microbiology.pptx biochem nothing don't use this

Biochemistry

1. PHYSIOLOGY OF MICROORGANISMS AND METHODS FOR STUDYING THE BIOCHEMICAL PROPERTIES OF BACTERIA BY:SIKKANDAR BATCHA BOWSANA      THANDAPANDI KEERTHI       SUDHARSHA BRITNY       NAGARAJAPANDIAN AMRITHA       GROUP NUMBER-M24 09B       GUIDED BY:PHD ASSOCIATE PROFESSOR                        SARSEMBAYEV KHUSSEIN SAMIR

2. MAIN PARTS NUTRTIONAL REQUIREMENT OF MICROORGANISMS UPTAKE OF NUTIENTS MICROBIAL METABOLISM ENZYMES AND THEIR ROLE IN MICROBIAL PHYSIOLOGY GROWTH AND REPRODUCTION OF MICROORGANISMS IMPORTANCE OF STUDYING MICROBIAL PHYSIOLOGY METHODS FOR STUDYING BIOCHEMICAL PROPERTIES CARBOHYDRATE FERMENTATION TEST INDOLE TEST METHYL RED TEST CONCLUSION

3. Introduction The physiology of microorganisms is the study of all vital life processes that occur within microbial cells, including their nutrition, metabolism, growth, reproduction, and response to environmental conditions. It explains how microorganisms live, obtain and utilize energy, synthesize cellular components, and adapt to various ecological and physiological environments. Though microorganisms are structurally simple, they perform all fundamental biological activities essential for life. The study of microbial physiology provides the foundation for understanding microbial ecology, industrial microbiology, and medical microbiology. It helps in explaining the mechanisms of infection, antibiotic action, and the biochemical basis of microbial growth and metabolism.

4. Nutritional Requirements of Microorganisms Every microorganism requires nutrients for survival, growth, and reproduction. Nutrients supply the energy and materials needed for building cellular structures and carrying out metabolic activities. The essential nutrients are classified as macronutrients and micronutrients. Macronutrients such as carbon, hydrogen, oxygen, nitrogen, sulfur , and phosphorus are required in large amounts because they form the basic building blocks of proteins, nucleic acids, carbohydrates, and lipids. Micronutrients like iron, manganese, zinc, copper, and cobalt are needed in very small amounts but are vital for enzyme function and electron transport. Microorganisms differ widely in their nutritional requirements. Based on the source of energy and carbon, they are classified as autotrophs and heterotrophs. Autotrophic microorganisms obtain carbon from carbon dioxide, while heterotrophic microorganisms derive carbon from organic compounds. Similarly, depending on their energy source, microorganisms may be phototrophs that utilize light energy or chemotrophs that derive energy from chemical compounds. Combining these two characteristics, four main nutritional types are recognized: photoautotrophs, photoheterotrophs, chemoautotrophs, and chemoheterotrophs. This diversity in nutritional requirements reflects the remarkable adaptability of microorganisms to various habitats.

5. Uptake of Nutrients The cell membrane of microorganisms plays a crucial role in the uptake of nutrients. Nutrients are transported from the external environment into the cell by several mechanisms such as passive diffusion, facilitated diffusion, active transport, and group translocation. In passive diffusion, molecules move from an area of higher concentration to lower concentration without the use of energy. Only small nonpolar molecules like oxygen and carbon dioxide can enter this way. Facilitated diffusion involves specific carrier proteins that increase the rate of transport but still operate along the concentration gradient. Active transport, in contrast, requires energy in the form of ATP to move substances against their concentration gradient. This allows the bacterial cell to accumulate essential nutrients even when their external concentration is low. Group translocation is a process unique to prokaryotes, where the transported substance is chemically modified as it enters the cell; for instance, glucose is phosphorylated during its uptake by bacteria. These mechanisms ensure that cells obtain nutrients efficiently even under unfavorable environmental conditions.

6. Microbial Metabolism Metabolism in microorganisms includes all the chemical reactions that occur within the cell to maintain life. It consists of two interrelated processes: catabolism and anabolism. Catabolism refers to the breakdown of complex molecules into simpler ones, releasing energy that is conserved in the form of adenosine triphosphate (ATP). This energy is then utilized in anabolic reactions, which are synthetic processes that build up complex cellular components from simpler precursors. The balance between catabolism and anabolism maintains the energy equilibrium of the cell.

7. Energy Production Microorganisms generate energy through several biochemical pathways, the most common being respiration and fermentation. In respiration, energy is produced by the oxidation of organic or inorganic compounds. When oxygen acts as the final electron acceptor, the process is called aerobic respiration, and it yields a high amount of energy. In the absence of oxygen, microorganisms may use other inorganic molecules like nitrate or sulfate as electron acceptors, a process known as anaerobic respiration. Fermentation is another form of energy metabolism that occurs in the absence of an external electron acceptor. During fermentation, organic compounds act as both electron donors and acceptors, resulting in the partial oxidation of the substrate. This process produces small amounts of ATP through substrate-level phosphorylation. The end products of fermentation vary with the organism and substrate and may include ethanol, lactic acid, butyric acid, or other organic acids. For example, Lactobacillus species produce lactic acid, while Saccharomyces cerevisiae produces ethanol and carbon dioxide.

8. Enzymes and Their Role in Microbial Physiology Enzymes are biological catalysts that accelerate biochemical reactions within microorganisms. They are mostly proteins and have a high degree of specificity for their substrates. Enzymes function by lowering the activation energy required for a reaction to proceed, thus facilitating metabolic processes. Some enzymes act inside the cell (endoenzymes), while others are secreted outside the cell (exoenzymes) to break down large molecules that cannot pass through the cell membrane. Examples of exoenzymes include amylase, lipase, and protease, which degrade starch, fats, and proteins, respectively. Enzyme activity is influenced by factors such as temperature, pH, substrate concentration, and the presence of inhibitors or cofactors. Many enzymes require cofactors, which may be metal ions or organic molecules known as coenzymes. Enzymes are regulated through mechanisms such as feedback inhibition and enzyme induction. Feedback inhibition occurs when the end product of a metabolic pathway inhibits the enzyme that catalyzes an early step in the pathway, preventing overproduction. Enzyme induction involves the synthesis of specific enzymes only in the presence of their substrates, as seen in the lac operon of Escherichia coli.

9. Growth and Reproduction of Microorganisms The growth of microorganisms refers to an increase in the number of cells rather than the size of individual cells. Most bacteria multiply by a process called binary fission, in which one cell divides into two identical daughter cells. Under favorable conditions, some bacteria can divide very rapidly, with E. coli having a generation time of only about 20 minutes. The growth of a bacterial population can be studied through the bacterial growth curve, which includes four distinct phases: lag phase, log or exponential phase, stationary phase, and death phase. During the lag phase, cells adjust to their new environment, synthesize enzymes, and prepare for active division. The exponential or log phase follows, during which cells divide at a constant and maximum rate. This is the phase where physiological and biochemical activities are most pronounced. The stationary phase occurs when nutrients become limited or toxic products accumulate, leading to a balance between cell division and death. Finally, the death phase sets in when the number of dying cells exceeds the number of new cells formed due to unfavorable conditions. The study of microbial growth helps in understanding infection dynamics, antimicrobial effects, and industrial fermentation processes.

10. Environmental Factors Affecting Microbial Physiology Microbial physiology is strongly influenced by external environmental factors such as temperature, pH, oxygen concentration, osmotic pressure, and light. Each microorganism has an optimum range of conditions for growth and metabolic activity. Based on temperature preference, microorganisms are classified into psychrophiles, which grow best at low temperatures (0–20°C); mesophiles, which thrive at moderate temperatures (25–40°C); and thermophiles, which prefer high temperatures (45–70°C). Most human pathogens are mesophilic as they grow optimally at body temperature. The hydrogen ion concentration or pH of the environment also plays a significant role in microbial growth. Most bacteria prefer a near-neutral pH, while fungi and yeasts grow better in acidic conditions. Some bacteria known as acidophiles can survive in very low pH environments, whereas alkaliphiles thrive at high pH. The availability of oxygen further influences microbial physiology. Microorganisms can be classified as obligate aerobes, facultative anaerobes, obligate anaerobes, microaerophiles, and aerotolerant anaerobes depending on their oxygen requirement. Aerobes use oxygen for energy production, while anaerobes grow in its absence. Osmotic pressure and water activity also affect microbial growth. Most microorganisms require isotonic or slightly hypotonic conditions, but some, called halophiles, can tolerate or even require high salt concentrations. Light plays an important role in the physiology of photosynthetic microorganisms such as cyanobacteria and algae, which use light energy for photosynthesis. Non-photosynthetic microorganisms may be inhibited by ultraviolet light, which damages DNA.

11. Physiological Adaptations of Microorganisms Microorganisms exhibit remarkable physiological adaptations that enable them to survive in extreme environments. Thermophilic bacteria possess heat-stable enzymes and membrane lipids that allow them to function at high temperatures. Psychrophiles have enzymes that remain active at low temperatures. Acidophiles maintain their internal pH close to neutrality despite acidic external conditions. Some bacteria, such as Bacillus and Clostridium species, produce endospores that allow them to survive adverse conditions such as heat, radiation, and desiccation. These adaptations demonstrate the physiological diversity and resilience of microorganisms.

12. Importance of Studying Microbial Physiology Understanding the physiology of microorganisms has immense importance in various fields of science and medicine. In medical microbiology, it helps explain how pathogens survive, cause disease, and resist antimicrobial agents. Many antibiotics act by interfering with essential physiological processes such as cell wall synthesis, protein synthesis, or metabolic pathways. In industrial microbiology, knowledge of microbial physiology is applied to optimize fermentation processes for the production of antibiotics, enzymes, vitamins, and organic acids. In environmental microbiology, it aids in understanding the role of microorganisms in nutrient cycling, biodegradation, and bioremediation. Thus, microbial physiology provides the fundamental scientific foundation for applied microbiology and biotechnology.

13. biochemical properties of bacteria The biochemical properties of bacteria reflect their metabolic activities — the ways in which bacteria utilize nutrients, derive energy, and produce specific end products. These properties form the basis for bacterial identification, classification, and understanding of pathogenic mechanisms.  Because most bacteria cannot be distinguished by morphology alone (many are just rods, cocci, or spirilla), the biochemical characteristics become the most reliable tools for differentiating closely related species. Hence, biochemical tests are designed to detect: • The enzymes bacteria produce, • The substrates they use, • The metabolic pathways they perform, and • The end products they form.

14. Aims of Studying Biochemical Properties 1. To identify bacterial species and differentiate between closely related organisms. 2. To determine metabolic pathways — carbohydrate, protein, and lipid metabolism.  3. To study enzyme production and toxin formation.  4. To detect fermentation and oxidation reactions.  5. To assist in antibiotic susceptibility testing and pathogenicity determination.

15. General Principle of Biochemical Tests Each biochemical test is based on:  1. Providing a specific substrate in the culture medium. 2. Allowing the bacteria to metabolize it. 3. Detecting the end product by a color change, pH indicator, or precipitation reaction.  For example: • Sugar fermentation produces acid → detected by pH indicator. • Protein breakdown produces ammonia → detected by alkalinity.  • Hydrogen sulfide production turns the medium black.

16. Major Methods for Studying Biochemical Properties Carbohydrate Fermentation Tests Purpose: To determine the ability of a bacterium to ferment various carbohydrates (e.g., glucose, lactose, sucrose, mannitol) and to identify acid and gas production. Medium used: • Peptone water or basal medium containing 1% carbohydrate and a pH indicator (usually Andrade’s or phenol red). • A Durham tube is placed inverted to collect gas. Principle: If bacteria ferment the sugar → acid is produced → indicator changes color (e.g., red → yellow for phenol red). If gas forms → a bubble appears in the Durham tube. Example results: • E. coli ferments lactose and produces acid + gas.  • Salmonella typhi does not ferment lactose (no color change). Reference: Ananthanarayan & Paniker , Chapter on “Identification of Bacteria.”

17. Indole Test Purpose: To detect the ability of bacteria to break down tryptophan into indole using the enzyme tryptophanase.  Medium:  • Peptone water containing tryptophan. Principle: Indole reacts with Kovac’s reagent (p- dimethylaminobenzaldehyde in amyl alcohol) → forms a red ring at the surface. Interpretation: • Positive: E. coli, Proteus vulgaris • Negative: Klebsiella pneumoniae, Enterobacter aerogenes Reference: Prescott’s Microbiology, Ch. 16 – Biochemical Identification of Bacteria.

18. Methyl Red (MR) Test Purpose: To detect mixed acid fermentation from glucose. Medium: • MR-VP broth (glucose phosphate broth).  Principle: Mixed acid fermenters produce stable acids that keep pH < 4.4. When methyl red indicator is added → red color appears.  Interpretation: • Positive (red): E. coli • Negative (yellow): Enterobacter aerogenes

19. Voges–Proskauer (VP) Test Purpose: To detect acetoin (acetylmethylcarbinol) production from glucose fermentation via the butylene glycol pathway. Principle: In the presence of α-naphthol and 40% KOH, acetoin is oxidized to diacetyl, which reacts with creatine to form a red color . Interpretation: • Positive: Enterobacter, Klebsiella • Negative: E. coli

20. Citrate Utilization Test Purpose:  To test whether bacteria can utilize citrate as the sole carbon source and ammonium salts as nitrogen source.  Medium: • Simmons citrate agar (contains sodium citrate and bromothymol blue indicator).  Principle: Citrate utilization → alkali formation → medium turns blue. Interpretation: • Positive: Klebsiella pneumoniae • Negative: E. coli

21. Urease Test Purpose: To determine the ability of bacteria to hydrolyze urea into ammonia and carbon dioxide using the enzyme urease. Medium: • Christensen’s urea agar or broth containing phenol red. Principle: Ammonia increases pH → medium turns pink.  Interpretation: • Positive: Proteus, Klebsiella • Negative: E. coli

22. Hydrogen Sulfide (H₂S) Production Test Purpose: To detect the ability to produce H₂S gas from sulfur -containing amino acids.  Medium: • Triple Sugar Iron (TSI) agar or SIM medium containing ferrous sulfate . Principle: H₂S reacts with ferrous ions → black precipitate of ferrous sulfide . Interpretation: • Positive: Salmonella, Proteus • Negative: Shigella

23. Catalase Test Purpose:  To detect the presence of the enzyme catalase, which decomposes hydrogen peroxide into water and oxygen.  Method: • A drop of 3% H₂O₂ placed on a bacterial colony.  Observation: • Bubbling (O₂ release) → positive. Interpretation: • Positive: Staphylococcus • Negative: Streptococcus

24. Oxidase Test Purpose: To detect the presence of cytochrome oxidase enzyme, part of the electron transport chain. Reagent: • Tetramethyl-p-phenylenediamine (oxidase reagent).  Principle: The reagent is oxidized → forms purple color .  Interpretation: • Positive: Pseudomonas, Neisseria • Negative: Enterobacteriaceae

25. Nitrate Reduction Test Purpose: To test whether bacteria can reduce nitrate (NO₃⁻) to nitrite (NO₂⁻) or further to nitrogen gas (N₂).  Medium: • Nitrate broth. Reagents: • Sulfanilic acid and α-naphthylamine (for nitrite detection). Principle: Red color → presence of nitrite → positive reduction. No color → test with zinc dust (if red after zinc, negative). Interpretation: • Positive: E. coli, Pseudomonas • Negative: Acinetobacter

26. Gelatin Liquefaction Test Purpose:  To detect the production of the enzyme gelatinase, which hydrolyzes gelatin . Medium: • Nutrient gelatin .  Principle: Liquefaction of gelatin after incubation → positive.  Interpretation: • Positive: Pseudomonas, Proteus • Negative: E. coli

27. Starch Hydrolysis Test Purpose:  To detect amylase enzyme production.  Medium: • Nutrient agar with starch. Procedure: After growth, flood plate with iodine solution.  Principle: Clear zone around colonies → starch hydrolysis → positive. Interpretation: • Positive: Bacillus subtilis • Negative: E. coli

28. Litmus Milk Test Purpose: To study multiple biochemical changes — lactose fermentation, clot formation, proteolysis, and litmus reduction.  Medium: • Litmus milk (milk + litmus indicator).  Interpretation: • Pink → acid formation • Blue → alkalinity • Coagulation → clot due to acid • Peptonization → digestion of casein

29. Automated and Modern Biochemical Methods In modern microbiology, manual tests are supplemented by automated systems, e.g.: • API (Analytical Profile Index) – small strip with dehydrated biochemical media for multiple tests simultaneously.  • VITEK 2 system – automated colorimetric analysis. • MALDI-TOF MS – identifies bacteria based on protein fingerprinting, complementing biochemical data.

30. Conclusion In conclusion, the physiology of microorganisms encompasses the study of all vital functions of microbial life, including nutrition, metabolism, energy production, enzyme activity, and environmental adaptation. It reveals how microorganisms survive, grow, and reproduce under diverse conditions. By understanding these physiological processes, scientists can exploit microorganisms for beneficial purposes in medicine, industry, and environmental management, as well as control their harmful effects in disease. The study of microbial physiology therefore remains one of the most important and dynamic aspects of microbiological science.

31. Reference 📘 Ananthanarayan & Paniker’s Textbook of Microbiology (12th Edition), 📗 Prescott’s Microbiology (11th Edition)  📙 Cappuccino & Sherman: Microbiology – A Laboratory Manual (12th Edition).

32. THANK YOU FOR YOUR ATTENTION