micro sheet # 4

Today our lecture will be about bacterial growth.

Bacterial Growth

*When we discuss bacterial growth & culture, we should study the physical & chemical processes which take place at the cellular level that end by growth of bacteria.
*Metabolism is influenced by physical & chemical processes that take place at the cellular level.
Grow: Increase in size & number which leads to division of the organism for production & finally increase in number of it.
*Bacteria Multiply by binary fission, where the chromosomal DNA divides into 2 chromosomes & each one of them are surrounded by cell membrane, then the cell wall divides & each half becomes a new daughter cell.
*Starting with one cell, binary fission theoretically results in 10 cells in 24 hours, that is 4 tones (4000Kg) of bacteria, but actually when the number reaches (10^8-10^9) bacteria do not divide any more due to many factors:
1. Depletion of (O2) & other nutrients.
2. Accumulation of toxic metabolites.
3. there are continuously dying bacteria.

Growth requirements:

- Chemical requirements:
1. Water:
*The most important element.
*Solvent that dissolve nutrients.
*Important in hydrolysis reactions as some compounds cannot be taken unless they are hydrolyzed or dissolved.
*Medium for reactions to occur especially Glycolysis.
*Providing precursors for growth.

2. Carbon:
*It's an essential component of proteins, lipids & other macromolecules.
*Depending on the source of carbon, bacteria are classified into 2 major groups:
A- Autotrophs: that utilize inorganic carbon in the form of CO2.
B- Heterotrophs: they require ready-made organic molecules of food as sugar. Heterotrophs are further classified based on the energy used in utilizing these organic molecules to:-
1. Photoheterotrophs: they use light as a source of energy.
2. Chemoheterotrophs: in which chemical energy is the source of energy.

Source of energy:

*Carbon may be obtained from proteins in the presence of suitable environment &certain proteases,( protein act as a source of carbon & nitrogen).

3- Nitrogen:
*Present in amino acid, purines & pyrimedins (nucleic acid).
*NO3 & NO 2 are the major sources for N2 but they must be converted into ammonia (NH3) before they can be utilized.
*Sometimes bacteria breakdown proteins (using proteases) to get carbon & nitrogen.

4- Sulfur and Phosphorous:
*obtained from inorganic sources & are integrated into sulfur containing amino acids & phosphate groups in nucleic acid.
*Phosphate acts as intracellular buffer due to its capability to absorb hydrogen ions which results from metabolism.
*Hydrogen ions are toxic & can raise acidic environment which neutralized by presence of phosphorus & mono or dipotassium salts (K2HPO4, KH2PO4).
*Sulfur is a reactive group especially in disulphide bonds.

5- Oxygen:
*It is a very important element.
*During normal respiration, molecular oxygen is reduced by addition of 4e to generate water & some reactive by-products (H2O2 & (superoxide) which are are produced inside the cell.
*These by-products create havoc by reacting with protein & DNA, thus inactivating them.
*It is the last e acceptor, so it is very important growth factor.
1) O2 + 2e O2 - superoxide.
2) O2- + e + 2H+  H2O2 Hydrogen peroxide.
3) H2O2 + e + H+  + H2O +OH. Hydroxyl radical.
4) OH. + e+ H+  H2O Water.

 Over all: O2+ 4e + 4H+  2H2O

*O2 will react in the cell to form two major toxic products, hydrogen peroxide & superoxide. these molecules can potentially wreck havoc on a cell by causing uncontrolled reductions of cellular components:
- Superoxide is detoxified by superoxide dismutase, which combines two superoxide molecules forming hydrogen peroxide (H2O2) & (O2).

*Hydrogen peroxide is dealt with the enzyme catalase which takes 2 hydrogen peroxides & forms one molecule of water & one molecule of oxygen.
*Peroxidase reduces hydrogen peroxide with NADH- + H- and produces water.

*Anaerobes have no cellular machinery (enzymes) to protect them from the oxidizing power of oxygen. Organisms that cannot deal with the problems presented by O2 cannot survive in the air & are killed.

*According to oxygen requirements bacteria are divided into:
1. Aerobic bacteria: must grow in the presence of O2, they can't carry out fermentation, & they live in atmospheric concentration of O2 = 20% "not 10% ;)"

2. Anaerobic bacteria: do not carry out oxidative phosphorylation . Furthermore, they are killed by O2 they lack enzymes which detoxify both hydrogen peroxide & oxygen free radicals (superoxide) produced as by-products during metabolism in the presence of O2 extremely sensitive organisms.

3. Aerotolerant anaerobes: are bacteria that respire anaerobically, but can survive in the presence of O2 .

*Between the 1st two categories there are:
4. Facultative anaerobes: can perform both fermentation (without O2) & aerobic respiration. In the presence of O2, anaerobic respiration is generally shut down & these organisms respire aerobically. Most human pathogens are of this type.

5. Microaerophilic bacteria: grow well in low concentration of O2 (less than the atmospheric O2), but are killed by higher concentrations.(about 5% atmosphere for its optional growth)

*O2 plays important rule as electron acceptor & its fully metabolism require 4 e to form H2O.

*During its metabolism, free radicals are formed like (singlet O2,superoxide, hydroxyl radical, & hydrogen peroxide) their hazardous effect will be on DNA.

6-Minerals:
Include: Ca,Mg,K,Fe,S,Na,Zn,Co,Cu,Mn,…..
These minerals are involved in metabolic pathway of different bacteria because they are required for the action on enzymes.

*some minerals have odd functions as in the case of some types of hot spring bacteria that depend on sulfur for respiration!.

7-Growh factor:
*Are supplied by the media in which bacteria is growing.
*Required but cannot be synthesized in the cell.
*They are taken as ready-made molecules.
*Bacteria do not grow unless provided with these factors by the media.

Physical requirements:


1-Hydrogen ions concentration (PH):
-It is one of the most important factors of normal cell activity, it's related to the acidity of the environment.
-Bacteria like other organisms require certain H concentration.
-Any deviation in this PH might be lethal to bacteria.
-Bacteria is classified according to PH they tolerate into:

-Neutrophills:
*almost all kinds of bacteria are involved in this class & they live in almost a neutral PH (7.2-7.4).
*Bacteria that cause human diseases are mostly neutrophills cz they accommodate with human PH.

-Basophills :
They require increased PH in order to live & grow, ex: Vibro cholera requires PH around 8.6.


-Acidophills:
Require decreased PH, ex: heleco bacter which lives in stomach & may cause gastritis, this one requires PH around 3.





2- Temperature:

*mesophills:
-the grp of bacteria that grow @ body temp. (35-37c), and most bacteria that cause human diseases are of this type ,as a result our body uses defense mechanism against such bacteria by elevating the body temp.

*Thermophills:
-minor category of bacteria that can live in a higher temp. (more than 40c) e.g: pseudomonas bacteria & tag polymerase enzyme which is used in poly chain reaction is produced by thermophills which take place in a high temp (90c).

*psychrophills:
-They can grow at lower temp (<30c) or even at refrigerated temp like staphyl cocci.
-Fungi also psychrophills.
-all bacteria can be preserved in frozen environment! They don't die but they stop metabolism.

3-Osmotic pressure:
It is the Concentration of solutes in a medium .
*most bacteria that cause human disease grow @ our body media which is mainly Isotonic (equal to Na+ conc.= 0.9mol/L).
*there are bacteria that live in hypertonic solution & they are called Halophills. E.g: Staphylcocci.
*gall bladder is a hypertonic solution.
*there is NO bacteria that live in Hypotonic media .
Hepertonic & hyptonic madia have adverse effects on baceria.but their cell wall protects them, so if it is absent they will undergo Lysis.



Cultivation of microorganisms:
We need a media that meets the chemical & physical needs.
*media can be classified according to its physical state to :
-broth, semisolid,solid.
*media can be solidified by addition of Ager.
*semisolid:few quantities of ager ,solid: more ager.
*Broth: liquid media.
*ager: is solid & melts @ high temp. (60-70c), then become solid again @ 45c.
*according to its composition, media can be either:
1- defined media : a medium that we prepare, so we know its exact composition.


2- Complex media: is the one that we obtain from a certain source like (meet), & we don't exactly know its exact composition.

*Media can be in different types according to the purpose of the medium. So there is:
1- All purpose media: used for all purposes & to grow all kinds of bacteria.

2- Enriched Media:
- a media that when an ingredient or more is added to it, we can achieve more bacterial growth.
Ex.: addition of blood, or adding alkaline peptone water for cholera, or salinate for salmonilla & shigalla .

3- Selective media:
- a media which enhances the growth of one organism & inhibits (suppresses) the growth of others. & this is achieved by addition of certain substances.
Ex.: addition of certain antibiotics or use dies like crystal violet which inhibits gram negative.
- Selectivity can be reduced to select individual genus only.

4- Differential media:
- Which allows the differentiation between different bacteria.
- Ex. we can differentiate between organisms that fermentate lactose from those that don’t by addition of lactose to the media & then using a pH indicator that indicates the presence of "Acid-products of fermentation" in the colonies that fermentate lactose.

5- Transport media:
- When there is a sample, that has to be studied, in a place where there are no lab. Facilities"like if u were in the desert!!"& u need to transport the sample to a lab, so u should use transport media.
- Transport media usually contains minimum requirements, (because we are not concerned her of growing & producing more bacteria).



Growth Phase:
- Organisms will grow if they were placed in a suitable media.
- The increase of population of vital bacteria with time could be plotted in "growth curve"  go to the pic in the slide!.
- Because the number of organisms is usually very high we study the log not the absolute number.
- The phases:
1. Log phase
2. Acceleration phrase
3. Exponential phase
4. Stationary phrase
5. Death phrase



Done by :Asma Hannoun 


This sheet is dedicated to Farah Ismail & Yasmeen Hzayen 