Factors Affecting Bacterial Growth

2. any organism can develop and grow in his optimal condition and this conditions are physical or nutritional Physical conditions include: • Ph • Temprature • Salinity ( content of NACL) • Osmolarity Nutritional: should have • Carbon source • Nitrogen source بعض الكائنات تستكفي بهم لصنع الفيتامينات والأحماض الأمينيه وصنع غذاءها ولكن بالمقابل هناك كائنات أخرى تحتاج لأن يكون غذاءها جاهز لا يتم تصنيعه ذاتيا Optimum growth when growth rate is maximum and doubling time is minimum We can calculate them from lock phase or exponintial phase

3. Factors Affecting Bacterial Growth • The kinds of organisms found in a given environment and the rates at which they grow can be influenced by a variety of factors, both physical and biochemical • Physical factors include: pH, temperature, oxygen concentration, moisture, hydrostatic pressure, osmotic pressure, and radiation • Nutritional factors include: availability of carbon, nitrogen, sulfur, phosphorus, trace elements and, in some cases, vitamins

4. pH Optimum pH: the pH at which the microorganism grows best (e.g. pH 7) Most microbes doesn’t grow at onr PH unit above or below the optimum PH(optimum PH +or -1) Not all organisms lives At PH=7 Some of them lives in acidic environment such as lactobacillus wich cause fermentation in yogurt to produce acidic conditions where it can live Also vibrio cholerae lives at high PH value Also H.pylori (Helicobacter pylori)wich cause peptic ulcer can withstand the acidic conditions in stomach the mechanism for withstanding this conditon by being embeded in mucus for protection against H+ and it has urease enzyme to convert the urine to ammonium wich nutralize H+ According to their tolerance for acidity/alkalinity, bacteria are classified as: 1. Acidophiles (acid-loving): grow best at pH 0.1-5.4 2. Neutrophiles: grow best at pH 5.4 to 8.0 • Alkaliphiles (base-loving): grow best at pH 7.0-11.5

5. Bacteria that oxidise sulphur and sulphuric acid wihstand very low PH and very strong acid media Miroorganism can spread in avery huge amount when they are capable To adapt conditions so then it cause epidemic But if it sensitive it cant spread and cause epidemic Any organism withstand extreme conditions should have certain mechanism When they live at extreme PH they have impervious means don’t allow the passage and protect the cell wall from being exposed to extreme PH in the medium. In lab to keep the optimum PH for the bacteria growth in the medium we put buffers and to prevent extreme PH

6. Temperature Obligate: organism must have specified environmental conditionلما يكونو مو موجودين هي الظروف لاتنمو Facultative: organism is able to adjust to and tolerate environmental condition, but can also live in other conditions يعني بوجود هاي الظروف بتعطي أحسن نمو These 2 terms we use them with oxygen and temprature According to their growth temperature range, bacteria can be classified as: psychrophiles: 15-20oC the problem here that it cause food poisoning Mesophiles: 25-40oC تقريبا درجة حرارة جسم الانسان =37 So they are pathogenic for human Thermophiles: 50-60oC extreme thermophiles lives at very high tempratue such in volcano and heat geyser They have special structure in the cell wall or special conistituen in cell wall to protect them against extreme temprature

7. Thermophiles: Thermophilic sulfur bacteria can live and grow in the runoff waters from such geysers despite the near-boiling temperatures

8. Growth rates of psychrophilic, mesophilic, and thermophilic bacteria

9. شرح للرسمه : عند القمه بتكون درجة الحراره المثلى للنمو زي درجة الحرارة المثلى للنمو 10 ...هلأ بعدها نجد psychrophile المنحنى يتناقص بشكل سريع يعني معدل النمو نزل بسرعه والميل هنا اقل من ميل لما كنا بدنا نوصل الى درجة الحراره المثلى يعني لما كنا بدنا نوصل لدرجة الحراره المثلى كانت بتزيد شوي شوي-بالتدريج- والميل عالي بس لما زدنا درجة الحرارة شويه زياده عن المثلى نزل معدل النمو فجأه بس مو تدريجي .

10. Obligate thermophites: they live only above 37 Bacillus stearothermophilus: Bacillus means: spherical Thermophilus: extreme temprature Remember that: bacillus and clostridium can produce endospores…. Bacillus stearothermophilus lives at temprature between 65 and 75 but it can display minimum growth at low temprature 30 so its facultative and cause food poisoning Facultative thermophites: can live above or below 37

11. Oxygen • Aerobes: require oxygen to grow • Obligate aerobes: must have free oxygen for aerobic respiration (e.g. Pseudomonas) • Anaerobes: do not require oxygen to grow • Obligate anaerobes: killed by free oxygen (e.g. Bacteroides) • Microaerophiles: grow best in presence of small amount of free oxygen • Capnophiles: carbon-dioxide loving organisms that thrive under conditions of low oxygen • Facultative anaerobes: carry on aerobic metabolism when oxygen is present, but shift to anaerobic metabolism when oxygen is absent • Aerotolerant anaerobes: can survive in the presence of oxygen but do not use it in their metabolism

12. Obligate aerobes: they need oxygen to live. But why microrganisms need oxygen? coz it acts as a final receptor in electron transport chain and in the absence of oxygen fermentation occurred . So obligate aerobes need O2 for aerobic respiration for production of ATP that is important for the growth. Obligate anaerobes: don’t need O2 such as Bacteroides wich is gram –ve anaerobes and clostridium wich is gram +ve anaerobes , bacteriods cause problems in JI tract specially in colon . Remember that: in JI tract 2 organisms normal flora and bacteroids. Microaerophiles: grow best in presence of small amount of free oxygen. Pour method is the best for them coz in spread organisms grow at the top of agar but microaerophiles needs small amount of O2 so they don’t grow at the top of agar …….such as campylobacter They are cnsidered as obligate but they need small amount of O2. How to prepare this medium? by anaerobic jars then we pot inside it sachet wich gives low oxygen environment or mixture of CO2 and O2

13. Capnophiles: it is anaerobe and carbon-dioxide loving organisms that thrive under conditions of low oxygen So they need 2 things: -low amount of O2 -carbon-dioxide Usually anaerobes use CO2 or N2 instead of O2 in case of Microaerophiles low concentration of oxygen , Capnophiles CO2 Facultative anaerobes: carry on aerobic metabolism when oxygen is present and shift to anaerobic metabolism in the absence of O2. Such as: staphylococcus and escherichia coli Aerotolerant anaerobes: can survive in the presence of oxygen but do not use it in their metabolism. It carries on fermentation process such as lactobacilus wich produce lactic acid If we have plate….now at the surface aerobes and fucltative anaerobes grow But capnophiles and anaerobes and microarophiles needs special conditions Anaerobes environment: co2 or N2 Microaerophites:Co2 with low oxygen this medium is suitable for capnophiles but not N2 with low O2 JustCo2 with low O2

14. اللي بينمو كتير على السطح Aerobes اللي بينمو شوي Capnophiles or microaerophites Why anaerobes killed by O2? Coz it is toxic for them and when it enters their bodies will form free radical and they don’t have enzymes for oxidising them (but aerobes have this enzyme for detoxification) so there is no metabolic detoxification process for converting them so they reach respiration cells as free radicals If we remove these enzymes from aerobes they will be converted to anaerobes Facultative anaerobes have more complex metabolic way than aerobes coz they can live in the presence or absence of O2(in the absence they use N2) super oxide formed by superoxide synthatase then converted to hydrogen peroxideH2o2 and O2 by superoxide dismutase then coverted to H2o and O2 by catalase That’s why we use H2O2 for sterilization process Catalase……is the detoxifying enzyme

15. We can classify bacteria depending on enzymes they own Catalase +ve: they have catalase Catalase –ve: no catalase how can I know if they are catalase –ve or +ve? We prepare two cultures then we put H2O2 in the 2 cultures catalase+ve one air puples appeares here due to converting H2o2 to o2 but catalase –ve die and no air pupples

16. Patterns of Oxygen Use

17. Hydrostatic Pressure • Water in oceans and lakes exerts pressure exerted by standing water, in proportion to its depth • Pressure doubles with every 10 meter increase in depth • Barophiles: bacteria that live at high pressures(due to their cell wall) but die if left in laboratory at standard atmospheric pressure(so they are obligate)

18. Osmotic Pressure • Environments that contain dissolved substances exert osmotic pressure, and pressure can exceed that exerted by dissolved substances in cells • Hyperosmotic environments: cells lose water and undergo plasmolysis (shrinking of cell) • Hypoosmotic environment: cells gain water and swell and burst

19. Halophiles • Salt-loving organisms which require moderate to large quantities of salt (sodium chloride) such as staphylococcus aureus • Membrane transport systems actively transport sodium ions out of cells and concentrate potassium ions inside • Why do halophiles require sodium? • Cells need sodium to maintain a high intracellular potassium concentration for enzymatic function • Cells need sodium to maintain the integrity of their cell walls

20. Responses to Salt

21. The Great Salt Lake in Utah

22. Moisture:bacteria needs water to live and to grow up مشان هيك لازم اي اشي أكل او جرح لازم نجففه مشان ما ينمو علي بكتيريا طبعا بيختلفو البكتيريا عن بعض اعتمادا على نسبة احتياجها للماء Gram-ve needs water more than gram+ve one Water activity: انو البكتيريا اي نوع منها بتحتاج كميه من الماء في النظام تبعها بيختلف عن الأنواع الأخرى Some microorganisms needs low conc of water بس ما في احياء دقيقه او بكتيريا يصل احتياجها للماء صفر لأنو في هي الحاله اما بتموت او بتتحول الى Endospores Radiation: some microorganisms can adabt radiation but they differ in adabtation depending on repair system they own . Some enzymes do repairing and some pigments protect them against radiation هيك خلصنا العوامل الفيزيائيه

23. Nutritional Factors • Carbon sources • Nitrogen sources • Sulfur and phosphorus • Trace elements (e.g. copper, iron, zinc, and cobalt) • Vitamins (e.g. folic acid, vitamin B-12, vitamin K) • Amino acids

24. Carbon: Microrganisms are either: Heterotrophs: they cant synthesise their carbon source it must be given as glycerol or glucose …..etc Autotrophs: can synthesise their own carbon source they look like chlorophyll organisms Photoautotrophic organisms reduce carbon dioxide to glucose and other organic molecules. Both autotrophic and heterotrophic organisms can obtain energy from glucose by glycolysis fermentation and krebs cycle they also synthesise some cell component from intermediates in these pathways. Nitrogen Essential for the synthesis of enzymes and proteins and nucleic acids. Some of then needs inorganic nitrogen sources such as ammonium sulphate and glutamate Trace elements+sulphur and phosphorus: Are needed At very low concentraion …. بنلاقيهم داخل الماء او بيكوكنو As a supplements

25. They are essential as cofactors in enzymatic reactions . Vitamens: They make their own vitamens But some of them you have to supply them So many microorganisms make their own vitamens from simpler sustances other microorganisms require several vitamens in their media because they the lack the enzymes to synthesize them .vitamens required by some microorganisms, include folic acid , vitamen B12,vitaminK Microbes that live in human intestine manufacture vitamin K for blood clotting and vitaminB .

26. A USDA scientist working on his microbial brew – a mix of some 80 ingredients to support growth of nutritionally fastidious spiroplasmas

27. Spiroplasma spp. responsible for hundreds of crop and animal diseases

28. Locations of Enzymes Microorganisms produce certain types of enzymes Exoenzymes: production of enzymes that are released through cell or plasma membrane(to the out side) Most exoenzymes are hydrolases the use water to remove covalent bonds 2 types of exo enzymes: Extracellular enzymes: usually produced by gram-positive rods, which act in the medium around the organism Periplasmic enzymes: usually produced by gram-negative organisms, which act in the periplasmic space Examples on exoenzymes: carbohydrase,amylase,cellulase,sucrase,lactase,maltase,lipase , protease ,caseinase ,gelatinase……table 6.2 مش حفظ These enzymes are essential in classification process.

29. Adaptation to limited nutrients: decrease in growth rate and organism size 1- increase the amount of synthesising enzymes for up take and metabolism of limited nutrients.this cause the microorganism to obtain and use larger proportion of the few nutrient molecules that are available. 2- synthesising enzymes needed to use different nutrient 3- adjusting the rate at wich they metabolise nutrient and the rate at wich they synthesise molecules they are required for growth Both metabolism and growth are slowed Example on adaptation ….endospores

30. Sporulation The formation of endospores, occurs in Bacillus, Clostridium and a few other gram-positive genera Protective or survival mechanism, not a means of reproduction Core(contain genetic material)>>then>>cortex>>then>>coat As endospore formation begins, DNA is replicated and forms a long, compact, axial nucleoid The DNA where the endospore will form directs endospore formation . Most the cells RNA and some cytoplasmatic proteins molecules gather around DNA to make the core or living part of endospore The core has low concentration of dipicolinic acid and calcium dipicolinate and calcium ions This protecting the microorganism against heat so protecting proteins against denatoration Septum: grows around the core, enclosing it in a double thickness of membrane

31. Cortex: consist of peptidoglycan lyer for withstanding hydroststic pressure Spore coat: consist of keratin like protein wich impervious to many chemicals ,is down around the cortex by the mother cell. Some of endospores are exosporium: alipid-protein membrane is formed out side the coat As endospore formation begins, DNA is replicated and forms a long, compact, axial nucleoid (Figure 6.17). The two chromosomes formed by replication separate and move to different locations in the cell. In some bacteria the endospore forms near the middle of the cell, and in others it forms at one end (Figure 6.18). The DNAwhere the endospore will form directs endospore formation. Most of the cell’s RNA and some cytoplasmic protein molecules gather around theDNAto make the core, or living part, of the endospore. The core contains dipicolinic (di-pik-olin0ik) acid and calcium ions, which probably contribute to an endospore’s heat resistance by stabilizing protein structure. An endospore septum, consisting of a cell membrane but lacking a cell wall, grows around the core, enclosing it in a double thickness of cell membrane (Figure 6.17). Both layers of this membrane synthesize peptidoglycan and

32. release it into the space between the membranes. Thus, a laminated layer called the cortex is formed. The cortex protects the core against changes in osmotic pressure, such as those that result from drying. A spore coat of keratinlike protein, which is impervious to many chemicals, is laid down around the cortex by the mother cell. Finally, in some endospores an exosporium, a lipid-protein membrane, is formed outside the coat by the mother cell. The function of the exosporium is unknown. Under laboratory conditions, sporulation takes about 7 hours. Once favorable conditions return, an endospore develops into a vegetative cell, which lacks the endospore’s resistant properties. Germination, in which a spore returns to its vegetative state, occurs in three stages. The first stage, activation, usually requires some traumatic agent such as low pH or heat, which damages the coat. Without such damage, some endospores germinate slowly, if at all. The second state, germination proper, requires

33. water and a germination agent (such as the amino acid alanine or certain inorganic ions) that penetrates the damaged coat. During this process, much of the cortical peptidoglycan is broken down, and its fragments are released into the medium. The living cell (which occupied the core) now takes in large quantities of water and loses its resistance to heat and staining, as well as its refractility (ability to bend light rays). Finally, outgrowth occurs in a medium with adequate nutrients. Proteins and RNA are synthesized, and in about an hour, DNA synthesis begins. The cell is now a vegetative cell and undergoes binary fission. Thus, bacterial cells capable of sporulation display two cycles—the vegetative cycle and the sporulation

34. cycle (Figure 6.17). The vegetative cycle is repeated at intervals of 20 minutes or more, and the sporulation cycle is initiated periodically. Endospores known to be 300 or more years old have been observed to undergo germination when placed in a favorable medium. They are a very good form of insurance against extinction.

35. Core (living part of endospore): most of cell’s RNA and some cytoplasmic protein molecules gather around DNA • Dipicolinic acid: contained in the core along with calcium ions • Endospore septum: grows around the core, enclosing it in a double thickness of membrane • Cortex: laminated layer forms when peptidoglycan is released into space between endospore septum membranes • Spore coat: keratin-like protein, impervious to chemicals is laid down around the cortex • Exosporium: found in some endospores, a lipid-protein membrane formed outside the coat

36. Vegetative and Sporulation Cycles in Bacteria capable of Sporulation

37. Germination • A spore returns to its vegetative state, occurs in three stages: • Activation • Germination proper • Outgrowth

38. Bacterial endospores in two Clostridium species

39. Culturing Bacteria • Culturing of bacteria in the laboratory presents two problems: • A pure culture of a single species is needed to study an organism’s characteristics • A medium must be found that will support growth of the desired organism • Pure culture: a culture that contains only a single species of organism

40. dilutions. Koch’s technique of spreading bacteria thinly over a solid surface was more effective because it deposited a single bacterium at some sites. However, he tried several different solid substances. Using the discovery of Angelina Hesse, the wife of an associate, he settled on agar as the ideal solidifying Agent We have 2 methods :

41. The Streak Plate Method Today, the accepted way to prepare pure cultures is the streak plate method, which uses agar plates. Bacteria are picked up on a sterile wire loop, and the wire is moved lightly along the agar surface, depositing streaks of bacteria on the surface. The inoculating loop is flamed, and a few bacteria are picked up from the region already deposited and streaked onto a new region Fewer and fewer bacteria are deposited as the streaking continues, and the loop is flamed after each streaking. Individual organisms are deposited in the region streaked last. After the plate is incubated at a suitable growth temperature for the organism, small colonies—each derived from a single bacterial cell— appear. The wire loop is used to pick up a portion of an isolated colony and transfer it to any appropriate sterile medium for further study. The use of sterile (aseptic) technique ensures that the new medium will contain organisms of only a single species.

42. The Streak Plate Method uses agar plates to prepare pure cultures

43. A Streak Plate of Serratia marcescens. Note the greatly reduced numbers of growth /colonies in each successive region

44. Because this method embeds some organisms in the medium, it is particularly useful for growing microaerophiles that cannot tolerate exposure to oxygen in the air at the surface of the medium Types of media for bacteria culture: Solid…..agar Liquid….broth The Pour Plate Method Another way to obtain pure cultures, the pour plate method, makes use of serial dilutions.

45. Types of Culture Media • Natural Media: In nature, many species of microorganisms grow together in oceans, lakes, and soil and on living or dead organic matter • Synthetic medium: A medium prepared in the laboratory from material of precise or reasonably well-defined composition(chemical defined media) such as glucose with ammonium sulphate so defined syntheticmedium is one that contains known specific kinds and amounts of chemical substances Complex medium: contains reasonably familiar material but varies slightly in chemical composition from batch to batch (e.g. peptone, a product of enzyme digestion of proteins)rich media oh nitrogen and carbon source such as agar so it is chemically nondefined Medium بنعرف الأنواع اللي فيها بس ما بنميز عددهم It contains peptone, a product of enzyme digestion of proteins. It provides small peptides that microorganisms can use