Biotechnology: Class 12 — Chapter 6: Microbial Culture

• Antonie van Leeuwenhoek: Developed early microscopes in the 1670s-1680s; first to observe microscopic organisms, coining the term animalcules.
• Lazzaro Spallanzani: Observed that boiled broth remained free of microscopic life when sealed, challenging early ideas of spontaneous generation.
• Louis Pasteur: Disproved the theory of spontaneous generation using his famous swan-neck flask experiment; established the germ theory of disease, proving that microorganisms cause infectious diseases.
• Robert Koch: Provided direct experimental proof for the germ theory of disease by isolating Bacillus anthracis and demonstrating its ability to cause anthrax in healthy mice.

• Louis Pasteur (1860): Formulated an early synthetic medium containing yeast, ash, and ammonium salts to supply carbon, nitrogen, and essential minerals.
• Ferdinand Cohn: Refined early media formulations by systematically varying sugar types to develop versatile basal media.
• Robert Koch: Introduced solid media alternatives to overcome the limitations of liquid broths; evaluated coagulated egg albumin, starch paste, and potato slices before developing meat extract media solidified with gelatin.
• Agar-Agar Transition: Replaced gelatin as the standard solidifying agent because it remains solid at incubation temperatures (up to 100°C) and is resistant to microbial degradation.
• Julius Richard Petri (1887): Improved culturing conditions by replacing flat glass plates with a shallow, circular glass dish with a loose-fitting cover, known as the Petri dish.
• Colony Definition: Coined by Robert Koch to describe a visible, discrete cluster of microorganisms growing on a solid medium, originating from a single mother cell.

• Macronutrients: Required in large amounts; includes Carbon (C), Oxygen (O), Hydrogen (H), Nitrogen (N), Sulfur (S), Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg), and Iron (Fe). C, O, H, N, S, and P form the structural backbone of carbohydrates, lipids, proteins, and nucleic acids.
• Micronutrients (Trace Elements): Required in trace amounts; includes Manganese (Mn), Zinc (Zn), Cobalt (Co), Molybdenum (Mo), Nickel (Ni), and Copper (Cu). These act as essential cofactors for enzymes and are typically present as impurities in regular media components.
• Carbon Source: Provides the carbon skeleton for organic molecules; common sources include glucose, lactose, sucrose, starch, glycogen, cellulose, cereal grain powders, and cane molasses.
• Nitrogen, Phosphorus, and Sulfur Sources: Nitrogen is supplied via ammonium salts, urea, animal tissue extracts, or plant-tissue extracts for amino acid and nucleic acid synthesis. Phosphorus is supplied as inorganic phosphate for nucleic acids and phospholipids. Sulfur is supplied as sulfate or sulfur-containing amino acids (cysteine, methionine).
• Growth Factors: Organic compounds (amino acids, purines, pyrimidines, vitamins) that target cells cannot synthesise de novo but are required for survival.
• Anti-foams: Non-nutritional agents (e.g., olive oil, sunflower oil, silicones) added to prevent excessive foaming caused by agitation and protein components in the media.
• Water: The essential solvent and base for all liquid and solid culture media.

• Boiling: Exposure to water at 100°C for 30 minutes; suitable for disinfecting syringes, rubber goods, and surgical instruments, but does not guarantee the destruction of all bacterial spores.
• Autoclaving (Moist Heat): The most common laboratory sterilisation method; utilizes saturated steam under pressure. Standard operating conditions are 121°C at 15 psi (pounds per square inch) pressure for a holding time of at least 30 minutes. Works by denaturing and coagulating essential cellular proteins.
• Pasteurisation: Controlled heat treatment used in the food and dairy industry to kill pathogens without sterilising the product. Methods include:
  • Low Temperature Long Time (LTLT): Heated at 63°C for 30 minutes.
  • High Temperature Short Time (HTST / Flash Method): Heated at 72°C for 15 seconds, followed by rapid cooling.
  • Ultra-High Temperature (UHT): Heated at 140–150°C for 1–3 seconds.
• Heating and Flaming (Dry Heat): Direct exposure to a Bunsen burner flame. Inoculation loops and wires are heated until red hot. Glassware mouths (flasks, test tubes) are passed through the flame. Hot air ovens are used for dry-heat sterilisation of glassware at 160–180°C.
• Radiation Sterilisation ("Cold Sterilisation"):
  • Non-ionizing Radiation (UV Rays): Operates in the 200–280 nm range (260 nm is most effective); induces thymine-thymine dimers in DNA, blocking replication. Used for surface disinfection of laminar flow hoods and culture rooms.
  • Ionizing Radiation: Includes particulate electron beams and electromagnetic gamma rays. High penetrative power; damages microbial nucleic acids. Used to sterilise disposable plasticware (Petri dishes, syringes), antibiotics, and hormones.
• Filtration: Physical separation of microbes from heat-labile liquids (serum, enzymes, sugars, urea) using membrane filters with pore sizes of 0.2–0.45 μm. Air is sterilised using High Efficiency Particulate Air (HEPA) filters, which remove 99.97% of particles larger than 0.3 μm.

• Alcohols: 70% ethyl alcohol and isopropyl alcohol; act by dehydrating cells, disrupting membranes, and coagulating proteins. Used as skin antiseptics and surface disinfectants.
• Aldehydes: 40% formaldehyde (formalin); used for surface disinfection and gaseous fumigation of cleanrooms, operating theatres, and biological safety cabinets.
• Heavy Metals: Mercury, silver, arsenic, zinc, and copper; act as germicides by binding to and precipitating cellular proteins.
• Hydrogen Peroxide: Used as an antiseptic for skin wounds and ulcers, and as a sterilising agent for medical equipment.

The standard method for isolating pure bacterial colonies from a mixed inoculum using mechanical dilution on an agar surface.

1. Sterilise an inoculation loop in a Bunsen burner flame until it glows red hot, then allow it to cool.
2. Aseptically pick up a small sample of the bacterial suspension or colony using the sterile loop.
3. Gently streak the loop back and forth across the first quadrant of a solidified agar plate.
4. Flame the loop and allow it to cool to prevent carrying over excess bacteria.
5. Rotate the plate 90 degrees, pass the loop through the end of the first streak pattern, and streak across the second quadrant.
6. Repeat the flaming, cooling, and streaking process for the third and fourth quadrants to progressively dilute the bacterial cells.

An isolation technique where the sample is diluted in liquid agar before solidification, resulting in colonies growing both on the surface and within the agar medium.

1. Prepare a series of tubes containing 9 mL of sterile saline solution.
2. Transfer 1 mL of the original mixed broth culture into the first tube (Tube 1) to make a 1:10 dilution, and mix thoroughly.
3. Transfer 1 mL from Tube 1 into Tube 2, repeating the serial dilution process across multiple tubes to reduce cell concentration.
4. Pipette 1 mL of each selected dilution into the bottom of empty, sterile Petri dishes.
5. Pour melted nutrient agar (cooled to approximately 45°C) into the plates.
6. Gently swirl the plates in a circular motion to mix the sample uniformly with the agar.
7. Allow the agar to solidify, incubate, and count the resulting surface and subsurface colonies.

An easy, direct method where a diluted bacterial mixture is spread uniformly over the surface of a pre-solidified agar plate.

1. Pipette a small volume (typically ~0.1 mL) of a diluted bacterial suspension onto the center of a solidified agar plate.
2. Sterilise an L-shaped glass spreader by dipping it in alcohol and passing it through a flame to burn off the alcohol.
3. Allow the glass spreader to cool completely.
4. Place the sterile spreader on the agar surface and rotate the plate to spread the liquid sample evenly across the entire surface.
5. Incubate the plate; the dispersed cells will grow into distinct, isolated colonies on the agar surface.

• Mesophiles: Microorganisms that grow best in moderate temperature ranges, typically between 20–45°C; includes most human pathogens and soil microbes.
• Thermophiles: Heat-tolerant microbes that grow within a range of 40–80°C, with an optimum temperature around 50–65°C.
• Extreme Thermophiles: Hyperthermophilic organisms capable of surviving and multiplying at temperatures above 100°C (e.g., near hydrothermal vents).
• pH Sensitivity: Most bacteria grow best near neutrality (pH 7.0), with some preferring slightly alkaline conditions. Fungi generally tolerate wider pH ranges and prefer slightly acidic environments (pH 5.0–6.0).

• Aerobes: Require atmospheric oxygen (21% $O_2$) for respiration and survival.
• Anaerobes: Capable of surviving and growing in the complete absence of oxygen.
• Obligate Anaerobes: Cannot tolerate the presence of oxygen; oxygen is toxic to them. They must be cultured in specialized anaerobic chambers.
• Facultative Anaerobes: Prefer oxygen for aerobic respiration but can switch to fermentation or anaerobic respiration to survive in its absence.
• Aerotolerant Anaerobes: Do not utilize oxygen for growth but are not harmed or inhibited by its presence in the atmosphere.
• Carbon Dioxide ($CO_2$): Autotrophs require $CO_2$ as a carbon source, supplied in culture media as bicarbonate or incubated in $CO_2$-enriched incubators.
• Light: Phototrophic microbes require specific wavelengths of light to drive photosynthesis.

1. Lag Phase: The initial period following inoculation into fresh medium. No immediate increase in cell number occurs; cells are metabolically active, synthesising enzymes, proteins, and essential cofactors required for cell division.
2. Exponential (Log) Phase: Cells divide at their maximal rate via binary fission. The population doubles at regular intervals, and the growth rate is constant. The population is highly uniform in chemical and physiological properties, making log-phase cells ideal for biochemical studies.
3. Stationary Phase: The growth curve flattens as the rate of cell division equals the rate of cell death. This phase is triggered by nutrient depletion, accumulation of toxic metabolic wastes, and limited space.
4. Death (Decline) Phase: The number of viable cells decreases logarithmically due to prolonged nutrient deprivation and toxic waste accumulation (e.g., lactic acid production by Streptococci).

Microbial growth kinetics during the exponential phase can be modeled using the following equations:

Specific Growth Rate ($\mu$):

$$\mu = \frac{2.303(\log_{10} X_t - \log_{10} X_0)}{t}$$

Where:

• $X_t$ = Cell concentration at time $t$
• $X_0$ = Initial cell concentration
• $t$ = Time elapsed

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Doubling Time ($t_d$):

$$t_d = \frac{0.693}{\mu}$$

$$t_d = \frac{t}{n}$$

Where $n$ is the number of generations during time $t$. For example, the doubling time of E. coli under optimal conditions is approximately 20 minutes.

Problem: A culture contains $10^4 \text{ cells/mL}$ initially ($X_0$) and reaches $10^8 \text{ cells/mL}$ ($X_t$) after $2 \text{ hours}$ ($t$). Calculate the specific growth rate ($\mu$) and doubling time ($t_d$).

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Solution:

$$\mu = \frac{2.303(\log_{10}(10^8) - \log_{10}(10^4))}{2}$$

$$\mu = \frac{2.303(8 - 4)}{2} = \frac{2.303 \times 4}{2} = 4.606 \text{ hr}^{-1}$$

$$t_d = \frac{0.693}{\mu} = \frac{0.693}{4.606} \text{ hr} \times 60 \text{ min/hr} \approx 9 \text{ minutes}$$