number Done by Corrected by Doctor Hamed Alzoubi

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1 number 4 Done by Tasneem Mountaj Saad Hayek Corrected by حسام أبو عوض Doctor Hamed Alzoubi

2 Bacterial growth, physiology and metabolism Bacteria, like all living species, need to grow and replicate in order to survive. In this sheet, we will discuss general properties and basic information regarding this topic. Bacterial Growth: It's known as any increase in the size and number of the organisms, but regardless of which process contributes more to the growth we call the net effect "increase in total mass (biomass) of the culture". Some Bacteria divide in a high rate they re called fast growing bacteria as they take min to divide e.g Vibreo Cholera, others divide in a slow rate as they take around 24 hours to divide and they re called slow growing bacteria e.g Mycobacterium Tuberculosis. Bacteria need nutrients (provide raw materials) and energy (allow reactions to occur) to grow. We'll focus on the increase of number as a diagnostic tool. In the lab, we can observe bacterial growth via three ways: a. Development of colonies : the microscopic product of cell divisions of a single cell. b. Transformation of a clear broth medium to a turbid suspension of cells per ml. c. Biofilm formation: which is growth of a bacterial community, in which its cells are attached to each other and often adhere to another surface (in the case of a lab; they adhere to a catheter or any other inert object). Colony formation. Increasing turbidity from left to right.

3 Biofilm formation Stages of Bacterial Growth: A. Lag phase Cells don't increase in number (or in slow rate), they use the available nutrients instead and adjust to the new "situation". like they re getting prepared to divide by making protein and so on (metabolic activity is high). B. Log (exponential) phase Most of the replication and growth occurs here. The bacterial population doubling occurs at constant rate (fastest rate). The rate of cell division is higher than the rate of cell death. C. Stationary phase Occurs when cell division decreases to a rate equal to that of cell death, i.e the number of cells remains constant. In this stage nutrients begin to deplete and toxins are produced (toxins are produced as a survival mechanism by bacteria). D. Death (decline) phase The number of deaths exceeds the number of newly formed cells.

4 Note: Sometimes you want to extend the log phase to perform further manipulation by replenish nutrients and harvest cells continuously. This process is called "Chemostat", and it s important for industrial and research purposes. Bacterial Division and Generation Time: Bacterial division method: binary fission which is the simplest way of reproduction, as the cell divides into 2 100% identical cells and the 2 becomes 4 and so on.(notice that binary fission doesn t alter the bacterial gene and how that s happen is explained in the previous lecture) The method of binary fission is by the development of constrictions which is made by the assembly of FTsZ (bacterial actin like protein) Constrictions proceeds from the periphery inwards and produce a transverse cell wall known as cross wall or septum. (some organisms reproduce by budding, aerial spore formation and fragmentation).

5 Generation time is the time required for a cell to divide or a population to double. o It could be as short as 13 minutes for Vibrio (I.e 150 cells need 8 hours to be 6x10^9 ), or as long as 24 hours for Mycobacterium o Division time affects the speed of getting diagnostic results. Culture Media: Is a nutrient material prepared for the growth of bacteria in a laboratory. Microbes that grow and multiply in it are known as a culture agar is an effective setting agent.(a solid substrate to contain culture media, and it is very common used because Agar is indigestible for many organisms so that microbial growth does not affect the gel used and it remains stable) agar media are either plates (called petri dishes) or test tubes Media Needed for Bacterial Growth: Features of this media: 1-It should contain basic bacterial needs of carbohydrates and proteins, which are usually obtained from Casein a protein present in cow milk -. 2-Air, salts and ph must also be present and controlled. Media Needed for Bacterial Growth Could be solid, plates, broth (liquidish), and many more. Bacteria can be classified into: 1-Fastidious: i.e. needs many specific requirements (many nutrients), (Like it only grows on chocolate agar) in other words they will only grow when specific nutrients are included in their diet. 2-Simple: requires basic proteins and carbohydrates. It can make up everything from scratch. 3-Selective/ enrichment with indicator: for example, I will know that a certain bacterial type is living in this culture because I noticed a decrease in a certain nutrient (I know that X bacteria needs Y nutrient to grow), or the media color has changed from X to Y also due to presence of a certain bacterial type.

6 Some bacteria Cannot be cultured in vitro (in the lab): such as Rickettsia and Chlamydia --> need tissue-like culture. And Mycobacterium Leprae which can't be cultured at all. Note: In the lab, you can't predict the virulence of a certain bacteria only by its growth, like the case of Mycobacterium Leprae which causes Leprosy (some slow or non-culturable bacteria can be fatal). Bacterial Physiology and Metabolism: Metabolism includes anabolism (building) and catabolism (degradation). Metabolism in Bacteria is very similar to that in mammalians, but with faster rate than our bodies metabolism. Bacterial metabolism involves around 2000 chemical reactions categorized according to their functions in metabolic processes (fueling, biosynthesis, polymerization and assembly). Energy is conserved, it's neither lost nor gained (energy production = energy consumption). Bacterial nutritional requirements are more diverse than our cells requirements. Some biosynthetic processes, such as those producing murein, lipopolysaccharides (LPS), and teichoic acid, are unique to bacteria. Physiology and Metabolism understanding is important in diagnosis and treatment (as it s important for bacterial identification and antibacterial agents). Nutritional requirements: Including many elements, such as: 1- carbon, hydrogen, O2, nitrogen, phosphorus & Sulfur (which are needed for the synthesis of structural components), 2- potassium, calcium magnesium and iron (which are needed for cellular functions). Nutritional requirements can be obtained from the nutrients provided in the growth media (obtained from simple element or by break down of macromolecules like protein into amino acids using bacterial enzymes).

7 **Many bacteria have to synthesize some nutrients such as folic acid which makes these bacteria susceptible to any kind of drug that interferes with that process. (so we make agents that interfere with the biosynthesis of folic acid,like trimethoprim and sulfonamides antibiotics) Nutritional requirements differ among bacteria and can be used for identification of bacteria type. Sources of nutritions: 1-Elements, such as: a)hydrogen and Oxygen: are obtained from water b)carbon: is obtained by either Oxidation of carbohydrates (Oxygen is the final acceptor of electron, and produces more energy) or Fermentation of carbohydrates (a less efficient pathway which uses an organic compound as a final electron acceptor, yields 2 ATP). c)nitrogen: could be obtained from ammonia in the environment or protein s Deamination using bacterial enzymes. Note: Carbon is the main component of a bacterial cell (also is the main energy source), followed by Nitrogen and Oxygen. 2-Organic factors: cannot be synthesized by the bacteria and have to be taken from exogenous source. a)amino acids (from protein breakdown). b)purines and Pyrimidines (nucleic acid precursors) **Must be converted to nucleotides and nucleosides before being incorporated into DNA and RNA. 3- Vitamins are also important and are used as co-factors. Energy Sources: Phototrophs: uses light as a source of energy. Chemotroph: uses chemical pathways such as oxidation and reduction of external chemical compounds.

8 Hydrogen donor: 1-if the source of Hydrogen is from organic compounds bacteria is an organotroph. 2-If the source of Hydrogen is inorganic compounds (like ammonia or hydrogen sulfide) bacteria is a lithotroph. Energy and hydrogen donor designations are referred to routinely by combining the two terms: chemo-organotrophs: most of the clinically significant bacteria is chemo-organotrophs (means energy from chemicals and hydrogen from organic compounds ) Chemo-lithotrophs: Some Pseudomonas species. Carbon Sources: Autotrophs: Carbon is obtained from CO2. Heterotrophs: Carbon is obtained from organic compounds. Mixotrophs: Carbon is obtained from both sources. Energy and carbon combination: Chemoheterotrophs: energy from chemical compounds and carbon from organic compounds. Note: Chemoheterotrophs is the most common group among energy and carbon sources as all protozoa, fungi and animals are from this group Metabolic Pathways: (similar to ours) 1- Embden-Meyerhof glycolytic pathway.(glycolysis) 2- The pentose phosphate pathway. 3- The Krebs Cycle (yield the carbon compounds needed for biosynthesis). For oxidation: 1- Electron transport chain (after Krebs Cycle) ends with a final electron acceptor which is usually oxygen, but in some cases other molecules do the job, either inorganic (eg, nitrate) or organic (eg, succinate), in such

9 cases the bacteria can survive without oxygen even if it cannot undergo fermentation. 2- It yields 38 ATP in prokaryotes and 34 ATP in eukaryotes. For fermentation: 1- the final acceptor of electrons in electron transport chain is an organic compound. 2- It yields 2 ATP. Note: CO2 is produced in both. Enviromental conditions governing growth: 1-Temperature (most of the clinically significant bacteria are mesophilic i.e. live in 37 C ). 2-Water (makes up around 80% of the bacterial cell). 3-Oxygen and carbon dioxide. Metabolic reactions that use oxygen can produce some toxins including superoxide anion (O² ) and hydrogen peroxide (H₂O₂) which are detoxified by superoxide dismutase and peroxidases (Catalase) respectively, bacteria with these enzymes can grow in the presence of oxygen. Classification of bacteria according to their response to oxygen: a. Strict (obligate) aerobic: must use O2 presence of CO2 is fatal. b. Strict (obligate) anaerobes: O2 presence is fatal. Therefore, these bacterial cells don t have catalases and Oxidases to neutralize the Oxygen free radicals. c. Micro-aerophilic: prefers low Oxygen levels. d. Capnophilic: prefers increased CO2 levels. e. Facultative anaerobes: grows best aerobically, but can grow anaerobically.

10 Acidity (ph): o Most Bacteria grow in a neutral or slightly alkaline ph ( 7-7.4). o Acidophiles: Bacteria which grows best at an acidic nature (below 7) such T.B-PH o Alkalophiles: Bacteria which grows best at an alkaline nature (more than 7) such as V.Cholera-PH ( it s also halophiles). Refer to summary in the last slide to chick your understanding. Please refer to the slides, just in case. Don't hesitate to contact me if you have any inquiry or comment. You are doing great, keep up the hard work <3