Microbial Culture Chapter Notes | Biotechnology for Class 12 - NEET PDF Download

Historical Perspective

• Microbiology studies small organisms, with significant applications in fundamental research, agriculture, pharmaceuticals, medicine, environmental science, food technology, and genetic engineering.
• The discovery of the microscope in the mid-1600s by Antonie van Leeuwenhoek laid the foundation for microbiology, allowing observation of microscopic organisms termed "animalcules."
• Post-Leeuwenhoek, microbiology progressed slowly due to limited microscope availability and lack of interest in microorganisms.
• In the 1700s, Lazzaro Spallanzani demonstrated that boiled broth contained no microscopic life, challenging the idea of spontaneous generation from lifeless matter.
• In the mid to late 1800s, Louis Pasteur conducted experiments proving the role of microorganisms in daily life and their link to human illnesses, encouraging further scientific exploration.
• Pasteur’s experiments involved boiling broth to sterilize it, showing that microorganisms grew only when exposed to air and dust, thus disproving spontaneous generation.
• Robert Koch advanced the germ theory by injecting pure Bacillus cultures into mice, demonstrating that Bacilli caused anthrax, and established principles linking microorganisms to diseases.
• The late 1800s to early 1900s, influenced by Pasteur and Koch, is known as the Golden Age of Microbiology, marked by discoveries of microbial disease causative agents.
• In the 19th century, bacteriologists used food-based media, with Pasteur using yeast, ash, and ammonium salts, and Ferdinand Cohn refining it with varied sugars.
• Robert Koch developed broths from beef serum or meat extracts for optimal bacterial growth and introduced solid media to isolate pure cultures.
• Koch tested solid media like coagulated egg albumin, starch paste, and potato slices, but found them less effective for pathogenic bacteria like Bacillus anthracis.
• Koch later used gelatin with meat extract, eventually replacing gelatin with agar, which provided solid support without serving as a nutrient source.
• Koch coined the term "colony" to describe pure, discrete bacterial growth on solid media.
• In 1887, Julius Richard Petri improved culturing by introducing the Petri dish, a shallow circular glass dish with a loose-fitting cover, replacing flat glass plates.
• By the early 20th century, selective media were developed to favor specific microorganisms, containing compounds to promote targeted growth.
• In the 1930s, researchers recognized the importance of growth factors for bacterial nutrition.
• The 1940s saw the invention of the electron microscope, advancing virus study and culture methods.
• By the 1950s, coenzymes were found to support bacterial growth, and antibiotics were used in media as selective agents by the 1960s.
• Post-World War II, microbiology advancements led to antibiotics for diseases like pneumonia, tuberculosis, meningitis, and syphilis.
• In the 1950s and 1960s, vaccines for viral diseases such as polio, measles, mumps, and rubella were developed.
• Modern microbiology contributes to pharmaceuticals, food and dairy products, disease control, and industrial microbial applications.
• Microorganisms produce vitamins, amino acids, enzymes, growth supplements, and foods like fermented dairy, pickles, breads, and alcoholic beverages.
• In biotechnology, microorganisms serve as living factories for pharmaceuticals like insulin, interferon, blood-clotting factors, clot-dissolving enzymes, and vaccines.
• Genetically engineered microorganisms are used as host vectors in recombinant DNA technology to develop genetically modified organisms with enhanced traits.

Nutritional Requirements and Culture Media

• Microorganisms require nutrients for energy, growth, and multiplication, categorized by their carbon and energy sources.
• Heterotrophs, including all fungi, protozoans, and most bacteria, obtain carbon from organic molecules derived from other organisms.
• Autotrophs, including many bacteria and nearly all algae, use carbon dioxide as their carbon source.
• Autotrophs are divided into chemoautotrophs, which derive energy and carbon from inorganic sources (e.g., sulfur or nitrite oxidation), and photoautotrophs, which use photosynthetic pigments to convert light into chemical energy.
• Most heterotrophs, termed chemoheterotrophs, gain energy by oxidizing organic nutrients, while photoheterotrophs (e.g., green and purple non-sulfur bacteria) use light for energy but rely on organic carbon sources.
• Lithotrophs use inorganic molecules (e.g., H₂O, H₂S, ammonia) as electron sources, while organotrophs use organic molecules for electrons.
• Macronutrients required in large amounts include carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron.
• Carbon, oxygen, hydrogen, nitrogen, sulfur, and phosphorus are components of carbohydrates, lipids, proteins, and nucleic acids.
• Potassium, calcium, magnesium, and iron serve as enzyme cofactors and play various cellular roles.
• Micronutrients or trace elements (e.g., manganese, zinc, cobalt, molybdenum, nickel, copper) are needed in small amounts and are typically present in regular media components.
• Nutritional components of culture media include:
• Carbon sources like glucose, lactose, sucrose, starch, glycogen, cellulose, cereal grain powders, or cane molasses, providing carbon, oxygen, and hydrogen.
• Nitrogen sources such as ammonium salts, urea, animal tissue extracts, amino acid mixtures, or plant-tissue extracts for amino acid, nucleic acid, and enzyme synthesis.
• Phosphorus, primarily from inorganic phosphate, is used in nucleic acids, phospholipids, nucleotides, and cofactors.
• Sulfur, usually from sulfate, is needed for amino acids (e.g., cysteine, methionine) and some carbohydrates.
• Growth factors, organic compounds like amino acids, purines, pyrimidines, and vitamins, are essential for cells unable to synthesize them.
• Anti-foams (e.g., olive oil, sunflower oil, silicones) prevent excessive foaming caused by starch, proteins, or cell growth products during media agitation.
• Water is a critical base for all culture media, with less used in solid media compared to liquid media.
• Culture media must provide all nutrients, growth factors, and energy sources to support microbial growth, tailored to the specific microorganism based on its natural habitat.
• Culture media are classified by chemical composition into:
• Synthetic or chemically defined media, with known chemical components, used for photolithotrophic autotrophs (e.g., cyanobacteria) with CO₂, nitrate, or ammonia, or heterotrophs with glucose and ammonium salts.
• Examples include M9 media for E. coli and BG11 media for cyanobacteria.
• Complex media, with some unknown chemical components like peptones, beef extract, and yeast extract, meet the nutritional needs of various microorganisms.
• Peptones are protein hydrolysates from meat, casein, or gelatin, providing carbon, energy, and nitrogen.
• Beef extract contains amino acids, peptides, nucleotides, organic acids, vitamins, and minerals.
• Yeast extract is rich in vitamins, nitrogen, and carbon compounds.
• Examples include nutrient broth, tryptic soy broth, MacConkey agar, and Lysogeny broth (LB), which contains 10% tryptone/peptone, 5% yeast extract, and 10% sodium chloride for E. coli.
• Potato dextrose agar (PDA) is used for fungi, yeast, and mold cultivation and identification.
• Based on consistency, culture media are:
• Liquid media or broth, without agar, where growth becomes visible after inoculation and incubation.
• Solid media, with 1.0–2.0% agar for surface cultivation, used for agar slants, slopes, and stabs, as agar is undegradable by most microorganisms.
• Semi-solid media, with 0.5% agar, which may be selective to promote specific organisms while retarding others.
• Based on application and function, culture media include:
• Selective media, favoring specific microorganisms by providing nutrients that enhance their growth while suppressing others, e.g., cellulose-only media for cellulose-digesting bacteria or Endo agar, eosin methylene blue agar, and MacConkey agar for E. coli.
• Media with bile salts or dyes (e.g., basic fuchsin, crystal violet) suppress Gram-positive bacteria while encouraging Gram-negative bacteria.
• Differential media distinguish microorganisms by appearance or biological characteristics, e.g., blood agar differentiates hemolytic from non-hemolytic bacteria.
• MacConkey agar, both selective and differential, contains lactose and a pH-sensitive dye, producing red colonies for lactose-fermenting bacteria and colorless colonies for non-fermenters.
• Enrichment media adjust the nutritional environment to enhance specific microorganisms, e.g., adding plant/animal tissue extracts to nutrient broth/agar for fastidious heterotrophs, with blood agar as an example.

Sterilisation Methods

• Sterilization is essential in microbial studies to ensure media, working surfaces, glassware, and plasticware are free from contaminants.
• Sterilization can be achieved through physical methods (heat, radiation, filtration) and chemical methods.
• Sterilization by heat is the most effective and rapid for heat-resistant items, acting by denaturing and coagulating proteins, exerting oxidative effects, and interfering with metabolic reactions, leading to cell death.
• Heat sterilization methods include:
• Boiling at 100°C for 30 minutes in a water bath, used for syringes, rubber goods, and surgical instruments.
• Autoclaving, a common lab method, uses steam at temperatures above 100°C under pressure, typically 121°C at 15 psi for over 30 minutes, in vertical or horizontal, jacketed or non-jacketed autoclaves.
• Jacketed autoclaves have steam surrounding the chamber, while non-jacketed ones expose items directly to steam.
• Pasteurization, used in food and dairy industries, heats milk to kill pathogenic microorganisms without full sterilization, using methods like holder (LTLT, 63°C for 30 minutes), flash (HTST, 72°C for 15 seconds), or Ultra-High Temperature (UHT, 140–150°C for 1–3 seconds), followed by quick cooling.
• Heating and flaming involve dry heat sterilization, where items like inoculation loops, forceps tips, or test tube mouths are heated in a Bunsen flame until red hot or briefly passed through the flame.
• Hot air ovens sterilize glassware using dry heat.
• Sterilization by radiation, termed "cold sterilization," uses ionizing (electron beams, gamma rays) or non-ionizing (UV rays) radiation without generating heat.
• UV rays (200–280 nm, most effective at 260 nm) induce thymine-thymine dimers, inhibiting DNA replication and causing mutations, used for surface disinfection in labs, laminar hoods, and operation theaters.
• UV disadvantages include skin and eye harm and potential DNA repair by bacterial enzymes.
• Ionizing radiations include electron beams for sterilizing syringes, gloves, and foods, and gamma rays, which have greater penetration but require longer exposure, damaging microbial nucleic acids.
• Gamma rays sterilize disposable Petri dishes, plastic syringes, antibiotics, vitamins, hormones, glassware, and fabrics.
• Sterilization by filtration separates microbes from heat-labile solutions (e.g., serum, antibiotics, sugars, urea) using membrane filters with 0.2–0.45 μm pores.
• High Efficiency Particulate Air (HEPA) filters, 99.97% efficient for particles >0.3 μm, are used in biological safety cabinets for air filtration.
• Chemical sterilization/disinfection uses alcohols, aldehydes, heavy metals, and hydrogen peroxide.
• Alcohols (70% ethyl alcohol for skin antisepsis, isopropyl alcohol for surfaces and thermometers) sterilize by cell dehydration, membrane disruption, and protein coagulation.
• Aldehydes like 40% formaldehyde (formalin) are used for surface disinfection and fumigation of rooms, chambers, and safety cabinets.
• Heavy metals (mercury, silver, arsenic, zinc, copper) act as germicides by precipitating proteins.
• Hydrogen peroxide is used for skin disinfection of wounds, ulcers, and deodorizing wound dressings.

Pure Culture Techniques

• Bacteria are cultured in liquid (broth) or solid media, with solid media used for isolation and long-term storage, and broth for rapid, large-scale production.
• In natural habitats, microorganisms exist in complex ecosystems with numerous other microbes, making pure cultures (single strain) essential for studying specific microbes.
• The streak plate method is the standard for obtaining pure bacterial cultures, using an inoculating loop to streak a bacterial suspension or colony on a solid agar plate, progressively diluting it.
• After incubation at an appropriate temperature, cell divisions form visible colonies, with isolated colonies near the streak’s end due to reduced cell density.
• Colonies form because progeny remain in place on solid surfaces, preventing daughter cell movement.
• The pour-plate method dilutes the microbial sample multiple times to reduce population density, then pours 1 mL of each dilution into a Petri dish, followed by melted agar, gently mixed, and incubated to form isolated colonies.
• The spread-plate technique transfers a small volume of diluted bacterial mixture to a solidified agar plate, spreads it evenly with a sterile L-shaped glass spreader (sterilized by alcohol and flaming), and incubates to develop isolated colonies.

Factors Affecting Microbial Growth

• Microbial growth is influenced by several factors, including temperature, pH, oxygen, carbon dioxide, and light.
• Microorganisms grow over a wide temperature range, typically 25–45°C, with growth rates increasing until the optimum temperature, then declining due to enzyme inactivity.
• Mesophiles grow optimally at 20–45°C, thermophiles at 40–80°C (optimum 50–65°C), and extreme thermophiles tolerate above 100°C.
• pH strongly affects microbial growth, with fungi tolerating a wider pH range than bacteria.
• Most microorganisms grow best near neutral pH (7), with some bacteria preferring slightly alkaline conditions and a few tolerating acidic conditions.
• Fungi prefer slightly acidic conditions, dominating bacteria in such environments.
• Media pH is adjusted with acid or alkali during preparation based on the microorganism’s requirements.
• Oxygen, a major atmospheric component (21%), is required by aerobes, while anaerobes survive without it.
• Aerobes in shallow media (e.g., Petri dishes) access dissolved oxygen, but in broth, shaking or stirring ensures uniform oxygen distribution.
• Obligate anaerobes, intolerant to oxygen, are cultured in anaerobic chambers excluding oxygen from media.
• Facultative anaerobes grow with or without oxygen, while aerotolerant anaerobes, generally anaerobic, are unaffected by atmospheric oxygen but do not use it.
• Autotrophic organisms use carbon dioxide as a carbon source, provided as bicarbonate in media or in a CO₂-enriched atmosphere during incubation.
• Phototrophic microorganisms require light for photosynthesis, needing specific wavelengths in laboratory cultures.

The Growth Curve

• When single-celled microorganisms are grown in nutrient-limited media, they grow maximally in number and size, influenced by nutritional and environmental factors.
• Unicellular organisms divide by binary fission, replicating genetic material and splitting into two identical cells, leading to exponential growth (e.g., 1 to 2, 2 to 4, 4 to 8).
• Growth is analyzed by plotting the logarithm of viable cell numbers versus incubation time, yielding a curve with four phases: lag, exponential, stationary, and death.
• In the lag phase, no immediate cell number increase occurs after inoculation into fresh media, as cells synthesize components for division, with no net mass increase.
• Lag phase duration varies with inoculum condition and media nature, being longer for old or refrigerated cultures and shorter or absent for young, growing cultures.
• In the exponential (log) phase, microorganisms grow and divide at the maximum rate, doubling at regular intervals with a constant growth rate.
• The time for population doubling, called generation time or doubling time, is, e.g., 20 minutes for E. coli, calculated as \( t_d = \frac{0.693}{\mu} \), where \(\mu\) is the specific growth rate.
• Specific growth rate (\(\mu\)) is calculated as \(\mu = 2.303 \frac{(\log X_s - \log X_o)}{t}\), where \(X_s\) is cell concentration at time \(t\), \(X_o\) is initial concentration, and \(t\) is time.
• Exponential phase cells are uniform in chemical and physiological properties, ideal for biochemical and physiological studies.
• In the stationary phase, growth ceases, and the curve becomes horizontal as cell division balances cell death, maintaining a constant viable cell count.
• Stationary phase occurs due to nutrient limitation or other factors, depending on the microorganism and media conditions.
• In the death phase, unfavorable conditions like nutrient depletion or toxic waste accumulation cause a logarithmic decline in viable cells.
• Metabolic waste, e.g., lactic acid from Streptococci sugar fermentation, can acidify the medium, inhibiting growth.
• Microbial growth is characterized by doubling time (\(t_d\)), the time for cell mass or number to double, calculated as \(t_d = t/n\), where \(n\) is the number of generations.
• Specific growth rate (\(\mu\)) measures biomass increase per unit biomass concentration, varying by microbe, temperature, pH, media composition, and dissolved oxygen.
• Higher \(\mu\) values indicate shorter doubling times, used to optimize media composition and fermentation batch times in commercial fermenters.