General Microbiology for 2nd year biology departments

2. Microbial Growth Conditions
1. Macronutrients
2. Micronutrients
3. Growth factors
4. Environmental factors: temperature; pH; Oxygen etc.
Nutrients:
Substances in the
environment used by
organisms for
catabolism and
anabolism.

3. 1. Macronutrients: required in large amounts, including: carbon,
oxygen, hydrogen, nitrogen, sulfur, phosphorus (Components of
carbonhydrates, lipids, proteins, and mucleic acids ); potassium,
calcium, magnesium and iron (cations and part of enzymes and
cofactors).
2. Micronutrients: Microbes require very small amounts of other
mineral elements, 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.
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.
Nutrient requirementsNutrient requirements

4. Amino acids are needed for protein synthesis,
purines and pyrimidines for nucleic acid synthesis.
Vitamins are small organic molecules that usually make up
all or part enzyme cofactors, and only very small
amounts are required for growth.

5. The Common Nutrient Requirements
 Macroelements (macronutrients)
• C, O, H, N, S, P, K, Ca, Mg, and Fe
• required in relatively large amounts
 Micronutrients (trace elements)
• Mn, Zn, Co, Mo, Ni, and Cu
• required in trace amounts
• often supplied in water or in media components

6. Microbial NutritionMicrobial Nutrition
A.A. Nutrient RequirementsNutrient Requirements
B.B. Nutritional types of microorganismsNutritional types of microorganisms
(Nutritional categories)(Nutritional categories)
C.C. Nutrient Transport ProcessesNutrient Transport Processes ((How doHow do
nutrient get into the microbial cell?)nutrient get into the microbial cell?)
D.D. Culture MediaCulture Media (How to cultivate(How to cultivate
microorganisms?)microorganisms?)
E.E. Isolation of Pure CulturesIsolation of Pure Cultures

7. Autotroph and Heterotroph
 All organisms require Carbon, Hydrogen, and
Oxygen. Carbon is needed for the backbone of all
organic molecules.
 In addition all organisms require a source of
electrons. Electrons are involved in oxidation-
reduction reactions in the cell, electron transport
chains, and pumps that drive molecules against a
concentration gradient on cell membranes.
 Organic molecules that supply, carbon, hydrogen,
and oxygen are reduced and donate electrons for
biosynthesis.

8. Autotrophs
 CO2 is used by many microorganisms as the
source of Carbon.
 Autotrophs have the capacity to reduce it ,
to form organic molecules.
 Photosynthetic bacteria and microalgae are
Photoautotrophs that are able to fix CO2
and use light as their energy source.

9. Heterotrophs
 Organisms that use organic molecules as their
source of carbon are Heterotrophs. The most
common heterotrophs use organic compounds for
both energy and their source of carbon.
 Microorganisms are versatile in their ability to use
diverse sources of carbon. Burkholderia cepacia
can use over 100 different carbon compounds.
 Methylotrophic bacteria utilize methanol,
methane, and formic acid.

10. Requirements for Nitrogen,
Phosphorus, and Sulfur
 Needed for synthesis of important molecules (e.g.,
amino acids, nucleic acids)
 Nitrogen supplied in numerous ways
 Phosphorus usually supplied as inorganic phosphate
 Sulfur usually supplied as sulfate via assimilatory
sulfate reduction

11. Requirements for Nitrogen
 Nitrogen is required for the synthesis of amino acids that
compose the structure of proteins, purines and pyrimidines the
bases of both DNA and RNA, and for other derivative
molecules such as glucosamine.
 Many microorganisms can use the nitrogen directly from amino
acids. The amino group ( NH2) is derived from ammonia
through the action of enzymes such as glutamate
dehydrogenase.
 Most photoautotrophs and many nonphotosynthetic
microorganisms reduce nitrate to ammonia and assimilate
nitrogen through nitrate reduction. A variety of bacteria are
involved in the nitrogen cycle such as Rhizobium which is able
to use atmospheric nitrogen and convert it to ammonia. ( Found
on the roots of legumes like soy beans and clover) These
compounds are vital for the Nitrogen cycle and the
incorporation of nitrogen into plants to make nitrogen
comounds.

12. Sources of nitrogen
Organic nitrogenOrganic nitrogen
• Primarily from the catabolism of amino acids
OxidizedOxidized forms of inorganicinorganic nitrogen
• Nitrate (NO3
2-
) and nitrite (NO2
-
)
Reduced inorganicReduced inorganic nitrogen
• Ammonium (NH4
+
)
Dissolved nitrogen gasDissolved nitrogen gas (N2
) (Nitrogen
fixation)

13. Requirements for Phosphorous
 Phosphorous is present in
phospholipids( membranes), Nucleic acids( DNA
and RNA), coenzymes, ATP, some proteins, and
other key cellular components.
 Inorganic phosphorous is derived from the
environment in the form of phosphates. Some
microbes such as E. coli can use
organophosphates such as hexose – 6-phosphates .
 Phosphate sourcePhosphate source
• Organic phosphate
• Inorganic phosphate (H2PO4
-
and HPO4
2-
)

14.  Sulfur sourceSulfur source
• Organic sulfur
• Oxidized inorganic sulfur
• Sulfate (SO4
2-
)
• Reduced inorganic sulfur
• Sulfide (S2-
or H2S)
• Elemental sulfur (So)
Requirements for Sulfur
 Synthesis of proteinSynthesis of protein
 Synthesis of vitaminsSynthesis of vitamins
 Involved in cellular respiration and phtosynthesisInvolved in cellular respiration and phtosynthesis

15. Growth Factors (Growth Factors (Special requirements)Special requirements)
 organic compounds
 essential cell components (or their
precursors) that the cell cannot synthesize
 must be supplied by environment if cell is
to survive and reproduce
e.ge.g..
•Amino acids
•Nucleotide bases
•Enzymatic cofactors or “vitamins”

16. Classes of growth factors
 amino acids
• needed for protein synthesis
 purines and pyrimidines
• needed for nucleic acid synthesis
 vitamins
• function as enzyme cofactors
Practical importance of growth factors
 Development of quantitative growth-response assays for
measuring concentrations of growth factors in a
preparation.
 Industrial production of growth factors by
microorganisms.

17. Nutritional types of microorganismsNutritional types of microorganisms
(Nutritional categories)(Nutritional categories)
 Energy SourceEnergy Source
• PhototrophPhototroph (Uses light as an energy source)
• ChemotrophChemotroph (Uses energy from the oxidation of
reduced chemical compounds)
 Electron (Reduction potential) SourceElectron (Reduction potential) Source
• OrganotrophOrganotroph (Uses reduced organic
compounds as a source for reduction potential)
• LithotrophLithotroph (Uses reduced inorganic
compounds as a source for reduction potential)

18. • PrototrophPrototroph
A species or genetic strain of microbe capable of
growing on a minimal medium consisting a simple
carbohydrate or CO2 carbon source, with inorganic
sources of all other nutrient requirements
• AuxotrophAuxotroph
A species or genetic strain requiring one or more
complex organic nutrients (such as amino acids,
nucleotide bases, or enzymatic cofactors) for growth
Chemical energy – source organic, Inorganic H/e- donor,
Organic carbon source
• MixotrophyMixotrophy
 Carbon sourceCarbon source
• AutotrophAutotroph (Can use CO2
as a sole carbon source)
• HeterotrophHeterotroph (Requires an organic carbon source;
cannot use CO2
as a carbon source)

19. Photolithotrophic autotrophs
 Use light energy and have CO2 as their carbon source.
 Cyanobacteria uses water as the electron donor and release
oxygen
 Purple and green sulfur bacteria use inorganic donors like
hydrogen and hydrogen sulfide for electrons
Chemoorganotrophic heterotrophs
 Use organic compounds as sources of energy,hydrogen,
electrons and carbon
 Pathogenic organisms fall under this category of nutrition

20. Photoorganoheterotrophs
 Common inhabitants of polluted streams. These bacteria
use organic matter as their electron donor and carbon
source.
 They use light as their source of energy
 Important ecological organisms
Chemolithotrophic autotrophs
 Autotrophs
 Oxidize reduce inorganic compounds such as iron,
nitrogen, or sulfur molecules
 Derive energy and electrons for biosynthesis
 Carbon dioxide is the carbon source

21. Uptake of Nutrients by the Cell
 Some nutrients enter by passive(simple) diffusion
 Most nutrients enter by:
• facilitated diffusion
• active transport
• group translocation
1. Phagocytosis – Protozoa
2. Permeability absorption – Most microorganisms
C. Nutrient Transport ProcessesC. Nutrient Transport Processes
How do nutrient get into the microbial cell?How do nutrient get into the microbial cell?

22. D. Culture MediaD. Culture Media
How to cultivate microorganisms?How to cultivate microorganisms?
 preparations devised to support the growth (reproduction)
of microorganisms
 can be liquid or solid
• Liquid medium
• Components are dissolved in water and sterilized
• Semisolid medium
• A medium to which has been added a gelling agent
• Agar (most commonly used)
• Gelatin
• Silica gel (used when a non-organic gelling agent is
required)
• solid media are usually solidified with agar
 important to study of microorganisms

23. Microbiological MediaMicrobiological Media
 Chemically defined vs. complex mediaChemically defined vs. complex media
• Chemically defined media
• The exact chemical composition is known
• e.g. minimal media used in bacterial genetics
experiments
• Complex media
• Exact chemical composition is not known
• Often consist of plant or animal extracts, such as
soybean meal, milk protein, etc.
• Include most routine laboratory media,
e.g., tryptic soy broth

24. Synthetic or Defined Media
 all components
and their
concentrations are
known

25. Complex Media
 contain some
ingredients of
unknown
composition
and/or
concentration

26. Some media components
 peptones
• protein hydrolysates prepared by partial digestion of
various protein sources
 extracts
• aqueous extracts, usually of beef or yeast
 agar
• sulfated polysaccharide used to solidify liquid media

27. Types of Media
 General purpose media
• support the growth of many microorganisms
• e.g., tryptic soy agar
 Enriched media
• general purpose media supplemented by blood or other
special nutrients
• e.g., blood agar

28. Types of media…Types of media…
 Selective media
• Favor the growth of some microorganisms and
inhibit growth of others
• MacConkey agar
• selects for gram-negative bacteria
• Inhibits the growth of gram-positive bacteria

29. Types of media…
 Differential media
• Distinguish between different groups of
microorganisms based on their biological
characteristics
• Blood agar
• hemolytic versus nonhemolytic bacteria
• MacConkey agar
• lactose fermenters versus nonfermenters

30.  Streak plate method
• Developed in the 1870s by Koch and his co-workers
• The objective: to obtain isolated colonies – spots of
microbial growth that come from a single parent cell
• The method: streak the sample on semisolid medium,
containing a gelling agent
• Agar: the most commonly used gelling agent
E. Isolation of Pure CulturesE. Isolation of Pure Cultures
 Spread plating & pour plating
 Limiting dilution

31. Pure Culture TechniquePure Culture Technique

32. The Spread Plate and Streak Plate
 Involve spreading a mixture of cells on an
agar surface so that individual cells are well
separated from each other
 Each cell can reproduce to form a separate
colony (visible growth or cluster of
microorganisms)

33. 1. dispense cells onto
medium in petri dish
2. - 3. sterilize spreader
4. spread cells
across surface
Spread-plate technique

34. inoculating
loop
Streak plate technique

35. Isolation of Pure Cultures
 Pure culture
• population of cells arising from a single cell
 Spread plate, streak plate, and pour plate are
techniques used to isolate pure cultures

36. The Pour Plate
 Sample is diluted
several times
 Diluted samples are
mixed with liquid agar
 Mixture of cells and
agar are poured into
sterile culture dishes

37. Colony growth
 Most rapid at edge of colony
• oxygen and nutrients are more available at edge
 Slowest at center of colony
 In nature, many microorganisms form
biofilms on surfaces

38. Terms
1. Colony shape and size: round, irregular, punctiform (tiny)
2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed)
3. Elevation: convex, umbonate, flat, raised
4. Color: color or pigment, plus opaque, translucent, shiny or dull
5. Texture: moist, mucoid, dry (or rough).

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