growth factor is an organic molecule that is required
by the bacteria but can not synthesize by them.
• Factor ‘V’ and factor ‘X’ necessary for the growth of
Haemophilus influenzae
tarekviro@yahoo.com
SSMC2017

19. PH
• Most bacteria grow at neutral PH
– Neutralophiles: Grows best at between 6.0-8.0
– Acidophiles: Grows best as below as 3.0, e.g., Lactobacillus
– Alkaliphiles: Grows best above 10.5, e.g., Vibrio cholerae
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SSMC2017

20. TEMPERATURE
• Psychrophilic: Grows best at low temperatures (15–
20 °C)
• Mesophilic: Grow best at 30–37 °C
• Thermophilic: Grow best at 50–60 °C.
• Hyperthermophilic : Grows at well above the
temperature of boiling water
Most of the bacteria are mesophilic
tarekviro@yahoo.com
SSMC2017

21. GROWTH ON THE BASIS OF O2
REQUIREMENT
• Aerobic
• Anaerobic
– Strict or obligate anaerobe
– Facultative anaerobe
• Microaerophillic
tarekviro@yahoo.com
SSMC2017

22. AEROBIC BACTERIA
• Bacteria who utilize O2 to generate ATP are aerobic
bacteria
• O2 generates TWO toxic metabolites:
– Super oxide
– H2O2
• Aerobic bacteria have TWO enzymes that neutralize
these reactive molecules
• EXAMPLE: Mycobacterium tuberculosis
tarekviro@yahoo.com
SSMC2017

23. HOW AEROBIC BACTERIA SURVIVE?
• 2O2 + 2H+ H2 O2 + O2
Superoxide desmutase
• 2H2 O2 2H2 O + O2
Catalase
• Anaerobic bacteria lack these enzymes, so they
can not survive in presence of O2
tarekviro@yahoo.com
SSMC2017

24. ANAEROBIC BACTERIA
• STRICT ANAEROBE:
– Bacteria that can not grow in presence of oxygen
– Some anaerobe can survive but can not grow
– EXAMPLE: Clostridium tetani
• FACULTATIVE ANAEROBE:
– They can utilize oxygen when available
– They can even survive in low oxygen concentration by
fermentation process to generate ATP
– EXAMPE: Escherichia coli
tarekviro@yahoo.com
SSMC2017

25. MICROAEROPHILIC
• Bacteria that grow in low oxygen concentration
• Many Microaerophilic bacteria are capnophilic (e.g.,
require 5%-10% CO2)
• Example: Campylobacter require 5% ,
Neisseria meningitidis, Haemophilus influenzae
tarekviro@yahoo.com
SSMC2017

26. • Bacteria need energy for metabolism
• Metabolism is necessary for bacterial growth,
multiplication, motility, toxin production and
luminescence
• In bacterial growth macromolecules are synthesized from
subunits (e.g., proteins from aminoacids)
tarekviro@yahoo.com
SSMC2017
BACTERIAL GROWTH AND
METABOLISM

27. MICROBIAL GROWTH
• Growth requires polymerization of basic biochemical
molecules to protein, nucleic acid, lipid and polysaccharide
• Polymerization requires transfer of anhydrate bonds from ATP
• Growth requires metabolic energy for:
– Synthesis of anhydrate bonds
– Maintenance of ionic gradient across the cell membrane
tarekviro@yahoo.com
SSMC2017

28. ATP and Anhydrate bond
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SSMC2017
Pyrophosphate bond

29. tarekviro@yahoo.com
SSMC2017
SOURCES OF ENERGY
ENERGY SOURCE
LIGHT
(Photosynthetic
bacteria)
OXIDATION OF
CHEMICAL SUBSTANCES
(Chemosynthetic
bacteria)
Inorganic substances
(Lithotrophs or
Autotrophs)
Organic substances
(Organotrophs or
Heterotrophs

30. METABOLIC ENERGY
• Sources:
– Fermentation
– Respiration
– Photosynthesis
• Uses:
– Formation of high energy phosphate bonds in the
macromolecules
– Generation of proton motive force
tarekviro@yahoo.com
SSMC2017

31. • FERMENTATION:
– Is a substrate phosphorylation
– Fermentable substrates are: Glucose, lactose, arginine
– ADP is converted to ATP with a pyrophosphate bond
– Pyrophosphate bond is donated by phosphorylated
metabolic intermediate
– Phosphorylated metabolic intermediates are formed by
metabolic rearrangement of the substrates
tarekviro@yahoo.com
SSMC2017
METABOLIC ENERGY

32. FERMENTATION: Substrate
phosphorylation
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SSMC2017
Fermentable Substrate
Metabolic rearrangement of the substrate
Hydrolysis of the ATP: Breakdown of
the terminal pyrophosphate /
anhydrate bond
Release of
energy
Generation of pyrophosphate bond
Conversion of ADP to ATP

33. • RESPIRATION:
– Energy dependant process
– Needs reductant, oxidant and electron carrier
– Results in movement of a proton (proton motive force)
across cytoplasmic membrane of the bacteria that
generate free energy
– Common reductant(donate electron): lactic acid, H2 gas
– Common oxidant(receive electron): O2 , CO2 , SO4
2- ,NO3
-
tarekviro@yahoo.com
SSMC2017
METABOLIC ENERGY

34. • PHOTOSYNTHESIS:
– Almost similar process as respiration
– Oxidant and reductant are produced by light energy
absorbed by pigments in the bacterial cell membrane
tarekviro@yahoo.com
SSMC2017
METABOLIC ENERGY

35. BACTERIAL METABOLISM
Superoxide dismutase, Catalase
Present in aerobic bacteriatarekviro@yahoo.com
SSMC2017

36. CULTURE OF BACTERIA
Artificial growth in the laboratory requires:
– Culture medium: an artificial food for the bacteria
– Inoculation: Seeding of bacteria in culture medium
– Incubation: Provision of proper condition for the growth of
bacteria
– Incubation time: Time require for incubation (usually 18-
24 hours)
– Incubation temperature: Temperature required for
bacterial growth during incubation (usually 370C)
tarekviro@yahoo.com
SSMC2017

37. CULTURE IN ARTIFICIAL MEDIUM
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SSMC2017

38. FERMENTATIONOBSERVEDIN
CULTURE
Lactose Acid +gas
E.coli
Phenol red Yellow
tarekviro@yahoo.com
SSMC2017

39. STUDY QUESTIONS
True/False
• A bacterial cell becomes 6 in number after its 3rd generation
• Generation time depends on bacterial growth curve
• Catalase is the only enzyme lacking in anaerobic bacteria
• Oxygen is responsible for the final acceptance of the electron
• Exotoxins are produce in the lag phase of the growth curve
• Penicillin will act best in the log phase of the growth curve
tarekviro@yahoo.com
SSMC2017

40. • CO2 is an oxidant
• Aerobic bacteria lack superoxide dismutase.
• Alkaliphiles grow best between PH 3-5
• Haemophilus influenzae is a microaerophillic bacteria
• Facultative anaerobes can grow in low oxygen tension
• Oxidative phosphorylation occurs in the bacterial cell
membrane
• Bacterial motility is independent of proton motive force
tarekviro@yahoo.com
SSMC2017
STUDY QUESTIONS
True/False

41. REFERENCE
• Warren Levinson. Review of Medical Microbiology and
Immunology, 11th edition (2010). Appleton and Lange.
• Geo. F. Brooks, Karen C. Carroll, Janet S. Butel, Stephen A.
Morse, Timothy A. Mietzner. Medical Microbiology, 27th
edition (2016). Appleton & Lange.
• Medical Microbiology, M.R Chowdhury
tarekviro@yahoo.com
SSMC2017

42. tarekviro@yahoo.com
SSMC2017