THE CULTIVATION OF BACTERIA Dhananjay Desai Student P. G.
Department of Microbiology N. A. C. & Sc. College Ahmednagar
[email protected]
THE CULTIVATION OF BACTERIA
Dhananjay DesaiStudentP. G. Department of MicrobiologyN. A. C.
& Sc. CollegeAhmednagar
[email protected]
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).
WHY CULTIVATION ?
WE NEED BACTERIAIndustrial Application.
Pharmacological Application.
Environmental Application.
Research Application.
OutlineNutritional Requirement.
Nutritional Type of Bacteria.
Bacterial Media.
Physical Condition required.
Cultural Characteristics.
Reproduction.
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.
NEEDMacronutrients-
Micronutrients-
Growth factors-
Environmental factors-
Macronutrientsrequired 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).
MicronutrientsRequire 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.
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.
Environmental factorsTemperature-
pH-
Oxygen Requirement-
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.
Major nutritional typeSources of energy,hydrogen/electrons, and
carbonRepresentative microorganismsPhotoautotroph
(Photolithotroph)Light energy, inorganic hydrogen/electron(H/e-)
donor, CO2 carbon sourceAlgae, Purple and green bacteria,
CyanobacteriaPhotoheterotroph (Photoorganotroph)Light energy,
inorganic H/e- donor, Organic carbon sourcePurple nonsulfur
bacteria, Green sulfur
bacteriaChemoautotroph(Chemolithotroph)Chemical energy source
(inorganic), Inorganic H/e- donor, CO2 carbon sourceSulfur-oxdizing
bacteria, Hydrogen bacteria,Nitrifying
bacteriaChemoheterotroph(Chenoorganotroph) Chemical energy source
(organic), Organic H/e- donor, Organic carbon sourceMost bacteria,
fungi, protozoa
Nutritional types of microorganisms
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
Passive transport (simple diffusion).
Facilitated diffusion.
Active transport.
Group translocation.
Passive transportmolecules 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.
Facilitated diffusion
The diffusion process is aided by a carrier.
Permeases.
Each carrier is selective and will transport only closely
related solutes
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.
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.
Proton gradientsSymport: linked transport of two substances in
the same direction.Antiport: linked transport of two substances in
the opposite direction.Uniport: one substance enter.
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.
Iron uptake - Siderophores (S)Microbial cell Fe 2+/S
ReceptorFe 2+/S
CULTURE MEDIA
CULTURE METHODS
History of Culture MediaThe 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.
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.
Colony? Macroscopically visible collection of millions of
bacteria originating from a single bacterial cell.
Types of culture media
Based on their consistency
Solid medium- contains 3% agarColony 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.
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 knownEg: 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
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
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Selective media
The inhibitory substance is added to a solid media.
Eg:Mac Conkeys medium for gram negative bacteriaTCBS for
V.cholerae.LJ medium M.tuberculosis.Wilson and Blair medium
S.typhi.Potassium tellurite medium Diphtheria bacilli.
Indicator media
These media contain an indicator which changes its color when a
bacterium grows in them.
Eg: Blood agarMac Conkeys mediumChristensens urease mediumXLD
Medium
Differential media
A media which has substances incorporated in it enabling it to
distinguish between bacteria.Eg: Mac Conkeys
mediumPeptoneLactoseAgarNeutral redTaurocholate Distinguish between
lactose fermenters & non lactose fermenters.
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 (Durhams tube) for the detection of gas by
the bacteria.
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: Stuarts medium non nutrient soft agar gel
containing a reducing agentBuffered glycerol saline enteric
bacilli
Anaerobic media
These media are used to grow anaerobic organisms.
Eg: Robertsons cooked meat medium, Thioglycolate medium.
Deep-freezing: 50to 95C.
Lyophilization (freeze-drying): Frozen (54 to 72C) and
dehydrated in a vacuum.
ReproductionBinary fission
Budding
Conidiospores (actinomycetes)
Fragmentation of filaments
Binary Fission
copyright@
Phases of Growth
Lag phase
Log (logarithmic) phase
Stationary phase
Decline phase or death phase
Phases of Microbial Growth
Measuring Bacterial Growth
Direct Measurements of Microbial Growth.
Plate counts: Perform serial dilutions of a sample.
Direct Measurements of Microbial Growth
Standard Plate CountInoculate Petri plates from serial
dilutionsTwo methods:Pour PlateSpread Plate
Plate Count
Other Methods: Estimating Bacterial Numbers by Indirect
MethodsTurbidity.
Factors Affecting Bacterial Growth
TemperatureMinimum growth temperature
Optimum growth temperature
Maximum growth temperature
Factors Affecting Bacterial GrowthpHMost bacteria grow between
pH 6.5 and 7.5.
Molds and yeasts grow between pH 5 and 6.
Acidophiles grow in acidic environments.
Factors Affecting Bacterial GrowthOsmotic pressure
Hypertonic environments, increase salt or sugar, cause
plasmolysis.
Extreme or obligate halophiles require high osmotic
pressure.
Facultative halophiles tolerate high osmotic pressure.
NOW TOPIC OPEN FOR DIOSCUSSION
