Topic: Nutritional requirements

Subject: Basic Techniques in Microbiology

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

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.

Requirements for Carbon, Hydrogen, and Oxygen often satisfied together

carbon source often provides H, O and electrons

autotrophs

use carbon dioxide as their sole or principal carbon source

heterotrophs

use organic molecules as carbon sources

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 are Photoautotrophs that are able

to fix CO2 and use light as their energy source.

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.

Comparison of Nutritional Modes

Major Nutritional Types of Microorganisms

Major Nutritional Type Source of Energy, Hydrogen,

electrons, and Carbon

Representative microorganisms

Photolithotrophic autotrophy Light energy

Inorganic hydrogen/electron( H+/e-

) donor

CO2 carbon source

Algae

Purple and green sulfur bacteria

Cyanobacteria

Photoorganotrophic heterotrophy Light energy

Organic H+/e- donor

Organic carbon source or CO2

Purple and Green non-sulfur

bacteria

Chemolithotrophic autotrophy Chemical energy source – inorganic

Organic H+/e- donor

CO2 carbon source

Sulfur oxidizing bacteria

Nitrifying bacteria

Iron oxidizing bacteria

Chemoorganotrophic

heterotrophy

Chemical energy source( organic)

Organic( H+/e-) donor

Organic carbon source

Most non photosynthetic bacteria

including pathogens.

Protozoans

FungiStudy table adapted from Microbiology, Prescott, Chapter Five.

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

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

Requirements for Nitrogen Nitrogen is required for the synthesis of amino acids that

compose the structure of proteins, purines and pyrimidines thebases of both DNA and RNA, and for other derivativemolecules such as glucosamine.

Many microorganisms can use the nitrogen directly fromamino acids. The amino group ( NH2) is derived fromammonia through the action of enzymes such as glutamatedehydrogenase.

Most photoautotrophs and many nonphotosyntheticmicroorganisms reduce nitrate to ammonia and assimilatenitrogen through nitrate reduction. A variety of bacteria areinvolved in the nitrogen cycle such as Rhizobium which is ableto use atmospheric nitrogen and convert it to ammonia. ( Foundon the roots of legumes like soy beans and clover) Thesecompounds are vital for the Nitrogen cycle and theincorporation of nitrogen into plants to make nitrogencomounds.

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 .

Sources of nitrogen Organic molecules

Ammonia

Nitrate via assimilatory nitrate reduction

Nitrogen gas via nitrogen fixation

Growth Factors 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

Classes of growth factors amino acids

needed for protein synthesis

purines and pyrimidines

needed for nucleic acid synthesis

vitamins

function as enzyme cofactors

Uptake of Nutrients by the Cell Some nutrients enter by passive diffusion

Most nutrients enter by:

facilitated diffusion

active transport

group translocation

What is a culture media? A growth or culture medium is a gel or liquid

substance designed to support the growth of micro organisms or cells.

There are different types of media used for different types of micro organisms or cells.

The most commonly used growth media for micro organisms are nutrient broths and agar plates.

Some fastidious organisms need specialized media for there growth.

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NEED FOR CULTURE MEDIA

LIQUID AND SOLID MEDIA

AGAR

TYPES OF CULTURE MEDIA

TYPES OF CULTURE MEDIA

TYPES OF CULTURE MEDIA

TYPES OF CULTURE MEDIA

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Contents

Peptone

Lab-Lemco powder

Yeast extract

Sodium chloride

Agar

Uses It is used for culturing almost every micro organism.

Non-fastidious organisms grows well on nutrient agar.

It is used for routine culturing in microbiology lab.

In the early 1900’s it is suggested for water testing.

TYPES OF CULTURE MEDIA

ENRICHED MEDIA

ENRICHED MEDIA

Selenite F Broth It is recommended as enrichment media for the

growth of Salmonella from feces, urine and other pathological specimens

It is a selective medium.

Cotents Part A

Casein enzymic hydrolysate

Lactose

Sodium phosphate

Part B

Sodium hydrogen selenite

ReactionsOrganism Recovery Color of colony

Escherichia coli No increase in number Pink with bile precipitation

Salmonella typhii Good - luxuriant Colorless

Salmonella typhimurium

Good - luxuriant Colorless

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SELECTIVE MEDIA

Selective Media

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INDICATOR MEDIA

INDICATOR MEDIA

Blood agar It is an enriched and differential media.

It is used to grow fastidious organisms.

It contains defibrinated mammalian blood (usually sheep or horse blood).

Note:

Sheep blood may contain inhibitors to Heamophilus Influenzae.

Expired, citerated, donor blood should not be used because this may contain substance inhibitory to the growth of some pathogens (e.g. β Hemolytic Streptococci).

Uses It is used for the growth of fastidious organisms.

It is a differential medium based on the hemolytic reaction.

There are 2 types of hemolytic reactions shown by the organisms o blood agar plate.

• α-Hemolysis

• β-Hemolysis

BLOOD AGAR

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Chocolate agar When blood agar is heated, the red cells are lysed and

medium becomes brown in color. It is referred to aschocolate agar and supplies the factors required forthe growth Haemophilus influenzae.

It is also used to culture nutritionally demandingpathogens such as Neisseria meningitides andStreptpcoccus pneumoniae

DIFFERENTIAL MEDIA

DIFFERENTIAL MEDIA

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SUGAR MEDIA

TRIPLE SUGAR IRON AGAR (TSI)

o It contains 3 sugars – Glucose, Lactose and Sucrose.

o It is a composite media used to study different

properties of a bacterium – sugar fermentation,

gas production and H2S production.

o It is an orange red medium with a slant and a butt.

o It is recommended for the identification of member of Enterobactriaceae and Salmonella.

TSI ReactionsYellow – Acid

Pink - Alkaline

Yellow slant / Yellow butt – Lactose fermenters.

Pink slant / Yellow butt – Non lactose fermenters.

Pink slant / no colour change – Non fermenters

Black colour – H2S production.

Gas bubbles or crack in the medium – gas production.

Note

LF – E.coli, Klebsiella

NLF – Salmonella, Shigella

H2S - Proteus

TSI Reactions

Orgaism Slant Butt H2S Gas

E. Coli Yellow Yellow -ve +ve

Klebsiella Yellow Yellow -ve +ve

Citrobacter Red/Yellow Yellow d +ve

Enterobacter Yellow Yellow -ve +ve

Salmonella Red Yellow Weakly +ve -ve

Shegella Red Yellow -ve -ve

Proteus Red Yellow +ve d

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PURE CULTURES METHOD

CULTURE METHOD

STREAK CULTURE

STREAK CULTURE

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LAWN CULTURE

LAWN CULTURE

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STROKE CULTURE

10

STAB CULTURE

10

PORE PLATE CULTURE

POUR PLATE CULTURE

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LIQUID CULTURE

ANAEROBIC CULTURE METHODS

ANAEROBIC CULTURE METHODS

ANAEROBIC CULTURE METHODS

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CHEMICAL METHOD

WORKING OF ANAEROBIC JAR

GASPAK

Figures:Fig 1 http://microbeonline.com/tag/types-of-culture-media-for-bacteria/Fig 2 http://www.medicotips.com/2011/07/culture-media-for-bacteria-types-of.htmlFig 3 http://en.wikipedia.org/wiki/Chocolate_agarFig 4 http://en.wikipedia.org/wiki/Growth_mediumFig 5 http://iws2.collin.edu/dcain/CCCCD%20Micro/hemolysis.htmFig 6 http://faculty.mc3.edu/jearl/ML/ml-8.htmFig 7 http://commons.wikimedia.org/wiki/File:Agar_tsi.JPGFig 8 http://jeremyrenners.blogspot.in/2009/01/streaking-agar-plates.htmlFig 9 http://en.wikipedia.org/wiki/Bacterial_lawnFig 10 http://textbookofbacteriology.net/structure_2.htmlFig 11 http://classes.midlandstech.edu/carterp/courses/bio225/chap06/lecture5.htmFig 12 http://www.ijmm.org/article.asp?issn=0255-

0857;year=2013;volume=31;issue=2;spage=173;epage=176;aulast=Maiti

References

Book:• Microbiology by pelczar

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