2. Nutrients are substances used in biosynthesis and
energy release and therefore are required for microbial
growth
INTRODUCTION
3. Common Nutrient Requirements
The first six (C, O, H, N, S and P) : components of carbohydrates, lipids and proteins
K+ : enzyme -protein synthesis
Ca2+ : heat resistance of bacterial endospores
Mg2+ : cofactors, stabilise the cell membrane and ribosomes
Fe2+ and Fe3+ : part of cytochrome and cofactors for enzymes and ECP
95%
cells dry
weight
Carbon
Oxygen
Nitrogen
Phosphorous
Calcium
Hydrogen
Potassium
Magnesium
Sulphur
Iron
Macroelements/
Macronutrients
4. Micronutrients/ Trace elements : Several nutrients which are required in small
amounts
Ubiquitous and normally part of enzymes and cofactors
Aid in catalysis of reaction and maintenance of protein structure
Mn2+: transfer of phosphate groups
Mo2+ : nitrogen fixation
Co2+ : component of vitamin B12
Hence microorganisms require a balanced mixture of nutrients
Micro
nutri
ents
Manganese
Zinc
Cobalt
Molybdenum
Nickel
Copper
5. Requirements for Carbon, Hydrogen, Oxygen and
Electrons
All organisms need carbon, oxygen, hydrogen and source of
electrons
CARBON : Skeleton/backbone of all organic molecules from which
the organism is built
HYDROGEN & OXYGEN: Important elements found in the organic
molecules
ELECTRONS
Electron transport chain and the oxidation reduction reactions-
energy for use in cellular work
Reduce molecules during biosynthesis (CO2 organic
molecules)
reduced
6. AUTOTROPHS : use only CO2 as a sole source of carbon
obtain energy from light or reduced inorganic molecule
Because CO2 cannot supply their energy needs, they must obtain energy
from other sources, such as light or reduced inorganic molecules
HETEROTROPHS : organisms that use reduced, preformed organic
molecules as their carbon source
Heterotrophic microorganisms has extraordinary flexibility with respect
to carbon sources
Actinomycetes : common soil bacteria
degrade the paraffin, amyl alcohol and even rubber
Burkholderia cepacia : use 100 different carbon compounds
Leptospira : use long chain fatty acids as the major source of carbon
7. Nutritional Types of Microorganisms
Source of Carbon, Energy and Electrons
CARBON SOURCES
Autotrophs CO2 sole or principle biosynthetic carbon
source
Heterotrophs Reduced, performed, organic molecules
from other organisms
ENERGY SOURCES
Phototrophs Light
Chemotrophs Oxidation of organic or inorganic
compounds
ELECTRON SOURCES
Lithotrophs Reduced inorganic molecules
Organotrophs Organic molecules
Microorganisms can be classified as autotrophs or heterotrophs on the
basis of the preferred source of carbon.
There are two sources of energy available to organisms
Light energy
Energy derived from oxidising organic or inorganic molecules
PHOTOTROPHS : Use light as energy source
CHEMOTROPHS : energy obtained from oxidation of chemical compound
LITHOTROPHS (rock eaters) : Reduced inorganic substance as energy source
ORGANOTROPHS : extract electrons from reduced organic source
Majority of the microorganisms studied so far are Photolithotrophic
autotrophs or chemoorganotrophic heterotrophs
8. Major nutritional types of microorganisms
NURTIONAL TYPE CARBON
SOURCE
ENERGY
SOURCE
ELECTRON
SOURCE
REPRESENTATIVE
MICROORGANISMS
Photolithography CO2 Light Inorganic e-
donor
Purple and green sulfur
bacteria, cynobacteria
Photoorganoheterophy Organic
carbon
Light Organic e-
donor
Purple nonsulfur
bacteria, green
nonsulfur bacteria
Chemolithoautotrophy CO2 Inorganic
chemicals
Inorganic e-
donor
Sulfur, iron, hydrogen-
oxidizing bacteria,
methanogens
Chemolithoheterotrophy Organic
carbon
Inorganic
chemicals
Inorganic e-
donor
Some sulfur oxidizing
bacteria
Chemoorganoheterotrophy Organic
carbon
Organic
chemicals
Organic e-
donor
Fungi, protists and
many archaea
9. Phototrophic bacteria play important roles in aquatic ecosystems, where they can
cause blooms
(a) A cyanobacterial and an algal bloom in a eutrophic pond
(b) Purple sulfur bacteria growing in a bog
(c) A bloom of purple sulfur bacteria in a sewage lagoon
Phototrophic Bacteria
10. Chemolithotrophic Bacteria
(a)Transmission electron micrograph of Nitrobacter winogradskyi, an organism that
uses nitrite as its source of energy
(b) Light micrograph of Beggiatoa alba, an organism that uses hydrogen sulfide as its
energy source and organic molecules as carbon sources. The dark spots within the
filaments are granules of elemental sulfur produced when hydrogen sulfide is oxidized
11. Requirements for Phosphorus, Nitrogen and
Sulfur
Nitrogen : synthesis of amino acids, purines, pyrimidines, some
carbohydrates and lipids, enzyme cofactor
Some incorporate ammonia directly through the action of enzymes such
as glutamate dehydrogenase or glutamine synthetase and glutamine
synthase
Phototrophs and chemotrophic microbes
Nitrogen Fixation :
Variety of bacteria (Cyanobacteria, Symbiotic bacterium Rhizobium)
Assimilate atmospheric nitrogen (N2) by reducing it to ammonium
Nitrate Ammonia Incorporated
Assimilatory
nitrate reduction
12. Phosphorus
Phosphorus present in nucleic acid, phospholipids, nucleotides (ATP),
cofactors, some proteins and other cell components
Use of inorganic phosphate as phosphorous source which is directly
incorporated
Aquatic environments : Low phosphate levels limits microbial growth
E.coli uses organic and inorganic phosphate
Organophosphate
Hexose 6-phosphate
Taken up directly
by the cell
Others
Inorganic
Phosphate
Transported across
plasma membrane
14. Growth Factors
Organic compounds that are essential cell components or precursors of such
components but cannot be synthesised by the organism
Protein synthesis
Nucleic acid
synthesis
All or some part of
enzyme cofactors
Vitamin Functions Examples
Biotin One carbon
metabolism, CO2
fixation
Leuconostoc mesenteroids
Saccharomyces cerevisiae
Acanthamoeba castellanii
Folic acid One carbon
metabolism
Tetrahymena spp.
Enterococcus fecalis
Riboflavin (B2) Precursor of FAD &
FMN
Caulobacter vibriodes
Dictyostellium spp.
Thiamine (B1) Aldehyde group
transfer
Bacillus anthracis
Colpidium campylum
Ochromonas malhamensis
Pantothenic acid Precursor of
coenzyme A
Proteus morganii
Paramecium spp.
Functions of some common vitamins in microorganism
16. A solid/liquid preparation used to grow, transport, and
store microorganisms
Complex liquid media (urine, chicken/ meat broth)- Louis
Pasteur
Solid media (Potato surface, gelatin): Robert Koch
• Gelatin melts at 24ºC
• Microbes used it as a substrate
Agar was first described for use in microbiology by Walter Hesse
Culture Media
17. Culture Media
Requirement
s of culture
media
Carbon
source Energy
source
Nitrogen
source
Salts
pH
Growth
factors
Indicator
s
Inhibitor
s
Oxidatio
n
reductio
n
potential
19. Liquid and solidified media are routinely used in microbiology labs, solidified media
are particularly important
Both defined and complex media can be solidified with the addition of 1.0 to 2.0%
agar; most commonly 1.5% is used
Agar –
Sulphated polymer (D-galactose, 3,6-anhydro-L-galactose, and D-glucuronic
acid)
Extracted from red algae
Melting temperature- about 90°C and Solidifying temperature- 45°C
Microbes growing on agar medium can be incubated at a wide range of
temperatures
Agar is an excellent hardening agent because most microorganisms cannot
degrade it
Other solidifying agents -silica gel is used to grow autotrophic bacteria
Culture Media
20. Defined or Synthetic medium
• All chemical components are known in defined medium.
• Can be in a liquid form (broth) or solidified by an agent such as
agar
• Widely used in research, as it is often desirable to know what
the experimental microorganism is metabolizing
• Culture photolithotrophic autotrophs (cyanobacteria and
photosynthetic protists), chemoorganotrophic heterotrophs
• All defined media are as simple, but may be constructed from
dozens of components
Culture Media
Medium for Escherichia coli Amount (g/litre)
Glucose 1.0
Na2HPO4 16.4
KH2PO4 1.5
(NH4)2SO4 2.0
MgSO4· 7H2O 200.0 mg
CaCl2 10.0 mg
FeSO4 · 7H2O 0.5 mg
Final pH 6.8–7.0
21. Complex media
Media that contain some ingredients of unknown chemical composition
Single complex medium may be sufficiently rich to completely meet the
nutritional requirements of many different microorganisms
The nutritional requirements of a particular microorganism are unknown, and
thus a defined medium cannot be constructed
Undefined components like peptones, meat extract, and yeast extract
Nutrient broth, tryptic soy broth, and MacConkey agar
Culture Media
Tryptic Soy Broth Amt (g/ltr)
Tryptone (pancreatic digest of
casein)
17
Peptone (soybean digest) 3
Glucose 2.5
Sodium chloride 5
Dipotassium phosphate 2.5
22. General purpose media or supportive media: they sustain the growth of
many microorganisms. Ex: tryptic soy broth and tryptic soy agar
Enriched media: Blood and other special nutrients may be added to general
purpose media to encourage the growth of fastidious microbes. These specially
fortified media (e.g., blood agar)
Selective media: favour the growth of particular microorganisms
Differential media: are media that distinguish among different groups of microbes
and even permit tentative identification of microorganisms based on their biological
characteristics (e.g., blood agar: hemolytic and non-hemolytic bacteria)
Functional types of media
23. Functional types of media
(a) Blood agar culture of bacteria from the human throat
(b) Chocolate agar, an enriched medium used to grow fastidious organisms such as
Neisseria gonorrhoeae
The brown color is the result of heating red blood cells and lysing them before adding
them to the medium
It is called chocolate agar because of its chocolate brown color
a
b
24. Pure culture: a population of cells arising from a single cell,
to characterize an individual species.
Pure culture techniques were developed by Robert Koch
Few common approach's to prepare the pure culture are
The spread plate and streak plate
The pour plate
Isolation of Pure Cultures
25. The spread plate is an easy, direct way of achieving this result.
The dispersed cells develop into isolated colonies
Isolation of Pure Cultures
Spread-Plate Technique
dilute microbial mixture (30 to 300 cells)
transferred
centre of an agar plate
a sterile bent-glass rod
spread evenly over the surface
26. Spread-Plate Technique
(a)The preparation of a spread plate.
(1) Pipette a small sample onto the centre of an
agar medium plate.
(2) Dip a glass spreader into a beaker of
ethanol.
(3) Briefly flame the ethanol-soaked spreader
and allow it to cool.
(4) Spread the sample evenly over the agar
surface with the sterilized spreader. Incubate.
(b)Typical result of spread-plate technique.
27. STREAK PLATE METHOD
Pure colonies also can be obtained from streak plates
The microbial mixture edge of an agar plate
streaked out over the surface in one of several patterns
Thus this is essentially a dilution process and single colonies are developed
Isolation of Pure Cultures
inoculating loop or swab
transferred
inoculating
loop is
sterilized
streaking the third sector
After the first sector is streaked
Inoculum for the second sector is obtained from the first sector
29. POUR PLATE
Isolation of Pure Cultures
Serial dilution of the originals sample
Small volume of the serially diluted sample
+ liquid agar (45ºC)
Mixture immediately transferred into the
sterile culture dishes
After the agar is hardened, each cell is fixed
in a place and forms a individual colony
Colonies growing on the surface can be
taken to prepare pure cultures
Solidified media can be used to isolate different microbes from each other in order to establish pure cultures
Because the number of colonies should equal the number of viable organisms in the sample, spread plates can be used to count the microbial population.
Eventually, very few cells will be on the loop, and single cells will drop from it as it is rubbed along the agar surface.
Extensively used with procaryotes and fungi, a pour plate also can yield isolated colonies. Plates containing between 30 and 300 colonies are counted. The total number of colonies equals the number of viable microorganisms in the sample that are capable of growing in the medium used.
