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Copyright © McGraw-Hill companies, Inc. Permission required for reproduction or display. 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
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Copyright © McGraw-Hill companies, Inc. Permission required for reproduction or display.
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
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Requirements for Carbon, Hydrogen, and Oxygen
• often satisfied together• autotrophs
– use carbon dioxide as their sole or principal carbon source
• heterotrophs– use organic molecules as carbon
sources
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Nutritional Types of Microorganisms
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Uptake of Nutrients by the Cell
Most common mechanisms are:– facilitated diffusion– active transport– group translocation
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Active Transport
• energy-dependent process– ATP or proton motive force used
• moves molecules against the gradient
• concentrates molecules inside cell• involves carrier proteins (permeases)
– carrier saturation effect is observed
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ABC transporters
• ATP-binding cassette transporters
• observed in bacteria, archaea, and eucaryotes
Figure 5.3
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Figure 5.4
antiport
symport
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• molecules are modified as they are transported across the membrane
• energy-dependent process
P-S-PT-SOr
PTS
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Iron Uptake• ferric iron is very
insoluble so uptake is difficult
• microorganisms use siderophoresto aid uptake
• siderophore complexes with ferric ion
• complex is then transported into cell
Figure 5.6
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Culture Media• preparations devised to support the
growth (reproduction) of microorganisms
• can be liquid or solid– solid media are usually solidified with
agar
• important to study of microorganisms
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Synthetic or Defined Media
all components and their concentrations are known
I media sintetici hanno una composizione chimica nota e relativamente semplice. Utili per i FOTOAUTOTROFI, per selezionare mutanti nutrizionali e per usare radioisotopi.
Esempio: desiderate non selezionare organismi che possano ottenere tutto l'azoto dall’ N2 atmosferico ? Preparate il media sintetico senza fonte di N.
MEDIUM BG-11 A modification of this medium, BG-110 (nitrate omitted) isemployed for nitrogen-fixing strains after addition of a solution of filter-sterilized NaHCO3 (5 or 10 mM final concentration, as specifiedin the strain description).
Pasteur Culture Collection of CyanobacteriaCulture media
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Complex Media
• contain some ingredients of unknown composition and/or concentration
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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
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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
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Types of media…
• selective media– favor the growth of some
microorganisms and inhibit growth of others
– e.g., MacConkey agar• selects for gram-negative bacteria
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Types of media…
• differential media– distinguish between different groups of
microorganisms based on their biological characteristics
– e.g., blood agar• hemolytic versus nonhemolytic bacteria
– e.g., MacConkey agar• lactose fermenters versus nonfermenters
Luria Broth
pH Conditions Adjust pH to 7.0 with 1N HCl
Remarks For solid media add 1.5% agar before sterilization
Tryptone 10 g
yeast extract 5 g
NaCl 5 g
water 1 l
Media Complessi: composizione non completamente nota.Spesso ricavati da materiali organici economici avanzi di carni (sottoposti a proteolisi
parziale: tryptone ecc.), avanzi di soie, lieviti (sorgente ricca di vitamine ad es), sangue animale, ecc.
I media standard usati in laboratorio sono di norma complessi
The basic growth media for bacteria is Nutrient Broth (NB), Nutrient Agar (NA), Tryptic Soy Broth (TSB), and TrypticSoy Agar (TSA). These preparations may be made in variousforms: broth, deep, slant or plates.
L’autoclave è attalmente lo strumento più usato in laboratorio per sterilizzare strumenti e terreni
Lavora ad una pressione di vapore di una atmosfera e 121°CTmin=15min routine ma in generale f(Vol da sterilizzare)->Altre forme di sterilizz. al calore
I terreni selettivi possono essere sintetici o complessi.
Esempio: i sali della bile inibiscono lo sviluppo della maggior parte dei batteri gram-positivi e di alcuni batteri gram-negativi, ma i batteri enterici crescono bene.Il terreno di MacConkey è un esempio molto importante di terreno seletivo
Il terreno di MacConkey è contemporaneamente un terreno differenziale.L' agar di MacConkey ha indicatore di pH che distingue la presenza di acido prodotto da
batteri che fermentano uno zucchero (che si coloreranno di rosa-rosso). I batteri che non possono fermentare lo stesso zucchero crescono ma rimangono bianchi.
MAC is selective:Gram negative organisms will grow, Gram positive organisms will not. This is due to the addition of bile salts and crystal violet.
MAC is differential:Lactose fermenters will appear pink, non-lactose fermenters will appearcolorless. This is due to the additionof the indicator, neutral red.
Enterobacter cloacaeGram Negative (growth)Lactose Fermenter (pink colonies)
Escherichia coliGram Negative: growthLactose Fermentation: positive: pink colonies
SalmonellatyphimuriumGramNegative: growthLactoseFermentation: negative (colorlesscolonies)
Notare alcuni patogeni si distinguono per questa caratteristica
Staphylococcus aureusGram Positive: did not growLactose fermentation: undetermined.
Fermentation for S. aureus or other gram positive organisms cannot be determined from this test since theydo not grow on MAC agar.
To determine lactose fermentationfor gram positive organisms, use the carbohydrate fermentation tests.
Carbohydrate Fermentation Tests (Phenol Red Broth)
LactoseFermentation
Organism Test Result
1. Control -
2. S. aureus +
3. P. vulgaris -
4. P. aeruginosa -
5. E. coli +
Metodi di isolamento di colture pure-1
Metodi di isolamento di colture pure-2
Metodi di isolamento di colture pure-3
Una tipica curva di crescita batterica di una coltura in batch
G0, ≠metab.
V max cresc.; no limit.nutr.Bioch. omogenee
Nutrienti or O2, metaboliticellule: In/out or stopresa+massa per cell
Lomg-termsurvival
conservazione
Gen(t) number log2N log10Nof bacteria
0(0’) 1 or 20 0 0
1(20’)2 or 21 1 .301
2(40’)4 or 22 2 .602
3(60’)8 or 23 3 .903
4 ... 16 or 24 4 ...
5 ... 32 or 25 5 …
n (t) 2n n ...
Vedi dip. lin.f(t)
La fase di crescita esponenziale
Gen(t) number log2N log10Nof bacteria
0(0’) 1 or 20 0 0
1(20’)2 or 21 1 .301
2(40’)4 or 22 2 .602
3(60’)8 or 23 3 .903
4 ... 16 or 24 4 ...
5 ... 32 or 25 5 …
n (t) 2n n ...
Gen(t) number of bacteria
0(0) 1 N0
n (t) 2n N0 2n
Nt = N0 2n
Vedi dip. lin.f(t)
