Micropbiology, Selective Media, Cultivated bacteria, blood agar, Effect of pH and Temperature on Bacterial growth

2. THE CULTIVATION OF
BACTERIA
Dhananjay Desai
Student
P. G. Department of Microbiology
N. A. C. & Sc. College
Ahmednagar
dhananjayashokdesai@gmail.com

3. INTRODUCTION
The process of growing microorganisms in culture by
taking bacteria from the infection site (in vivo or
environment) and grow them in artificial environment
in the laboratory (in vitro).

4. WHY CULTIVATION ?

5. WE NEED BACTERIA
Industrial Application.
Pharmacological Application.
Environmental Application.
Research Application.

6. Outline
 Nutritional Requirement.
 Nutritional Type of Bacteria.
 Bacterial Media.
 Physical Condition required.
 Cultural Characteristics.
 Reproduction.

7. NUTRITIONAL REQUIREMENT
Microorganisms require about ten elements in large
quantities, because they are used to construct
carbohydrates, lipids, proteins, and nucleic acids.
Several other elements are needed in very small
amounts and are parts of enzymes and cofactors.

8. NEED
 Macronutrients-
 Micronutrients-
 Growth factors-
 Environmental factors-

9. Macronutrients
 required in large amounts, including.
 carbon, oxygen, hydrogen, nitrogen, sulfur,
phosphorus (Components of carbohydrates, lipids,
proteins, and nucleic acids ).
 potassium, calcium, magnesium and iron (part of
enzymes and cofactors).

10. Micronutrients
 Require very small amounts.
 Such as iron, copper, molybdenum, and zinc;
these are referred to as trace elements.
 Most are essential for activity of certain enzymes,
usually as cofactors.

11. Growth Factors
 Amino acids- protein synthesis.
 Purines and Pyrimidines- nucleic acid synthesis.
 Vitamins- small organic molecules that usually
make up all or part enzyme cofactors, and only
very small amounts are required for growth.

12. Environmental factors
 Temperature-
 pH-
 Oxygen Requirement-

13. Phototrophs: use light as energy source.
Chemotrophs: obtain energy from the oxidation
of chemical compounds.
Lithotrophs: use reduced inorganic substances as
their electron source.
Organotrophs: extract electrons from organic
compounds.

14. Major nutritional
type
Sources of energy,
hydrogen/electrons,
and carbon
Representative
microorganisms
Photoautotroph
(Photolithotroph)
Light energy, inorganic
hydrogen/electron(H/e-)
donor, CO2 carbon source
Algae, Purple and green
bacteria, Cyanobacteria
Photoheterotroph
(Photoorganotroph)
Light energy, inorganic
H/e- donor,
Organic carbon source
Purple nonsulfur bacteria,
Green sulfur bacteria
Chemoautotroph
(Chemolithotroph)
Chemical energy source
(inorganic), Inorganic H/e-
donor, CO2 carbon source
Sulfur-oxdizing bacteria,
Hydrogen bacteria,
Nitrifying bacteria
Chemoheterotroph
(Chenoorganotroph)
Chemical energy source
(organic), Organic H/e-
donor, Organic carbon
source
Most bacteria, fungi,
protozoa
Nutritional types of microorganisms

15. Nutrient molecules frequently cannot cross
selectively permeable plasma membranes through
passive diffusion and must be transported by one of
three major mechanisms involving the use of
membrane carrier proteins.
Uptake of nutrients

16.  Passive transport (simple diffusion).
 Facilitated diffusion.
 Active transport.
 Group translocation.

17. Passive transport
 molecules move from a region of higher concentration to
one of lower concentration as a result of random thermal
agitation.
 No carrier protein.
 No energy.
 Glycerol, H2O, O2.

18. Facilitated diffusion
 The diffusion process is aided by a carrier.
 Permeases.
 Each carrier is selective
and will transport only
closely related solutes

19. The membrane carrier can change
conformation after binding an external
molecule and subsequently release the
molecule on the cell interior. It then
returns to the outward oriented position
and is ready to bind another solute
molecule.
Model of Facilitated Diffusion
Because there is no energy input,
molecules will continue to enter only as
long as their concentration is greater on
the outside.

20. Active Transport
 Active transport is the transport of solute molecules to
higher concentrations, or against a concentration gradient,
with the use of metabolic energy input.
 Permeases.
 Energy need.
lower concentration  higher concentration.

21. Proton gradients
 Symport: linked transport of
two substances in the same
direction.
 Antiport: linked transport of
two substances in the opposite
direction.
 Uniport: one substance enter.

22. Group Translocation
 Transported into the cell while being chemically
altered.
 The best-known group translocation system is the
phosphoenolpyruvate: sugar phosphotransferase
system (PTS), which transports a variety of sugars
into procaryotic cells while Simultaneously
phosphorylating them using phosphoenolpyruvate
(PTS), (PEP) as the phosphate donor.

24. Iron uptake - Siderophores (S)
Microbial cell Fe 2+/S
Receptor
Fe 2+/S

25. CULTURE MEDIA
CULTURE METHODS

26. History of Culture Media
 The original media used by Louis Pasteur – urine or
meat broth.
 Liquid medium – diffuse growth.
 Sold medium – discrete colonies.
 Cooked cut potato by Robert Koch – earliest solid
medium.
 Gelatin – not satisfactory
 - liquefy at 24oC.

27. Agar Powder
 Used for preparing solid medium.
 Obtained from seaweeds.
 No nutritive value.
 Not affected by the growth of the bacteria.
 Melts at 98oC & sets at 42oC.
 3% agar is employed in solid medium.

28. Colony?
 Macroscopically visible collection of millions of
bacteria originating from a single bacterial cell.

30. Types of culture media

31. Based on their consistency
 Solid medium- contains 3% agar
• Colony morphology, pigmentation, hemolysis can be
appreciated.
• Eg: Nutrient agar, Blood agar.
 Liquid medium- contains no Agar.
• For inoculums preparation, Blood culture, for the isolation of
pathogens from a mixture.
• Eg: Nutrient broth.
 Semi solid medium- contains 0.5% agar.
• Eg: Motility medium.

33. Based on the constituents/
ingredients
• simple medium- Eg: NB, NA
• - NB consists of peptone, meat extract, NaCl,
• - NB + 2% agar = Nutrient agar
• complex medium- Media other than basal media.
• They have added ingredients.
• Provide special nutrients .
• synthetic or defined medium- Media prepared from pure chemical substances and its exact
composition is known
• Eg: peptone water – 1% peptone + 0.5% NaCl in water
• Special media- Substances like blood, serum, egg are added to the basal medium.
• Used to grow bacteria that are exacting in their nutritional needs.
• Eg: Blood agar, Chocolate agar

35. Enrichment media
 Liquid media used to isolate pathogens from a mixed
culture.
 Media is incorporated with inhibitory substances to
suppress the unwanted organism.
 Eg:
– Selenite F Broth – for the isolation of Salmonella,
Shigella.
– Alkaline Peptone Water – for Vibrio cholera

36. Selective media
 The inhibitory substance is added to a solid media.
 Eg:
• Mac Conkey’s medium for gram negative bacteria
• TCBS – for V.cholerae.
• LJ medium – M.tuberculosis.
• Wilson and Blair medium – S.typhi.
• Potassium tellurite medium – Diphtheria bacilli.

38. Indicator media
 These media contain an indicator which changes its
color when a bacterium grows in them.
 Eg:
• Blood agar
• Mac Conkey’s medium
• Christensen’s urease medium
• XLD Medium

40. Differential media
 A media which has substances incorporated in it
enabling it to distinguish between bacteria.
 Eg: Mac Conkey’s medium
• Peptone
• Lactose
• Agar
• Neutral red
• Taurocholate
 Distinguish between lactose fermenters & non lactose
fermenters.

42. Sugar media
 Media containing any fermentable substance.
 Eg: glucose, arabinose, lactose, starch etc.
 Media consists of 1% of the sugar in peptone water.
 Contain a small tube (Durham’s tube) for the
detection of gas by the bacteria.

44. Transport media
 Media used for transporting the samples.
 Delicate organisms may not survive the time taken
for transporting the specimen without a transport
media.
 Eg:
– Stuart’s medium – non nutrient soft agar gel
containing a reducing agent
– Buffered glycerol saline – enteric bacilli

46. Anaerobic media
 These media are used to grow anaerobic organisms.
 Eg: Robertson’s cooked meat medium, Thioglycolate
medium.

50.  Deep-freezing: –50°to –95°C.
 Lyophilization (freeze-drying): Frozen (–54° to –
72°C) and dehydrated in a vacuum.

51. Reproduction
 Binary fission
 Budding
 Conidiospores (actinomycetes)
 Fragmentation of filaments

52. Binary Fission
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53. Phases of Growth
 Lag phase
 Log (logarithmic) phase
 Stationary phase
 Decline phase or death phase

54. Phases of Microbial Growth

55. Measuring Bacterial Growth
 Direct Measurements of Microbial Growth.
 Plate counts: Perform serial dilutions of a sample.

56. Direct Measurements of Microbial
Growth

57. Standard Plate Count
 Inoculate Petri
plates from serial
dilutions
 Two methods:
• Pour Plate
• Spread Plate

58. Plate Count

59. Other Methods: Estimating Bacterial Numbers
by Indirect Methods
 Turbidity.

60. Factors Affecting Bacterial Growth
 Temperature
• Minimum growth temperature
• Optimum growth temperature
• Maximum growth temperature

62. Factors Affecting Bacterial Growth
 pH
• Most bacteria grow between pH 6.5 and 7.5.
• Molds and yeasts grow between pH 5 and 6.
• Acidophiles grow in acidic environments.

63. Factors Affecting Bacterial Growth
 Osmotic pressure
• Hypertonic environments, increase salt or sugar,
cause plasmolysis.
• Extreme or obligate halophiles require high
osmotic pressure.
• Facultative halophiles tolerate high osmotic
pressure.

66. NOW TOPIC OPEN FOR
DIOSCUSSION