Growth of bacteria

Growth & Measurement of bacteria

1. Growth of Bacteria
Dr. Pulipati Sowjanya
Professor & HOD
Dept. of Pharm. Biotechnology
Vignan Pharmacy College
Guntur

2. Reproduction & Growth
of Bacteria
•Whenbacteriaareinoculatedintoasuitablemediumandincubated
underappropriateconditions,atremendousincreaseinthenumberof
cellsoccurswithinarelativelyshorttime.
•Determinationofgrowthrequiresquantitativemeasurementofthe
totalpopulationofthecellsatthetimeofinoculationandagainafter
incubation.
•Reproduction:Themostcommonmodeofcelldivisionintheusual
growthcycleofbacterialpopulationsistransversebinaryfission,in
whichasinglecelldividesafterdevelopingatransverseseptum

3. Modes of Cell Division
A.Transverse binary fission in Bacillus
subtilis
B.Transverse binary fission in
Streptococcus faecalis
C.Transverse binary fission in
Prosthecatebacterium Prosthecobacter
fusiformis
D.Budding in Rhodopseudomonas
acidophila
E.Budding in Hyphomicrobiumvulgare
F.Fragmentation in Nocardia sps.
G.Formation of conidiospores by
Streptomyces species

4. Binary Fission
❑Transverse binary fission is an asexual reproduction.
❑The cells increase in size and doubled its components.
❑One cell divides into two, those two cells divide into four and four
cells become eight, the increase in cell numbers is exponential.
❑Microbial growth is defined as an increase in the number of cells in a
population.
❑The time it takes for the population to double in number is the
generation,or doubling time.
❑This varies depending on species of the organism and the conditions
in which it is grown.
❑Ex: E.coli can double in 20 minutes. Mycobacterium requires 12
hours to double.

5. Binary Fission

6. Binary Fission

7. Bacterial Growth Curve
1.Intense activity preparing for
population growth, but no
increase in population
2.Logarithmic or exponential
increase in population.
3.Period of equilibrium;
microbial deaths balance
production of new cells.
4.Population is decreasing at a
logarithmic rate.

8. Bacterial Growth Curve
Bacterial Growth Curve : When bacteria are inoculated into a liquid growth
medium, we can plot ofthenumberofcellsinthepopulationovertime.
Four phases of BacterialGrowth:
1. LagPhase:
✓Period of adjustment to newconditions.
✓Little or no cell division occurs, population size doesn’tincrease.
✓Phase of intense metabolic activity, in which individual organisms grow in
size.
✓May last from one hour to severaldays.

9. Bacterial Growth Curve
2. LogPhase:
✓Cells begin to divide and generation time reaches a constantminimum.
✓Period of most rapidgrowth.
✓Numberofcellsproduced>Numberofcellsdying
✓Cells are at highest metabolicactivity.
✓Cells are most susceptible to adverse environmental factors at this
stage.
✓Radiation
✓Antibiotics

10. Bacterial Growth Curve
3. StationaryPhase:
✓Population size begins tostabilize.
✓Numberofcellsproduced=Numberofcellsdying
✓Overall cell number does notincrease.
✓Cell division begins to slowdown.
✓Factors that slow down microbialgrowth:
▪Accumulation of toxic wastematerials
▪Acidic P
H
ofmedia
▪Limitednutrients
▪Insufficient oxygensupply

11. Bacterial Growth Curve
4. Death or DeclinePhase:
✓Population size begins todecrease.
Numberofcellsdying>Numberofcellsproduced
✓Cell number decreases at a logarithmicrate.
✓Cells lose their ability todivide.
✓A few cells may remain alive for a long period of time.

12. Synchronous Growth
✓Maintaining the growth of cultures in the same
stage of their growth cycle, i.e., growing
synchronously.
✓It lasts only for few generations.
✓A population can be synchronized by
manipulating the physical environment or the
chemical composition of the medium.
✓Ex:the cells may be inoculated into a medium at
a suboptimal temperature; if they are kept in this
condition for sometime they will metabolize
slowly but will not divide.
✓When the temp is subsequently raised, the cells
will undergo a synchronized division
The Synchronous Growth of Microorganism

13. Continuous Culture
Chemostat
Theconcentrationofanessentialnutrient
(substrate)withinculturevesselwillcontrolthe
growthrateofthecells.
Itiscontrolledbydilutionrate,i.e.,therateat
whichfreshmediumisbeingaddedtotheculture
(flowrate)dividedbythevolumeoftheculture
vessel.
Therefore,byadjustingthedilutionratethegrowth
rateiscontrolled.
Ifthedilutionrateislow,thecellsreachhigh
density.
Ifthedilutionrateishigh,thecelldensityislow.
A Continuous Culture System:
The Chemostat

14. Continuous Culture
Turbidostat
Inthismethodaphotoelectricdevicecontinuouslymonitorsthecell
densitywithintheculturevesselandcontrolsthedilutionrateto
maintainthecelldensityataconstantrate.
Ifthedensitybecomestoohighthedilutionrateisincreased;ifthe
densitybecomestoolow,thedilutionrateisdecreased.

15. Measuring Microbial Growth
1. Platecount:
✓Most frequently used method of measuring
bacterial populations.
✓Inoculate plate with a sample and count number of
colonies.
Assumptions:
•Eachcolonyoriginatesfromasinglebacterialcell.
•Original inoculum ishomogeneous.
•No cell aggregates arepresent.
Advantages:
•Measures viablecells
Disadvantages:
•Takes24hoursormoreforvisiblecolonies
toappear.
•Onlycountsbetween30and300colonies
areaccurate.
•Must perform serial dilutions to get
appropriatenumbers/plate.
Direct Methods ofMeasurement

16. Serial Dilution
Measuring Microbial Growth

17. Measuring Microbial Growth
1.Plate count(continued):
A. PourPlate:
✓Introducea1.0or0.1mlinoculum intoanempty
Petridish.
✓Add liquid nutrient medium kept at50
o
C.
✓Gently mix, allow to solidify, andincubate.
Disadvantages:
•Not useful for heat sensitiveorganisms.
•Colonies appear under agarsurface.
Direct Methods ofMeasurement

18. Measuring Microbial Growth
B. SpreadPlate:
✓Introduce a 0.1 ml inoculum onto the
surfaceof Petridish.
✓Spread with a sterile glassrod.
Advantages:Colonies will be grown on the
surface and not exposed to meltedagar.
Direct Methods ofMeasurement

19. Measuring Microbial Growth
2.Filtration:
✓Used to measure small quantities ofbacteria.
•Example: Fecal bacteria in a lake or in oceanwater.
✓A large sample (100 ml or more) is filtered to retain bacteria.
✓Filtrate is transferred onto a Petridish.
✓Incubate and countcolonies.
Direct Methods ofMeasurement

20. Measuring Microbial Growth
The Membrane Filtration Procedure –Membranes with different pore sizes are used to trap different
microorganisms. Incubation times for membranes also vary with the medium and microorganism
Membrane Filtration

21. Measuring Microbial Growth
3. Most Probable Number(MPN):
✓Used mainly to measure bacteria that will not grow on solidmedium.
✓Dilute a sample repeatedly and inoculate several broth tubes for each dilution
point.
✓Countthenumberofpositivetubesineachset.
✓Statistical method: Determines 95% probability that a bacterial population
falls within a certain range.
Direct Methods ofMeasurement

22. Measuring Microbial Growth
4.DirectMicroscopicCount:
✓Aspecificvolumeofabacterial
suspension(0.01ml)isplacedona
microscopeslidewithaspecialgrid
(PetroffHausser’sChamber).
✓Stainisaddedtovisualizebacteria.
✓Cellsarecountedandmultipliedbya
factortoobtainconcentration.
Direct Methods ofMeasurement
Advantages:
•Noincubationtimerequired.
Disadvantages:
•Cannotalwaysdistinguishbetween
liveanddeadbacteria.
•Motilebacteriaaredifficulttocount.
•Requiresahighconcentrationof
bacteria(10million/ml).

23. Measuring Microbial Growth
Direct MicroscopicCount

24. Measuring Microbial Growth
1.Turbidity:
✓As bacteria multiply in media, it
becomesturbid.
✓Use a spectrophotometer to determine
% transmission or absorbance.
✓Multiply by a factor to determine
concentration.
Indirect Methods ofMeasurement
Advantages:
•No incubation timerequired.
Disadvantages:
•Cannot distinguish between live and
deadbacteria.
•Requires a high concentration of
bacteria (10 to 100 million cells/ml).

25. Measuring Microbial Growth
2.Electronic Enumeration of Bacteria:
✓Such as coulter current
✓Microbial suspension forced through a small
hole or orifice
✓Movement of microbe through orifice impacts
electric current that flows through orifice.
Advantages:
Quick and easy to use
Useful for large volume of microbial suspension.
Disadvantages:
Cannot distinguish dead from living cells.
Indirect Methods ofMeasurement

26. Measuring Microbial Growth
3. MetabolicActivity:
✓As bacteria multiply in media, they
produce certain products:
•Carbondioxide
•Acids
✓Measure metabolicproducts.
✓Expensive
Indirect Methods ofMeasurement
4. DryWeight:
✓Bacteria or fungi in liquid media are
centrifuged.
✓Resulting cell pellet isweighed.
✓Doesn’t distinguish live and dead
cells.

27. REFERENCES
1.Prescott,HarleyandKlein's,Microbiology.5thedition.113-121.
2.GerardJ.Tortora,BerdellR.Funke,ChristineL.Case.Microbiology–An
Introduction.10
th
edition.Pearson.171-180.
3.MichaelJ.Pelczar,JR.,E.C.S.Chan,NoelR.Krieg.Microbiology.5
th
edition.115–132.
4.Ashutoshkar,PharmaceuticalMicrobiology.146-153.

28. THANK YOU