Bacteria require specific nutrients, temperature, pH, oxygen, and other physical conditions to grow. A culture medium provides bacteria with nutrients for growth and can be solid, liquid, or semi-solid. It typically contains a carbon source, nitrogen source, minerals, and other growth factors. Culture media are classified based on consistency, nutritional components, oxygen requirements, and intended use such as selection, differentiation or storage of bacteria. Bacteria primarily reproduce through binary fission but some bud. Their growth follows distinct phases of lag, log/exponential, stationary, and death. Growth is measured directly through microscopy, plate counts, or indirectly through turbidity.

1. Nutrition Requirement
• Nutrients must contain all the elements necessary
for the synthesis and growth of new organisms.
• Bactria requires nutrition, pH, temperature, oxygen
for growth.
For growth of media it consist of following-
• Hydrogen donors and acceptors
• Carbon source
• Nitrogen source
• Minerals : sulphur and phosphorus
• Growth factors: amino acids, purines, pyrimidines;
vitamins
• Trace elements: Mg, Fe, Mn.

3. Bacteriological Media
• It is an artificially prepered mixture of nutrients for
growth of m.o.
• A culture medium contains water, a source of carbon &
energy source of nitrogen, trace elements and some growth
factors.
• ThepHof the medium must be setaccordingly.
Uses:
• Enrichthe number ofbacteria.
• Selectfor certain bacteriaandsuppressothers.
• Differentiate amongdifferent kinds ofbacteria.

4. Purposeofculturing
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Isolation of bacteria.
Properties of bacteria i.e. culturing bacteria isthe initial step
in studying its morphology anditsidentification.
Maintenance of stockcultures.
Estimate variable counts.
Totest for antibioticsensitivity.
Tocreate antigens for laboratoryuse.
Certain genetic studiesand manipulations of the cells
also needthat bacteriato becultured in vitro.
Culturing on solid media is another convenient way of
separating bacteria inmixture.
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5. Common ingredient of culture media
1. Water:
 Tap, pure distilled water used for preparation of media by
dissolving all contents.
 It provide flow of nutrient.
2. Peptone:
 Complex mixture of partially digested protein obtain from
meat, heart mussels, soya meal, fibrin etc.
 It is important constituent of amino acid, inorganic salt,
potassium, magnesium ,Phosphorus growth factors etc.
 It mainly supply nitrogenous material and acts as buffer.
 Stored in tightly close container because it is hygroscopic in
nature & get sticky when exposed to air.

6. 3. Yeast extract:
 Prepare from backers yeast.
 Contains carbohydrates, amino acid, inorganic salts &
growth factors.
 It is mainly used as source of vitamins .
4.Meat extract:
 Prepare by fresh lean meat by hot water extraction.
 Contains gelatin, peptones proteos, amino acid, etc.
5. Agar:
 Agar,apolysaccharide extracted from marine seaweed
algae,Itreleaseagarcalledaggarophyte.
 Itcontainsmixtureoftwopolysaccharidesuchas
• Aggarose(70%) &aggaropectin(30%).

7. • Also contains calcium chlorides, magnesium, iron etc. It used to
solidify aspecificnutrientsolution.
• Unlike other gelling agent, it is not easily degraded by many
bacteria.
• It is not easily destroyed at higher temperatures, and
therefore it canbe sterilized by heating, the process which also
liquefiesit.
• Oncesolidified, agarmedium will remain solid.
• T h e culture media is contained in a Petri dish, a twopart, glass
or plastic coveredcontainer.
Properties of Agar:
 Prepare from seaweed algae
 Act as good solidifyig agent
 No nutritional values

8.  Bacteriologically inert.
 Resistant to all m.o.
 Stable a different temperature.
 Melts at 95-98oC .
 Solidify below 40oC
 Easily available &economical

9. ClassificationofCultureMedia
• Bacterialculture mediacanbeclassifiedinat leastthree
ways.
1. Consistency
2. Nutritional component
3. Functionaluse
4. OxygenRequirment

10. Classificationbasedonconsistency:
A. Liquidmedia:
• Available for usein test-tubes, bottles or flasks.
• Liquid media are sometimes referred as “broths” (e.g nutrient
broth).
• Bacteria grow uniformly producing generalturbidity.
• No agarisadded.
• Mostly usedfor inoculumspreparation.
B. Solidmedia:
• Available for usein Petri dish.
• 2%of agarisadded.
• Agaristhe most commonly usedsolidifying agent.
• Colony morphology, pigmentation, hemolysis can be appreciated.
Examples include Nutrient agarandBloodagar.

11. C.Semi-solidmedia:
• Suchmedia are fairly soft
• Useful in demonstrating bacterial motility separating motile
from non-motile strains.
• Examplesof Semi-solid media (Hugh& Leifson’s oxidation
fermentation).
• 0.5%agaris added.

12. ClassificationbasedonNutritional Components
1. Simplemedia.
2. Complexmedia.
3. Syntheticor chemicallydefinedmedia.

13. ClassificationbasedonNutritional Components
1.Simplemedia:
•Simplemedia suchaspeptone water, nutrient agar
•It is alsocalled asbasalmedia.
•Eg: NB,NA.
•Nutrient Broth consists of peptone, yeast extract and NaCl.
When 2% of agar is added to Nutrient Broth it forms Nutrient
agar.
2.Complexmedia.
•Theyhavespecialingredients in them for the growth of m.o.
•These special ingredients like yeast extracts or casein
hydrolysate
•consists of a mixture of many chemicals in an unknown
proportion.

14. 3. Synthetic media/Chemically defined media:
• Specially prepared media for research purposes where
the composition of every component is well known.
• It is prepared from pure chemical substances.
• Eg: peptone water (1% peptone + 0.5% NaCl in water).

15. 3.ClassificationbasedonFunctionalUse
1. Enrichedmedia.
2. Selectivemedia.
3. Differential media.
4. Transportmedia.
5. Indicatormedia
6. Assay media
7. Storagemedia
8. Sugar media
9. Enrichment media

16. 3.ClassificationbasedonOxygen requirement
1.AerobicMedia-allAbovemedia
2.Anaerobicmedia
• Anaerobic bacteria need special media for growth
because they need low oxygen content,
• Reduced oxidation –reduction potential and extra
nutrients.
• Media for anaerobes may have to be supplemented
with nutrients like hemin and vitamin K.
• Boiling the medium serves to expel any dissolved
oxygen.
• E.g. Thioglycollate media.

17. Physical Condition require for growth of Bacteria
• Apart from media many physical conditios affect growth
of bacteria
1. Temperature
2. pH
3. Oxygen
5. Light
6.Hydrostatic pressure

18. • Microorganisms are sensitive to temperature changes
• Temprature at which m.o. shows rapid growth called as
Optimum Growth Temprature
• Highest temprature at which m.o. shows growth called as
Maximum Growth Temprature
• Lowest temprature at which m.o. shows growth called as
Minimum Growth Temprature.
• A Enzymes have temperature optima
– If temperature is too high, proteins denature, including
enzymes, carriers and structural components
Temperature

19. Depend on temperature condition & growth
bacteria classified as
Psychrophiles can grow well at 0oC, have optimal
growth at 15oC or lower, and usually will not grow
above 20oC
• Arctic/Antarctic ocean
• Protein synthesis, enzymatic activity and transport
systems have evolved to function at low
temperatures
• Cell walls contain high levels of unsaturated fatty acids
(semi-fluid when cold)
Mesophiles have growth minima of 15 to 20oC, optima of 20
to 45oC, and maxima of about 45oC or lower
• Majority of human pathogens

20. Thermophiles have growth minima around 45oC, and
optima of 55 to 65oC
• Hot springs, hot water pipes, compost heaps
• Lipids in PM more saturated than mesophiles.
Hyperthermophiles have growth minima around 55oC
and optima of 80 to 110oC
• Sea floor, sulfur vents
• pH is the negative logarithm of the hydrogen ion conc.
– Acidophiles grow best between pH 1and .5
– Neutrophiles grow best between pH 6.5 and 8.0
– Alkalophiles grow best between pH 8.5 and 11.5
– Extreme alkalophiles grow best at pH 10.0 or higher
pH

21. Oxygen concentration
– Obligate aerobes are completely dependent on
atmospheric O2 forgrowth
• Oxygen is used as the terminal electron acceptor for
electron transport in aerobic respiration
– Facultative anaerobes (Non- Stringent) do not require O2
for growth, but do grow better in its small presence
–Obligate (strict) anaerobes do not tolerate O2 and die in
itspresence.
–Microaerophiles are damaged by the normal
atmospheric level of O2 (20%) but require lower levels (2
to 10%) for growth

22. Pressure
–Barotolerant organisms are adversely
• affected by increased pressure, but not as severely as
are nontolerant organisms
• –Barophilic organisms require, or grow more rapidly in
the presence of increased pressure
Light
• Optimum condition for growth is darkness.

23. Cultivation of Anaerobic bacteria
• Strict anaerobic can be grown only in absence of oxygen.
• Such enviornment prepare by following method:
A. Displacement of oxygen with gases such as hydrogen,
nitrogen, helium, or cabon dioxide is employed.
 A popular method is Candle Jar method
 All innoculated plates are inside large air tight container
& lightened candles .
 Keep it into jar before lead is sealed.
 Burning of candle expected to use all oxygen.

24. B. Reduction of oxygen in medium achieved by reducing
agents.
• E.G. 1% glucose, 0.1% thioglycollate, 0.1% Ascorbic acid.
C. Cultivation in vacuum desiccator
D. Chemical or Biological method
 Alkaline pyrogallol absorb atmospheric oxygen.
 Pyrogallic acid added to solution of NaOH in test tube
placed inside air-tight jar to provide anaerobiosis.
E. Gas-Pack System
 It is available as disposable envelope, containing
chemicals which generate hydrogen and carbon dioxide
on addition of hydrogen.

25. Fig. Gas Pack System Presence of cold
catalyst in envelop
permit combination
of hydrogen, oxygen
to produce anaerobic
environment.
 Reduced methylene
blue used for
verifying anaerobic
condition in jar.
 It remains colourless
anaerobically .
 Turns blue on
exposure to oxygen.

26. Bacterial reproduction
• Cell growth and reproduction by cell division are tightly
linked in unicellular organisms.
• Bacteria grow to a fixed size and then reproduce through
binary fission, a form of asexual reproduction
• Under optimal conditions, bacteria can grow and divide
extremely rapidly, and bacterial populations can double as
quickly as every 9.8 minutes.
• In cell division, two identical clone daughter cells are
produced.

28. Binary fission
• Most prokaryotes reproduce by a process of binary fission,
in which the cell grows in divides in half to yield two
identical daughter cells.
• Each daughter cell can continue to grow at the same rate
as its parent.
• For this process to occur, the cell must grow over its entire
surface until the time of cell division, when a new
hemispherical pole forms at the division septum in the
middle of the cell.
• In order for the cell to divide in half, the peptidoglycan
structure must be different in the hemispherical cap than in
the straight portion of the cell wall, and different wall-
cross-linking enzymes must be active at the septum than
elsewhere.

29. • Binary fission begins with the single DNA molecule
replicating and both copies attaching to the cell
membrane.
• Next, the cell membrane begins to grow between
the two DNA molecules. Once the bacterium just
about doubles its original size, the cell membrane
begins to pinch inward.
• A cell wall then forms between the two DNA
molecules dividing the original cell into two
identical daughter cells

30. Budding
• A group of environmental bacteria reproduces by budding.
• In this process a small bud forms at one end of the mother
cell
• As growth proceeds, the size of the mother cell remains
about constant, but the bud enlarges.
• When the bud is about the same size as the mother cell, it
separates. This type of reproduction is analogous to that in
budding fungi, such as brewer’s yeast (Saccharomyces
cerevisiae).
• One difference between fission and budding is that, in the
latter, the mother cell often has different properties from the
offspring.
• Ex: In some strains, mother cells have a flagellum and are
motile, whereas the daughter buds lack flagella.

32. GROWTH OF BACTERIA
• Growth of Bacteria is the orderly increase of all the
chemical constituents and increase in no. of bacteria.
• Multiplication is the consequence of growth.
• Death of bacteria is the irreversible loss of ability
to reproduce.
• Population increases geometrically or exponentially
• 1  2 2223----2n

33. Growth Kinetics
• When bacterial cell count and the logarithm of
cell plotted against time on graph paper gives
typical curve that curve called Growth Curve.
• Bacterial growth follows four phases.
• lag phase
• log phase
• stationary phase
• death phase

35. Lag phase
• When bacteria inoculated into a fresh culture medium.
• No. of bacteria remains constant at initial phase.
• Period between inoculation & start of multiplication known
as lag Phase.
• In this period bacteria cells adapt to their new
environment
• cells are adapting to the high-nutrient environment and
preparing for fast growth.
• The lag phase has high biosynthesis rates, as proteins and
metabolic intermediates are synthesized in adequate
quantities for rapid growth & multiplication to proceed.
• New enzymes are synthesized.

36. Log/Exponential growth phase
• In this phase, the cells have adjusted to their new
environment and multiply rapidly (exponentially) constant
rate,.
• When plotted graph as log of no. of cells plotted against
time found straight line.
• Bacteria multiply fast at maximum rate.
• Time require for one bacterial cell division call generation
time (g).
g=t/n
• Where,
n- no. of generation
t- time
Growth rate (R) is reciprocal of generation time.
R=1/g = n/t

37. Stationary Phase
• In this phase a constant high population of cell is
maintained by balace between cell death & cell
division.
• Rate of multiplication reduce due to depletion of
nutrients, accumulation of toxic waste product, high
conc. of cells & low oxygen .
• The growth rate equals the death rate
• There is no net growth in the organism population – The
viable count remains stationary as an equilibrium exists
between the dying cells and newly formed cells.
• In this phase m.o. use reserved food material for growth.

38. Death Phase
- Phase of decline
- The living organism population decreases with time,
due to a lack of nutrients and accumulation of toxic
metabolic by-products.
- Cell death may also be caused by autolytic enzymes.
- Between every phase curve was observed it is
called as transition period.

39. • Bacterial growth is of different type
1. Synchronous Growth
2. Diauxic Growth
3. Continuous Growth
1. Synchronous Growth
• In this growth growing of m.o. occurs in such way that
they all grow at same time
with respect to each other.
• Figure shows growth pattern
of bacteria.

40. 2. Diauxic Growth Curve
• It is characterized by two separate phases due to
use of carbon source over another.
• e.g E.coli first utilize glucose after use of glucose it
use lactose.

41. 3. Continuous Growth Curve
• Technique by which microbial population maintained
always in exponential phase by providing constant
environment, media etc. called continuous culture
technique.
• This method is used
in industry for research
process.

42. • Nutrient Broth preparation
• https://www.youtube.com/watch?v=_xY4Xbky
KPs
• Nutrient Agar preparation
• https://www.youtube.com/watch?v=Csp1NTf
P7kY

43. Measurement of Bacterial Growth
1. Determination of cell number:
A. DIRECT METHODS
• Direct microscopic method
• Heamocytometer
• Proportional Count method
• Electronic counter method
B. INDIRECT METHODS
• Plate count technique
• Membrane filter count
2. Determination of cell mass:
• Dry weight measurement
• Turbidometry

44. DIRECT METHODS
1. Microscopic Methods
• Known volume 0.01ml
Bacterial suspension
• Spread uniformly on
glass slide
• Observe few microscopic
Area and average of all area
Taken
Total cell per square meter calculated .

45. 2. Heamocytometer
• Bacteria count easily & accurately by counting chamber
method.
• Minute drop of culture placed on Neubers slide
This slide ruled into square that are 1/400mm3
area
So we can count microorganisms

46. 3. Electronic Counter method
• Electronic instrument coulter counter is used.
• This technique bacterial suspension passed through
capillary tube.
• This diameter of tube is microscopic. It allow to pass one
at one time.
• Instrument counts no. of cells in few seconds.
• But it is having disadvantage it count dust particles also.
• Hence, the preparation
Should be free from
Foreign particle.

47. INDIRECT METHOD
1. Pour Plate Method
• In this method measured amount of bacterial suspension
added into test tube,then agar medium is added.
• Immediately mix agar medium with innoculum.
• Then add into petriplates
• After solidification of media plates incubated at 37oC for
24 hours in inverted position & then kept in normal
position.
Disadvantage
• Suspension contains different microbial species all of
them not grow,
• Coloneis grow aggregately.

48. INDIRECT METHOD
1. Pour Plate Method

49. Membrane count techniques
• It follow same principle like
Above technique.
• Diluted suspension of m.o.
Passed through Millipore filter
• Then filter disc removed &
Placed on culture medium.
• Petriplates inoculated and
Coloneis Counted .

50. Determination of cell mass
Direct Method
1.Direct weight measurement
• Simple and direct method of measuring cell mass.
• The suspension is centrifuged and pellets repeatedly
washed to remove foreign particles.
• Residue is dried & weighted.
• This method mainly is used in industry.
2.Measurment of cell Nitrogen
• Chemical constituent of cell is protein in which nitrogen
is imp constituent.
• So bacterial population counted by using cell nitrogen.
• It is measured by chemical analysis.

51. Indirect methods
Turbidometry methods
• Cells act like large particles
that scatter visible light
• A spectrophotometer
sends a beam of visible
light through a culture
and measures how much
light is scattered
• Scales read in either
absorbance or %
transmission
• Measures both live and
dead cells