Types of bacteriological culture media

Culture media are classified in several ways:

A. Based on physical state

1. Solid media — contain 1.5–2% agar (e.g., nutrient agar, blood agar). Used for isolation of colonies and pure culture.
2. Semi-solid media — contain 0.2–0.5% agar (e.g., motility test medium, Stuart's transport medium). Used to detect motility.
3. Liquid (broth) media — no agar (e.g., nutrient broth, peptone water). Used for enrichment and biochemical tests.

B. Based on chemical composition

1. Synthetic (defined) media — exact chemical composition known (e.g., Czapek–Dox agar, minimal medium).
2. Non-synthetic (complex) media — composition not exactly known, made from peptone, beef extract, yeast extract (e.g., nutrient agar).

C. Based on functional use

1. Basal (general purpose) media — support growth of most organisms (nutrient agar).
2. Enriched media — basal medium plus blood, serum, egg (blood agar, chocolate agar).
3. Enrichment media — liquid media that favour one organism and inhibit others (selenite F broth for Salmonella).
4. Selective media — inhibit unwanted organisms (MacConkey agar, bismuth sulphite agar).
5. Differential media — distinguish organisms by colony appearance (MacConkey agar, blood agar).
6. Transport media — preserve viability during transit (Cary–Blair, Amies medium).
7. Anaerobic media — for anaerobes (Robertson's cooked meat medium).
8. Assay media — for vitamin and antibiotic assays.

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Enumeration by the Most Probable Number (MPN) method

Principle. The MPN method is a statistical, multiple-tube dilution method used to estimate the number of viable organisms (especially coliforms) in a sample when the count is too low to be detected by plate count. It is based on the probability that the sample contains at least one viable cell in the inoculated volume. Results are read from probability (McCrady) tables and expressed as MPN per 100 mL of sample.

Materials required. Water sample, lactose broth (single and double strength), brilliant green lactose bile (BGLB) broth, MacConkey broth, Durham's tubes, inoculating loop, incubator, MPN tables.

Procedure

1. Presumptive test

   • Set up a series of fermentation tubes: 5 tubes of double-strength lactose broth (10 mL each) and 5 tubes each of single-strength lactose broth (10 mL) in two sets, each containing an inverted Durham's tube.
   • Inoculate 10 mL of the water sample into each double-strength tube, 1 mL into each of the first set of single-strength tubes, and 0.1 mL into each of the second set (diluted if necessary).
   • Incubate at 35–37 °C for 24 hours; if no gas, re-incubate up to 48 hours.
   • Gas production in the Durham's tube indicates a positive presumptive test (lactose fermented → coliforms present).

2. Confirmed test

   • Subculture from every gas-positive tube onto a differential medium (e.g., eosin methylene blue agar) or into BGLB broth.
   • Incubate at 35–37 °C for 24–48 hours.
   • Growth of typical colonies or gas in BGLB confirms the presence of coliforms.

3. Completed test

   • Pick a typical colony, inoculate into lactose broth with a Durham's tube and onto a nutrient agar slant.
   • Incubate at 35–37 °C for 24–48 hours.
   • Gas in the lactose broth plus Gram-negative, non-sporing rods on the slant completes the test.

4. Reading the result

   • Record the number of positive tubes in each of the three sets (10 mL, 1 mL, 0.1 mL) as a three-digit code, e.g., 5–3–1.
   • Compare this code with the standard MPN probability table to obtain the MPN index per 100 mL.
   • If the code is not listed, use the next higher or nearest value and note it as approximate.

Example. If the code is 4–2–0, the MPN table gives 22 MPN per 100 mL, meaning about 22 coliforms are present in every 100 mL of the sample.

Significance. MPN is useful for drinking water quality testing (WHO standard: MPN < 1 per 100 mL for potable water), for foods and for samples with very few organisms. Its limitations are that it is laborious, gives only a statistical estimate (not an exact count), and has wide confidence limits.

Structure of the Cell Wall of Gram-Positive Bacteria

The cell wall of gram-positive bacteria is thick, rigid, and relatively simple in organisation. It lies outside the cytoplasmic membrane and is composed mainly of peptidoglycan (murein), along with teichoic acids and, in some species, polysaccharides and proteins.

1. Peptidoglycan (Murein) Layer

• It forms the major component, constituting about 60–90% of the cell wall by weight.
• The layer is thick, about 20–80 nm, and may consist of up to 40 sheets of peptidoglycan.
• The glycan backbone is made of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues joined by β-1,4 glycosidic bonds.
• Tetrapeptide side chains are attached to NAM residues. The tetrapeptide typically contains L-alanine, D-glutamic acid, L-lysine (or diaminopimelic acid, DAP), and D-alanine.
• Adjacent tetrapeptide chains are cross-linked by a pentaglycine bridge (as in Staphylococcus aureus), giving the wall great mechanical strength.
• Because of the thick, highly cross-linked peptidoglycan, the wall retains crystal violet–iodine complex during gram staining, so the cells appear purple/violet.

2. Teichoic Acids

• These are negatively charged polymers of glycerol or ribitol phosphate, unique to gram-positive bacteria.
• Wall teichoic acids are covalently linked to NAM; lipoteichoic acids are anchored in the cytoplasmic membrane and extend through the peptidoglycan.
• Functions: provide negative charge, help in adherence, maintain cell wall rigidity, and act as antigenic determinants and virulence factors.

3. Other Components

• Some species have surface proteins (e.g., protein A in S. aureus, M protein in Streptococcus pyogenes).
• Polysaccharides may be present (e.g., teichoic acid–associated sugars).
• The wall is not surrounded by an outer membrane, and the periplasmic space is very narrow or absent.

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Differences Between Gram-Positive and Gram-Negative Bacteria

Reason for the difference in staining: The thick peptidoglycan layer of gram-positive bacteria traps the crystal violet–iodine complex on alcohol decolourisation, whereas the thin peptidoglycan and lipid-rich outer membrane of gram-negative bacteria allow the complex to be washed out, so they take up the safranin counterstain and appear pink/red.

Passive and Active Transport Systems in Bacteria

Bacteria are surrounded by a plasma membrane (cytoplasmic membrane) that is selectively permeable. Since bacteria lack membrane-bound organelles, all transport of nutrients, ions, and waste products across the membrane occurs through transport systems located in this membrane. These are broadly classified into two types: passive transport (does not require energy) and active transport (requires energy).

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1. Passive Transport System

In passive transport, substances move across the membrane down their concentration gradient (from higher to lower concentration) without the expenditure of metabolic energy (ATP). It is driven by the inherent kinetic energy of molecules.

a) Simple Diffusion

• Small, non-polar molecules such as O₂, CO₂, H₂O (slowly), and small hydrophobic molecules pass directly through the lipid bilayer.
• The rate depends on the concentration gradient and the permeability of the membrane.
• No carrier protein is involved.

b) Facilitated Diffusion

• Polar or charged molecules (e.g., glycerol, some sugars, ions) cannot pass through the lipid bilayer directly. They require specific carrier (transport) proteins.
• The carrier undergoes a conformational change to shuttle the molecule across the membrane.
• It is saturable (carriers become saturated at high substrate concentration), specific, and shows competitive inhibition.
• Examples: glycerol uptake in E. coli via GlpF (aquaglyceroporin channel), and water transport via aquaporins.

c) Osmosis

• Movement of water across the semipermeable membrane from a region of higher water potential (lower solute concentration) to lower water potential (higher solute concentration).
• In bacteria, water movement is facilitated by aquaporins (e.g., AqpZ in E. coli).

Key features of passive transport:

• No energy (ATP) required
• Down the concentration gradient
• Carrier proteins may or may not be involved
• Not affected by metabolic inhibitors (e.g., cyanide, DNP)

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2. Active Transport System

In active transport, substances are moved against their concentration gradient (from lower to higher concentration). This requires metabolic energy, usually from ATP hydrolysis or from the proton motive force (PMF). Active transport is of two main types: primary and secondary.

a) Primary Active Transport

Here, energy is supplied directly by ATP hydrolysis. The transport proteins are called ABC transporters (ATP-Binding Cassette transporters).

Structure of ABC transporter:

• Two hydrophobic membrane-spanning domains — form the translocation channel.
• Two nucleotide-binding domains (NBDs) — bind and hydrolyze ATP.

Mechanism:

1. The substrate binds to the periplasmic binding protein (in Gram-negative bacteria) or directly to the membrane domain.
2. ATP binds to the NBD, causing a conformational change.
3. The substrate is pushed across the membrane into the cytoplasm.
4. ATP is hydrolyzed to ADP + Pi, and the transporter returns to its original state.

Examples:

• Histidine permease in Salmonella typhimurium
• Maltose transport in E. coli (MalEFGK system)
• Phosphate (Pst) system in E. coli

b) Secondary Active Transport

Here, energy is not directly from ATP but from the proton motive force (PMF) — the electrochemical gradient of protons (H⁺) across the membrane. The transport of one substance is coupled to the movement of another.

Types:

• Symport (Cotransport): Both substances move in the same direction.
  • Example: Lactose permease (LacY) in E. coli — H⁺ and lactose enter together.
• Antiport (Countertransport): The two substances move in opposite directions.
  • Example: Na⁺/H⁺ antiporter in E. coli — Na⁺ is expelled while H⁺ enters.

c) Group Translocation

A special type of active transport found only in bacteria, where the substrate is chemically modified during transport.

Example: Phosphotransferase System (PTS)

• Used for uptake of glucose, mannose, fructose in E. coli.
• The sugar is phosphorylated to sugar-phosphate as it enters the cell.
• Energy comes from phosphoenolpyruvate (PEP), not directly from ATP.
• Components: Enzyme I, HPr, Enzyme II.
• The phosphorylated sugar cannot leave the cell, so it is effectively trapped.

Key features of active transport:

• Requires energy (ATP or PMF)
• Moves against the concentration gradient
• Involves specific carrier proteins
• Inhibited by metabolic poisons (e.g., cyanide, DNP, azide)
• Can be saturated and shows specificity

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Comparison Table

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Conclusion

Bacteria use passive transport for the simple, energy-free movement of small molecules and water down their concentration gradients. For the uptake of essential nutrients against a gradient, they employ active transport systems such as ABC transporters, secondary symport/antiport, and group translocation (PTS). Together, these systems allow bacteria to maintain internal homeostasis, accumulate nutrients, and survive in diverse environments.

Methods for the Cultivation of Viruses

Viruses are obligate intracellular parasites — they cannot multiply on their own and must be grown inside living host cells. Since they cannot be cultured on ordinary artificial media (like bacteria on nutrient agar), special methods are used. The main methods are:

1. Inoculation into Embryonated Eggs (Embryonated Egg Culture)

A fertilised hen's egg is incubated for 8–12 days so that the embryo develops. The virus is inoculated into different sites of the egg, each supporting different viruses:

• Chorioallantoic membrane (CAM) — for poxviruses, which produce visible pocks.
• Allantoic cavity — for influenza and mumps viruses, which multiply in the allantoic fluid.
• Amniotic cavity — for influenza and mumps viruses.
• Yolk sac — for herpes simplex and some rickettsiae.

Advantages: cheap, sterile, easy to maintain, large surface area, and a wide range of viruses can be grown. Disadvantages: eggs may carry their own viruses, and not all viruses grow well.

2. Animal Inoculation (Laboratory Animals)

Living animals are used as hosts for virus cultivation. Common animals include mice, rats, guinea pigs, rabbits, and monkeys. The virus is inoculated by routes such as intracerebral, intranasal, intraperitoneal, or subcutaneous. The animal is observed for signs of disease, and the virus is recovered from its tissues.

• Suckling mice are especially useful for arboviruses and coxsackieviruses.
• Monkeys were historically used for poliovirus.

Advantages: useful when no other method works and for studying pathogenesis and immune response. Disadvantages: costly, difficult to maintain, ethical concerns, and animals may carry latent viruses.

3. Tissue (Cell) Culture — the Most Widely Used Method

This is the most important and most commonly used method today. Host cells are grown in vitro in a suitable medium (e.g., Eagle's medium with serum) and the virus is inoculated onto them. There are three types:

(a) Organ culture — fragments of organs (e.g., trachea, intestine) are maintained in vitro with their original architecture. Used for respiratory and intestinal viruses.

(b) Explant (tissue) culture — small pieces of tissue are embedded in plasma clot or placed on a surface and allowed to grow out. Cells grow out from the explant.

(c) Cell culture — cells are dispersed (by trypsin) and grown as a monolayer. This is the most widely used type and is of three kinds:

• Primary cell culture — freshly obtained cells (e.g., monkey kidney cells); survive only a few passages. Susceptible to a wide range of viruses.
• Diploid (semi-continuous) cell culture — fibroblasts (e.g., human embryonic lung) that can be subcultured 50–100 times; retain diploid chromosome number. Used for many viruses.
• Continuous (established) cell line — immortalised cells (e.g., HeLa, Hep-2, Vero) that can be subcultured indefinitely. Convenient but may lose some original characters.

Detection of viral growth in cell culture: cytopathic effect (CPE), haemadsorption, interference, metabolic changes (colour change of phenol red indicator), and plaque formation.

Advantages: wide range of viruses, easy observation, large-scale production, and use for vaccine and diagnostic work. Disadvantages: risk of contamination, need for strict aseptic technique, and some viruses still fail to grow.

4. Chick Embryo Cell Culture (a special case)

Cells from chick embryos can be grown in vitro; this overlaps with cell culture but is often listed separately because it is a common source of primary cells.

Summary Table

Conclusion: Among all methods, cell culture is the method of choice today because it is sensitive, convenient, and suitable for both diagnosis and vaccine production. Eggs and animals are used mainly when cell culture is unsuitable.

Sterilization is the process of completely destroying or removing all forms of microbial life, including bacteria, bacterial spores, fungi, viruses, and other microorganisms, from an object, surface, or medium. A sterilized item is free from viable microorganisms and is termed sterile.

Filtration Sterilization

Principle

Filtration sterilization works on a physical (mechanical) principle rather than by killing microorganisms. The liquid or gas to be sterilized is passed through a filter having pores smaller than the microorganisms to be removed. Microorganisms are trapped on the surface or within the matrix of the filter by:

• Sieve retention — particles larger than the pore size are physically blocked.
• Adsorption — microorganisms adhere to the filter material due to electrostatic and van der Waals forces.

Since the pore size (usually 0.22 µm or 0.45 µm) is smaller than most bacteria (typically 0.5–5 µm), bacteria and fungi are retained, and the filtrate emerging on the other side is sterile. Filtration is especially valuable because it does not involve heat, so heat-labile solutions (sugars, vitamins, antibiotics, sera, enzymes) can be sterilized without denaturation or chemical change.

Procedure

1. Selection of filter: A membrane filter of pore size 0.22 µm (for sterilization) or 0.45 µm (for clarification) is chosen. Common types are cellulose nitrate, cellulose acetate, PTFE, and polyvinylidene difluoride (PVDF) membranes, or sintered glass/ceramic filters.

2. Assembly and pre-sterilization: The filter holder (e.g., a syringe filter, vacuum filtration unit, or cartridge filter) is assembled aseptically. The apparatus is itself pre-sterilized by autoclaving or by using pre-sterilized disposable units.

3. Filtration: The solution is forced through the membrane either by:

   • Positive pressure (syringe or pump), or
   • Negative pressure/vacuum (vacuum flask assembly).

   The liquid passes through the pores while microorganisms are retained on the membrane surface.

4. Collection: The sterile filtrate is collected into a pre-sterilized container under aseptic conditions (usually in a laminar flow hood).

5. Post-filtration handling: The filter is discarded or, in some protocols, the membrane is placed on nutrient medium to check for retained organisms (sterility testing). The filtrate is stored properly and used within a limited period.

6. For gases/air: Air is passed through HEPA (High-Efficiency Particulate Air) filters, which remove microorganisms and particulate matter by interception, impaction, and diffusion. This is used in laminar flow cabinets and operating theatres.

Applications

1. Heat-labile solutions: Sterilization of antibiotics, vitamins, sera, vaccines, enzymes, and sugar solutions that would be destroyed by autoclaving.

2. Bacteriological media: Filtration of serum, urea, and other heat-sensitive components that are added aseptically to autoclaved basal media.

3. Pharmaceutical industry: Production of sterile injections, ophthalmic solutions, and biological products; large-scale cartridge filtration is used in manufacturing.

4. Air and gas sterilization: HEPA filters in laminar flow hoods, operation theatres, and clean rooms to provide sterile air.

5. Water purification: Membrane filtration for obtaining sterile or microorganism-free water in laboratories and industries.

6. Vaccine and biological fluid production: Removal of bacteria and viruses from culture fluids without affecting their antigenic or biological properties.

7. Sterility testing and quality control: Used to test sterility of pharmaceutical products and to obtain cell-free filtrates of toxins and enzymes.

Limitations: Filtration cannot remove viruses smaller than the pore size, and it is unsuitable for turbid or viscous solutions that clog the membrane.

Bacterial Recombination

Definition: Bacterial recombination is the process by which genetic material (DNA) from one bacterium (the donor) is transferred to another bacterium (the recipient) and becomes integrated into the recipient's chromosome or exists as an independent replicon, producing a recombinant cell with a new combination of genes. Unlike sexual reproduction in eukaryotes, it is not associated with reproduction or the formation of a zygote; it is a unidirectional transfer of a fragment of DNA. It occurs by three main mechanisms: transformation, conjugation, and transduction.

Transduction

Transduction is the transfer of bacterial DNA from one bacterium to another by a bacteriophage (bacterial virus). It was discovered by Zinder and Lederberg (1952) in Salmonella typhimurium. It is of two types:

1. Generalized transduction

• Occurs during the lytic cycle of a virulent (lytic) phage.
• The phage infects the donor bacterium, replicates, and in the process the host chromosome is broken into fragments.
• Occasionally, a fragment of host DNA (instead of phage DNA) is packaged into the phage protein coat, forming a transducing particle.
• This defective phage injects the donor DNA into a recipient cell.
• The donor DNA fragment then undergoes homologous recombination with the recipient chromosome, replacing the corresponding segment.
• Any gene of the donor can be transferred, hence "generalized."
• Example: phage P22 in Salmonella, phage P1 in E. coli.

2. Specialized (restricted) transduction

• Occurs only with temperate phages (e.g., phage lambda, λ) that integrate into the host chromosome as a prophage.
• On induction (excision), the prophage sometimes excises incorrectly, carrying along a few adjacent host genes (e.g., gal or bio genes in λ).
• The resulting phage carries both phage genes and a small piece of host DNA.
• When it infects a new recipient, the host genes are transferred and integrated, but the phage is defective (cannot replicate independently).
• Only genes adjacent to the prophage attachment site are transferred, hence "specialized."

Steps in transduction (generalized)

1. Infection: Phage adsorbs to and injects its DNA into the donor bacterium.
2. Replication and fragmentation: Phage DNA replicates; host chromosome is degraded into fragments.
3. Packaging: A host DNA fragment is mistakenly packaged into a phage head.
4. Release: Cell lyses; transducing particles are released.
5. Infection of recipient: The transducing particle injects donor DNA into the recipient.
6. Recombination: Donor DNA integrates into the recipient chromosome by homologous recombination, producing a transductant with recombinant characters.

Significance

• It is a natural means of genetic exchange and variation in bacteria.
• It is widely used in genetic mapping (cotransduction frequency indicates gene distance).
• It explains the spread of genes such as antibiotic-resistance genes among bacteria.

Basis of Classification of Bacteria

Bacteria are classified on the following bases:

1. Morphological classification (shape)

• Cocci – spherical (e.g., Streptococcus)
• Bacilli – rod-shaped (e.g., Escherichia coli)
• Spirilla – spiral/curved (e.g., Spirillum)
• Vibrio – comma-shaped (e.g., Vibrio cholerae)
• Spirochetes – long, coiled, flexible (e.g., Treponema pallidum)
• Pleomorphic – variable shape (e.g., Mycoplasma)

2. Arrangement of cells

• Diplococci (pairs), Streptococci (chains), Staphylococci (clusters), Tetrads (groups of four), Sarcinae (cubes of eight).

3. Staining behaviour (Gram stain)

• Gram-positive – retain crystal violet (thick peptidoglycan layer)
• Gram-negative – decolourised, counterstained pink (thin peptidoglycan, outer membrane)
• Acid-fast – retain carbol fuchsin (e.g., Mycobacterium)

4. Oxygen requirement

• Aerobic – require oxygen
• Anaerobic – grow only without oxygen
• Facultative anaerobe – grow with or without oxygen
• Microaerophilic – need low oxygen tension

5. Temperature requirement

• Psychrophiles – cold-loving (below 20 °C)
• Mesophiles – moderate (20–45 °C)
• Thermophiles – heat-loving (above 45 °C)

6. Nutrition

• Autotrophs – synthesize own food (photoautotrophs, chemoautotrophs)
• Heterotrophs – depend on organic matter (saprophytes, parasites, symbionts)

7. Flagellation

• Atrichous (no flagella), Monotrichous (single), Lophotrichous (tuft at one end), Amphitrichous (both ends), Peritrichous (all over the body).

8. Capsule and spore formation

• Capsulated vs non-capsulated; spore-forming (e.g., Bacillus, Clostridium) vs non-spore-forming.

9. Genetic and molecular basis

• DNA base composition (G + C content)
• DNA hybridization and 16S rRNA sequencing — the most reliable modern basis, used in Bergey's classification.

10. Pathogenicity

• Pathogenic (disease-causing) vs non-pathogenic bacteria.

Applications of microorganisms in food and pharmaceutical industries

A. In the food industry

1. Fermented foods and beverages — Saccharomyces cerevisiae ferments sugars to produce bread, beer, wine and jand; lactic acid bacteria (Lactobacillus, Streptococcus) curdle milk into yoghurt, cheese, butter and mahi; Acetobacter aceti oxidises ethanol to acetic acid in vinegar production.
2. Food preservation — fermentation produces lactic acid, acetic acid and alcohol, which lower pH and inhibit spoilage organisms, thus preserving pickles, sauerkraut and silage.
3. Food additives and supplements — Aspergillus niger produces citric acid (used in soft drinks, jams); Acetobacter produces vinegar; Corynebacterium glutamicum produces monosodium glutamate (MSG); algae and yeasts give vitamins (B₁₂, riboflavin) and single-cell protein.
4. Enzymes — Aspergillus and Bacillus yield amylase (starch processing), protease (meat tenderising, cheese making), pectinase (fruit juice clarification) and rennin (cheese clotting).
5. Dairy products — starter cultures give curd, cheese, ghee flavour and probiotics that improve gut health.
6. Food spoilage control and safety — microorganisms are used as bio-preservatives (nisin from Lactococcus lactis) and in food quality testing.

B. In the pharmaceutical industry

1. Antibiotics — Penicillium notatum gives penicillin; Streptomyces gives streptomycin, tetracycline, erythromycin and chloramphenicol.
2. Vaccines — attenuated or killed pathogens (Mycobacterium bovis BCG, Salmonella typhi, polio virus) are used to prepare vaccines against TB, typhoid and polio.
3. Vitamins and enzymes — Ashbya gossypii produces riboflavin (B₂), Pseudomonas denitrificans produces vitamin B₁₂; microbial enzymes are used as medicines (streptokinase as a blood clot dissolver).
4. Hormones and therapeutic proteins — recombinant E. coli and yeast produce human insulin, growth hormone and interferon through genetic engineering.
5. Steroids and other drugs — Rhizopus and Aspergillus carry out biotransformation to produce corticosteroids and sex hormones.
6. Vaccines, sera and diagnostics — microbial antigens are used in diagnostic kits (Widal test, ELISA) and in producing antitoxins.

Conclusion — Microorganisms are thus indispensable in both industries: they act as fermenting agents, enzyme and metabolite factories in food production, and as sources of antibiotics, vaccines, hormones and diagnostics in pharmacy.

DNA (deoxyribonucleic acid) is the hereditary material of most organisms. Its main characteristics are:

1. Double-stranded helix — DNA consists of two polynucleotide strands wound around a common axis to form a right-handed double helix (Watson–Crick model).

2. Sugar–phosphate backbone — Each strand has a backbone of alternating deoxyribose sugar and phosphate groups, joined by 3′–5′ phosphodiester bonds.

3. Nitrogenous base pairing (complementarity) — The two strands are held together by hydrogen bonds between purine and pyrimidine bases: A pairs with T (2 H-bonds) and G pairs with C (3 H-bonds). Thus A = T and G = C.

4. Antiparallel strands — The two strands run in opposite directions: one is 5′→3′ and the other 3′→5′.

5. Constant base composition (Chargaff's rule) — In a given species, the ratio (A + T)/(G + C) is constant, though it varies between species.

6. Ability to replicate (self-replication) — DNA can make exact copies of itself by semi-conservative replication, so genetic information passes to daughter cells.

7. Stability and mutability — DNA is chemically stable, but occasional changes (mutations) in base sequence produce variation.

8. Carrier of genetic information — The sequence of bases forms the genetic code that controls protein synthesis and hence all traits.

9. Uniform width — Because a purine always pairs with a pyrimidine, the diameter of the helix is constant (about 2 nm).

10. Denaturation and renaturation — DNA can be denatured by heat or alkali (strands separate) and can renature on cooling, showing its stable yet dynamic nature.

The TCA Cycle (Tricarboxylic Acid Cycle / Krebs Cycle / Citric Acid Cycle)

The TCA cycle is the final common oxidative pathway in which acetyl-CoA is completely oxidized to CO₂, with the released energy conserved as NADH, FADH₂ and GTP. It occurs in the mitochondrial matrix in eukaryotes. One turn of the cycle yields 3 NADH, 1 FADH₂, 1 GTP, and 2 CO₂.

Steps of the cycle:

1. Formation of Citrate (C₆): Acetyl-CoA (2C) condenses with oxaloacetate (4C) to form citrate (6C). Enzyme: citrate synthase. Irreversible, rate-limiting step.

2. Isomerization to Isocitrate (C₆): Citrate is converted to isocitrate via cis-aconitate. Enzyme: aconitase.

3. Oxidative Decarboxylation to α-Ketoglutarate (C₅): Isocitrate loses CO₂ and is oxidized. Enzyme: isocitrate dehydrogenase. Yields 1 NADH and the first CO₂.

4. Oxidative Decarboxylation to Succinyl-CoA (C₄): α-Ketoglutarate loses CO₂ and is oxidized. Enzyme: α-ketoglutarate dehydrogenase complex. Yields 1 NADH and the second CO₂.

5. Conversion to Succinate (C₄): Succinyl-CoA is converted to succinate. Enzyme: succinyl-CoA synthetase. Yields 1 GTP (substrate-level phosphorylation).

6. Oxidation to Fumarate (C₄): Succinate is oxidized. Enzyme: succinate dehydrogenase (Complex II of ETC). Yields 1 FADH₂.

7. Hydration to Malate (C₄): Fumarate + H₂O → malate. Enzyme: fumarase.

8. Oxidation to Oxaloacetate (C₄): Malate is oxidized. Enzyme: malate dehydrogenase. Yields 1 NADH, regenerating oxaloacetate to start the next turn.

Significance:

• Major source of NADH and FADH₂ for oxidative phosphorylation (ATP production).
• Amphibolic in nature — provides precursors for gluconeogenesis, fatty acid synthesis, and amino acid metabolism.
• Links carbohydrate, fat, and protein metabolism.

Aseptic techniques are the set of practices and procedures used to prevent contamination of cultures, sterile media, equipment, and the worker by unwanted microorganisms, and to prevent the escape of pathogens from the laboratory. Since microorganisms are ubiquitous in air, on surfaces, and on the human body, every microbiological procedure must be carried out aseptically to obtain pure cultures and reliable results.

Major aseptic techniques used in the microbiology laboratory:

1. Sterilization of media and equipment — Culture media, glassware, and instruments are sterilized before use, usually by autoclaving at 121°C and 15 psi for 15–20 minutes. Heat-sensitive materials are sterilized by filtration (membrane filters) or by exposure to UV radiation/chemical disinfectants.

2. Flaming of instruments — Inoculating loops, needles, and forceps are held in a flame until red hot before and after each transfer. This kills all organisms on the instrument and prevents cross-contamination between cultures.

3. Flaming the mouth of containers — The neck/mouth of test tubes, culture bottles, and flasks is passed briefly through a flame after the cap or cotton plug is removed. This creates convection currents that keep airborne contaminants away and kills organisms on the rim.

4. Working near a flame or in a laminar flow hood — Transfers are performed in the updraft of a Bunsen burner flame or inside a laminar airflow cabinet (HEPA-filtered air), where the air is either sterile or moving away from the open container.

5. Proper handling of culture vessels — Plates are opened only slightly (just enough to insert the loop), held with the lid facing downward, and never left open. Tubes are held nearly horizontal, and caps are never placed on the bench.

6. Personal hygiene and protective measures — Hands are washed with soap/disinfectant before and after work; laboratory coats, gloves, and sometimes masks are worn. Talking, coughing, or sneezing over open cultures is avoided.

7. Surface disinfection — The working bench is wiped with 70% alcohol or other disinfectant before and after work, and spills are immediately treated with disinfectant.

8. Transfer techniques — Inoculation is done quickly and with minimal exposure; the loop is cooled before touching the medium; subculturing is done from pure cultures using sterile loops.

Conclusion: Together, these techniques maintain a sterile working environment, ensure pure cultures, protect the worker and the environment from infection, and are therefore fundamental to all microbiological work.

Types of Staining Methods for Bacteria

Staining is the technique of applying dyes to bacterial cells to make them visible under the microscope and to differentiate them. The main types are:

1. Simple Staining

A single basic dye (e.g., methylene blue, crystal violet, carbol fuchsin) is used.

• Purpose: To show the size, shape, and arrangement of bacteria.
• Procedure: Smear → heat fix → stain → wash → dry → observe.
• Result: All bacteria appear the same colour; no differentiation.

2. Differential Staining

Uses more than one dye to divide bacteria into groups based on cell wall or cellular properties.

(a) Gram Staining

• Reagents: Crystal violet (primary), Gram's iodine (mordant), alcohol/acetone (decolouriser), safranin (counterstain).
• Result: Gram-positive bacteria retain crystal violet and appear purple (thick peptidoglycan layer); Gram-negative bacteria lose the dye and take safranin, appearing pink/red (thin peptidoglycan, outer lipid membrane).

(b) Acid-Fast Staining (Ziehl–Neelsen)

• Reagents: Carbol fuchsin (with heat/phenol), acid-alcohol (decolouriser), methylene blue (counterstain).
• Result: Acid-fast bacteria (e.g., Mycobacterium tuberculosis) resist decolourisation and appear red; non-acid-fast bacteria appear blue.

3. Special Staining

Used to demonstrate specific bacterial structures.

4. Structural Staining

A subtype of special staining that highlights a particular cell part (capsule, spore, flagella, cell wall) rather than the whole cell.

Conclusion: Simple staining gives morphology only, while differential and special staining allow identification and classification of bacteria, which is essential in diagnosis and research.

Techniques for Isolation and Enumeration of Yeast

Isolation Techniques

1. Streak plate method A loopful of diluted sample is streaked in a zig-zag pattern over the surface of a solidified medium (e.g. YEPD / Sabouraud agar). Serial streaking progressively dilutes the cells so that isolated single colonies develop after incubation at 25–30 °C for 2–3 days. Each well-separated colony is presumed to arise from a single cell and is picked for purification.

2. Pour plate method The sample is serially diluted and 1 mL of the appropriate dilution is mixed with molten agar (cooled to ~45 °C) and poured into a sterile Petri dish. Colonies develop both on the surface and within the medium. This method both isolates and enumerates yeast.

3. Spread plate method 0.1 mL of the diluted sample is spread evenly over the surface of a pre-dried agar plate with a sterile L-shaped glass spreader. After incubation, well-distributed isolated colonies appear on the surface.

4. Enrichment / selective isolation Yeasts are selectively favoured by using media of low pH (pH 3.5–4.5) or by adding antibiotics (e.g. chloramphenicol, streptomycin) or acid (e.g. tartaric acid) to suppress bacterial growth. Media such as Sabouraud dextrose agar, YEPD agar and wort agar are commonly used.

5. Single-cell isolation Individual cells or colonies are picked under a microscope using a micromanipulator or a fine capillary, ensuring a pure culture derived from one cell.

Enumeration Techniques

1. Viable count (plate count) Serial dilutions of the sample are plated (pour or spread plate). After incubation, plates showing 30–300 colonies are counted. The number of colony-forming units (CFU) per mL is calculated as:

$\text{CFU/mL} = \frac{\text{Number of colonies}}{\text{Dilution} \times \text{Volume plated}}$

2. Direct microscopic count A known volume of sample is placed in a counting chamber (haemocytometer or Neubauer chamber) and the cells are counted under the microscope. It gives the total cell count (both living and dead) rapidly, but does not distinguish viable from non-viable cells.

3. Membrane filter technique A known volume of sample is passed through a membrane filter (pore size ~0.45 µm) that retains the yeast cells. The filter is placed on an appropriate agar medium and incubated; colonies growing on the filter are counted.

4. Turbidimetric (spectrophotometric) method The turbidity of a cell suspension is measured with a spectrophotometer (usually at 600 nm) and compared with a standard curve to estimate cell mass/concentration. It is rapid but measures total biomass, not viable cells.

5. Dry weight / packed cell volume method Cells from a known volume are harvested by centrifugation and the packed cell volume or dry weight is measured as an indirect measure of cell mass.

Conclusion

For routine work, the streak plate is best for obtaining pure isolates, while the plate count (viable count) is the standard method for enumeration, since it counts only living, reproducing cells. Direct microscopic count and turbidimetry are used when a rapid estimate of total cells is required.

Morphological Characteristics of Intestinal Protozoan Parasites

Intestinal protozoan parasites are unicellular eukaryotes that inhabit the human intestinal tract. Their morphology is studied mainly in the trophozoite (motile, feeding, pathogenic stage) and cyst (round, non-motile, infective stage) forms.

1. Entamoeba histolytica (causes amoebiasis)

• Trophozoite: 10–60 µm (average 15–20 µm); pleomorphic (shape changes with pseudopodia); cytoplasm differentiated into clear ectoplasm and granular endoplasm; single spherical nucleus with a central karyosome and peripheral chromatin; red blood cells often seen in the endoplasm (hallmark of invasive amoebiasis).
• Cyst: 10–15 µm; spherical; uninucleate to quadrinucleate (mature cyst has 4 nuclei); blunt, cigar-shaped chromatoid bars and glycogen mass present in immature cysts.

2. Giardia lamblia (causes giardiasis)

• Trophozoite: 12–15 µm long, 6–8 µm wide; pear-shaped (pyriform); bilaterally symmetrical; two large oval nuclei at the anterior end (resembling a face); four pairs of flagella; two median bodies (parabasal bodies); a ventral sucking disc for attachment to the duodenal mucosa.
• Cyst: 8–14 µm; oval; thick wall; four nuclei; remains of flagella and median bodies seen inside.

3. Trichomonas hominis (non-pathogenic, large intestine)

• Trophozoite: 5–15 µm; pear-shaped; single nucleus at the anterior end; undulating membrane extending the whole body length; 3–5 anterior flagella and one posterior flagellum; no cyst stage.

4. Balantidium coli (causes balantidiasis)

• Trophozoite: largest protozoan parasite of humans, 50–100 µm (up to 200 µm); oval; covered with cilia; kidney-shaped macronucleus and a small micronucleus; contractile vacuoles present; cytostome (mouth) and cytopyge (anal pore).
• Cyst: 50–70 µm; spherical; thick, double-layered wall; cilia not visible.

5. Cryptosporidium parvum (causes cryptosporidiosis)

• Oocyst: 4–6 µm; spherical to oval; thick, smooth wall; contains four sporozoites; acid-fast positive; no tissue-dwelling trophozoite seen in routine stool.

6. Isospora belli (causes isosporiasis)

• Oocyst: 20–33 µm; oval/ellipsoidal; transparent, smooth, thin wall; immature oocyst contains a single sporoblast, mature oocyst contains two sporocysts each with four sporozoites.

7. Cyclospora cayetanensis

• Oocyst: 8–10 µm; spherical; thick wall; contains two sporocysts, each with two sporozoites; requires special staining (modified acid-fast) for detection.

Summary Table

Key points: All intestinal protozoa are unicellular; most have both trophozoite and cyst stages (except Trichomonas); cysts are the infective forms and are resistant to gastric acid; trophozoites are the motile, feeding, pathogenic forms; motility is by pseudopodia, flagella or cilia; and the nucleus (number, karyosome position) is an important identifying feature.

Contributions of Louis Pasteur and Robert Koch in Microbiology (5 marks)

Louis Pasteur (1822–1895) — Father of Microbiology

1. Disproved spontaneous generation — Through his famous swan-neck flask experiments, he proved that microorganisms do not arise spontaneously from non-living matter but come from pre-existing microbes (biogenesis).
2. Germ theory of disease — Established that specific microorganisms cause specific diseases (e.g., anthrax, chicken cholera, rabies).
3. Pasteurization — Developed a heat-treatment method (heating at 63 °C for 30 minutes) to kill microbes in wine, beer and milk without spoiling the taste.
4. Vaccine development — Produced vaccines against chicken cholera, anthrax and rabies (the first human rabies vaccine was given in 1885).
5. Pure culture techniques — Introduced methods for growing microorganisms in nutrient media, laying the foundation for pure culture isolation.

Robert Koch (1843–1910) — Founder of Medical Bacteriology

1. Koch's postulates — Established a set of four criteria to prove that a specific microorganism causes a specific disease.
2. Discovery of causative agents — Identified Bacillus anthracis (anthrax, 1876), Mycobacterium tuberculosis (tuberculosis, 1882) and Vibrio cholerae (cholera, 1883).
3. Solid media culture — Introduced solid media (using agar and gelatin) for obtaining pure cultures of bacteria.
4. Staining techniques — Developed staining methods (e.g., methylene blue, Ziehl–Neelsen acid-fast stain) for observing bacteria under the microscope.
5. Petri dish — Popularized the use of the Petri dish (designed by his assistant Julius Petri) for culturing microorganisms.

Conclusion — Pasteur laid the foundation of microbiology through sterilization, pasteurization and vaccination, while Koch advanced it into a precise medical science through pure culture methods, staining and the germ theory proof. Together they established microbiology as a distinct scientific discipline.

Beneficial role of normal flora in the human body

Normal flora (indigenous microflora) are the microorganisms that permanently reside on the skin and mucous membranes of a healthy person without causing disease. They play several beneficial roles:

1. Protection against pathogens (microbial antagonism): They occupy attachment sites and consume available nutrients, so invading pathogens cannot colonize. They also produce inhibitory substances such as fatty acids (skin), lactic acid (vagina), and bacteriocins that kill or suppress harmful microbes.

2. Production of vitamins: Gut bacteria such as Escherichia coli and other colonic flora synthesize vitamin K and several B-complex vitamins (biotin, folic acid, riboflavin, pyridoxine), which are absorbed and used by the host.

3. Aid in digestion and absorption: They help break down complex carbohydrates, cellulose, and other indigestible food residues by fermentation, and assist in the absorption of nutrients and minerals.

4. Development and maturation of the immune system: Exposure to normal flora stimulates the lymphoid tissue (e.g., Peyer's patches) and the production of natural antibodies, helping the body develop immune tolerance and resistance.

5. Maintenance of physiological conditions: Vaginal lactobacilli keep the pH acidic (about 4–4.5) and prevent growth of Candida and other pathogens. Skin flora keep the skin surface slightly acidic.

6. Anticarcinogenic effect: Intestinal flora convert harmful substances and certain carcinogens into non-toxic forms, thus reducing the risk of colon cancer.

7. Recycling of body substances and waste: They decompose dead cells, urea, and other waste products, and help in the recycling of nitrogen and carbon compounds.

Thus, normal flora live in a mutually beneficial (commensal or symbiotic) relationship with the host and are essential for health, digestion, immunity, and protection against disease.

(a) RNA (Ribonucleic Acid)
RNA is a single-stranded polynucleotide made of ribonucleotides, each consisting of a nitrogenous base (adenine, guanine, cytosine or uracil), a ribose sugar and a phosphate group. It is mainly found in the cytoplasm and carries out protein synthesis (mRNA, tRNA, rRNA); in some viruses (e.g. HIV, influenza virus) it acts as the genetic material.

(b) Neutralization test
It is an immunological (antigen–antibody) test in which a specific antibody binds to and neutralizes the biological effect of a toxin or virus. Example: the toxin produced by Clostridium tetani is neutralized by its antitoxin, and this is used for diagnosis and for testing antitoxin potency.

(c) Metabolism
Metabolism is the sum total of all the chemical reactions occurring in a living cell, by which nutrients are broken down to release energy (catabolism) and simple substances are converted into complex cell components (anabolism). It is catalysed by enzymes.

(d) Photolithotrophic bacteria
These are bacteria that obtain their energy from sunlight (photo-) and their carbon from carbon dioxide, i.e. an inorganic source (litho-), using inorganic substances such as H₂S, H₂ or thiosulphate as electron donors. Example: green sulphur bacteria (Chlorobium) and purple sulphur bacteria (Chromatium).

(e) Methods of culture of anaerobic bacteria

1. Anaerobic jar (McIntosh and Fildes jar) — air is replaced by H₂/CO₂.
2. Gas-pak method — hydrogen and palladium catalyst remove oxygen.
3. Robertson's cooked meat medium.
4. Thioglycollate broth (fluid thioglycollate medium).
5. Anaerobic chamber / glove box.
6. Culture in deep agar tubes (shake culture) and by pyrogallol–NaOH method.

(f) Plasmid
A plasmid is a small, circular, extrachromosomal, double-stranded DNA molecule present in bacteria (and some yeasts) that replicates independently of the chromosome. It usually carries non-essential but useful genes such as those for antibiotic resistance, fertility (F factor) and toxin production, and is widely used as a vector in genetic engineering.

(g) Uses of dark field microscope

1. To observe living, unstained microorganisms, especially spirochaetes such as Treponema pallidum (syphilis).
2. To study motility and shape of motile bacteria.
3. To examine specimens like blood, urine and spinal fluid for live organisms.
4. To observe internal structures (e.g. granules) in living cells.

(h) Bacterial capsule with examples
The capsule is a well-organized, thick, gelatinous layer of polysaccharide (sometimes polypeptide) lying outside the cell wall, which protects the bacterium from phagocytosis and drying and helps in adherence. Examples: Streptococcus pneumoniae, Klebsiella pneumoniae, Bacillus anthracis (polypeptide capsule).

(i) Characteristics of nematodes

1. Triploblastic, bilaterally symmetrical, unsegmented roundworms with a cylindrical body tapering at both ends.
2. Body covered by a tough, non-chitinous cuticle; pseudocoelomate body cavity.
3. Digestive tract is a complete tube (mouth to anus); no circulatory or respiratory system.
4. Sexes usually separate (dioecious) with sexual dimorphism; excretion by renettes.
5. Many are free-living in soil/water, others are parasites of plants and animals (e.g. Ascaris, Wuchereria).

(j) Glycogenolysis
Glycogenolysis is the breakdown of stored glycogen into glucose-6-phosphate (and free glucose) by the enzyme glycogen phosphorylase. It occurs mainly in the liver and muscle, is stimulated by glucagon and adrenaline, and helps to raise blood glucose level during fasting or exercise.

Mutation is a heritable, sudden and permanent change in the nucleotide sequence of DNA (or in the genetic material of an organism) that alters the genotype and often the phenotype of the cell. In bacteria, mutations may arise spontaneously during DNA replication or be induced by physical and chemical mutagens. A mutation is transmitted to daughter cells (vertical inheritance) and, in bacteria, can also spread horizontally by transformation, transduction and conjugation.

Types of Bacterial Mutation

Bacterial mutations are classified in several ways.

A. On the basis of origin

1. Spontaneous mutation Occurs naturally without any external mutagen, due to errors in DNA replication (tautomeric shifts, base mispairing), depurination, deamination or the action of transposons. The frequency is low, about $10^{-6}$ to $10^{-9}$ per gene per generation. Example: mutation giving streptomycin resistance in E. coli.

2. Induced mutation Produced deliberately by exposing bacteria to mutagens:

• Physical mutagens — UV light (forms pyrimidine dimers), X-rays and $\gamma$-rays (strand breaks).
• Chemical mutagens — base analogues (5-bromouracil), deaminating agents (nitrous acid), alkylating agents (EMS, nitrosoguanidine) and intercalating agents (ethidium bromide).

B. On the basis of the change in DNA (molecular type)

1. Point mutation (substitution) Change of a single base pair:

• Silent (synonymous) — new codon codes for the same amino acid; no phenotypic change.
• Missense — new codon codes for a different amino acid, e.g. sickle-cell type change (GAG → GUG).
• Nonsense — codon changed into a stop codon (UAA, UAG, UGA), giving a truncated protein.

2. Frameshift mutation Insertion or deletion of one or two base pairs shifts the reading frame, so all downstream codons are misread. Usually produces a completely non-functional protein. Example: insertion of a base in the lac gene.

3. Deletion and duplication Loss or repetition of a segment of DNA. Large deletions may remove several genes (multi-gene deletion).

4. Inversion A segment of DNA is reversed end to end within the chromosome; gene order changes but gene number stays the same.

5. Insertion mutation A transposon or insertion sequence (IS element) inserts into a gene and inactivates it — called insertional inactivation.

C. On the basis of phenotypic effect

1. Auxotrophic mutation — the mutant loses the ability to synthesize an essential metabolite (amino acid, vitamin) and therefore requires it in the medium. Example: E. coli mutants requiring histidine or tryptophan.

2. Prototrophic (reversion) mutation — a mutant regains the ability to synthesize the metabolite and grows on minimal medium.

3. Resistance mutation — the mutant becomes resistant to an antibiotic, drug or phage. Example: rifampicin-resistant mutants of M. tuberculosis (RNA polymerase gene).

4. Fermentation / biochemical mutation — loss of ability to ferment a sugar, e.g. lactose-negative (lac⁻) mutants of E. coli.

5. Morphological (colonial) mutation — change in colony form, colour or cell shape, e.g. smooth (S) to rough (R) dissociation in Pneumococcus.

6. Virulence mutation — loss or gain of disease-producing ability, e.g. attenuated vaccine strains such as BCG.

D. On the basis of direction

1. Forward mutation — wild type changes to mutant type. 2. Reverse (back) mutation — mutant reverts to wild type; it may be a true reversion (original base restored) or a suppressor mutation (second mutation elsewhere restores function).

E. Special bacterial mutation systems

1. Conditional lethal mutation — the mutant survives only under a permissive condition, e.g. temperature-sensitive mutants grow at 30 °C but die at 42 °C.

2. Suppressor mutation — a second mutation at a different site masks the effect of the first.

3. Adaptive (directed) mutation — mutation arising under strong selective pressure in a starving population, as seen in E. coli lactose system.

Significance: mutations are the raw material of evolution, are used to produce attenuated vaccines, antibiotic-resistant strains (a clinical problem), and improved industrial strains in biotechnology.

Factors Affecting Bacterial Growth

Bacterial growth is the increase in the number of bacterial cells, not in cell size. It depends on several physical, chemical and biological factors. These factors are grouped as follows:

1. Physical Factors

(a) Temperature Temperature is one of the most important factors. Every bacterium has a minimum, optimum and maximum growth temperature.

• Psychrophiles – grow best at 0–15 °C (e.g., Pseudomonas).
• Mesophiles – grow best at 25–40 °C (e.g., E. coli); most pathogens belong here.
• Thermophiles – grow best at 50–70 °C (e.g., Bacillus stearothermophilus, Thermus aquaticus).
• Hyperthermophiles – grow best above 80 °C (e.g., Pyrolobus fumarii).

Low temperature slows metabolism (bacteria become dormant but not dead), while high temperature denatures proteins and enzymes, killing the cell.

(b) pH Most bacteria grow best at neutral pH (6.5–7.5). Acidophiles grow at low pH (e.g., Lactobacillus), while alkaliphiles grow at high pH. Extreme pH denatures enzymes and disrupts the cell membrane.

(c) Oxygen

• Obligate aerobes – require oxygen (e.g., Mycobacterium tuberculosis).
• Obligate anaerobes – killed by oxygen (e.g., Clostridium).
• Facultative anaerobes – grow with or without oxygen (e.g., E. coli).
• Microaerophiles – need low oxygen.
• Aerotolerant anaerobes – tolerate oxygen but do not use it.

(d) Moisture / Water Activity Water is essential for all metabolic activities. Drying (desiccation) stops growth but spores survive. High salt or sugar (low water activity) causes plasmolysis and inhibits growth.

(e) Osmotic Pressure Bacteria in hypertonic solutions lose water and shrink (plasmolysis). Halophiles tolerate high salt. Most bacteria are inhibited by high osmotic pressure.

2. Chemical Factors

(a) Nutrients Bacteria need carbon, nitrogen, phosphorus, sulphur, trace elements and growth factors. Absence of any essential nutrient limits growth.

(b) Oxygen and Carbon Dioxide CO₂ is needed by capnophilic bacteria. Oxygen acts as a terminal electron acceptor in aerobes but forms toxic superoxide in anaerobes.

(c) Inhibitory Substances Antibiotics, disinfectants, heavy metals, dyes and preservatives inhibit or kill bacteria.

3. Biological Factors

(a) Symbiosis and Antagonism Bacteria may grow faster in association with other organisms (symbiosis) or may be inhibited by products of other microbes (antibiosis), e.g., penicillin from Penicillium.

(b) Host Factors In the body, growth depends on host immunity, normal flora and availability of nutrients.

4. Other Factors

• Radiation – UV light and ionizing radiation cause mutations and death.
• Pressure – high hydrostatic pressure kills most bacteria; barophiles tolerate it.
• Generation time – determines the rate of growth.

Conclusion

Bacterial growth is thus controlled by a combination of temperature, pH, oxygen, moisture, osmotic pressure, nutrients, inhibitory substances and biological interactions. Understanding these factors is essential in medicine, food preservation and industrial microbiology.

Dry Heat Sterilization — Principle and Methods

Principle

Dry heat sterilization kills microorganisms by oxidation of cell constituents, protein denaturation, and burning (incineration) rather than by moist heat coagulation. Because there is no water or steam, the process requires higher temperatures and longer exposure times than moist heat sterilization to achieve the same lethal effect. The absence of moisture means heat is transferred to the article mainly by conduction and convection through hot air. Microbial death follows first-order kinetics, so the D-value (time to reduce the population tenfold) at a given temperature determines the required holding time. Dry heat is effective against bacterial spores, fungi, and viruses, and it also destroys pyrogens (endotoxins) at high temperature, which moist heat cannot do. It is therefore preferred for glassware, metal instruments, oils, powders, and heat-stable materials that would be damaged or corroded by steam.

Methods of Dry Heat Sterilization

1. Red heat (incineration to redness) The article is held directly in a flame until it glows red. Used for platinum loops, wires, needles, and forceps in the microbiology laboratory. Immediate and absolute, but limited to non-combustible metal items.

2. Flaming (burning) The article is passed through a flame without being heated to redness, after being dipped in a flammable liquid such as alcohol. Used for glass slides, mouths of culture tubes, scalpels, and glass spreaders. Quick, but only the exposed surface is sterilized.

3. Hot air oven (the standard method) A thermostatically controlled, electrically heated, fan-assisted chamber in which hot air is circulated. The usual standard cycle is 160 °C for 2 hours (or 170 °C for 1 hour, or 180 °C for 30 minutes). Articles must be dry, loosely packed, and arranged so that hot air circulates freely; they should not be overloaded. Used for glassware (test tubes, petri dishes, pipettes, flasks), metal instruments, glass syringes, oils, fats, waxes, and heat-stable powders (talc, sulphadiazine). The oven is fitted with a thermocouple or thermometer and a thermostat, and spore strips of Bacillus subtilis var. niger or Clostridium tetani are used as biological controls. Advantages: no corrosion of metal, no moisture damage. Limitations: slow, uneven penetration, unsuitable for rubber, plastic, cloth, or aqueous solutions.

4. Incineration (burning to ashes) The material is completely burnt in an incinerator. Used for contaminated dressings, animal carcasses, bedding, and disposable plastic or paper waste. It both sterilizes and disposes of the waste.

5. Infrared radiation (infra-red conveyor sterilizer) Infrared rays are directed onto the articles on a moving conveyor belt. The heat is absorbed and converted to thermal energy. Used for metal instruments and glassware in large-scale hospital practice; exposure is short (seconds to minutes) but only the directly irradiated surface is treated.

6. Dry heat in a sealed container / hot oil or sand bath Small instruments or glassware may be immersed in hot oil or sand at about 160 °C for 1 hour. Used occasionally for cutting instruments and small metal parts; messy and rarely preferred.

Factors affecting efficiency

• Temperature and time — inversely related; higher temperature needs shorter time.
• Nature of the load — density, packing, and material affect heat penetration.
• Moisture — even slight moisture converts the process to moist heat and shortens the required time.
• Type and number of organisms — spores are far more resistant than vegetative cells.
• Presence of organic matter — blood, pus, or grease protects organisms and must be removed first.

Summary

Dry heat sterilization works by oxidation and incineration of microbial protoplasm and needs higher temperature and longer time than moist heat. The main methods are red heat, flaming, hot air oven (160 °C for 2 hours), incineration, infrared radiation, and hot oil/sand bath, of which the hot air oven is the most widely used for glassware, metal instruments, oils, and powders.

General Structure of Virus

Viruses are obligate intracellular parasites that are inert outside the host cell. They are much smaller than bacteria (20–300 nm) and are not true cells — they lack cytoplasm, ribosomes and a metabolic machinery of their own. A complete, infectious virus particle is called a virion, and it consists of two essential parts — a nucleic acid core and a protein coat — with an outer envelope in some viruses.

1. Nucleic acid core (genome): The central core is made of a single type of nucleic acid, either DNA or RNA, never both. The nucleic acid may be single-stranded (ss) or double-stranded (ds), linear or circular, and segmented or non-segmented. It carries the genetic information and is the infectious part of the virion. Examples: dsDNA in bacteriophages, ssRNA in tobacco mosaic virus (TMV), ssDNA in φX174.

2. Capsid (protein coat): The nucleic acid is surrounded by a protein coat called the capsid, made of repeating protein subunits known as capsomeres. The capsid protects the genome from nucleases and environmental damage, and helps in attachment to the host cell. The combination of nucleic acid and capsid is called the nucleocapsid.

3. Envelope (in some viruses): Many animal viruses (e.g. influenza virus, HIV) have an additional outer envelope derived from the host cell membrane. It is a lipoprotein layer in which virus-specific glycoprotein spikes (peplomeres) are embedded. These spikes help in attachment and penetration. Enveloped viruses are generally more sensitive to ether, alcohol and detergents, whereas naked (non-enveloped) viruses such as adenovirus and poliovirus are more resistant.

4. Symmetry of capsid: The capsid shows three types of symmetry — helical (e.g. TMV), icosahedral/cubical (e.g. adenovirus) and complex (e.g. poxvirus, bacteriophage T4 with head, tail and tail fibres).

Classification of Viruses

Because viruses are not cellular, they cannot be classified by the binomial system of Linnaeus. They are classified on the basis of several criteria:

A. On the basis of host range (host tropism):

1. Bacteriophages (bacterial viruses): attack bacteria, e.g. T4 phage, λ phage.
2. Phytophages (plant viruses): attack plants, e.g. TMV, potato virus X.
3. Zoophages (animal viruses): attack animals and humans, e.g. polio virus, rabies virus.
4. Mycophages: attack fungi; cyanophages: attack cyanobacteria.

B. On the basis of nucleic acid type:

1. DNA viruses: contain DNA as genetic material, e.g. Poxvirus, Herpesvirus, Adenovirus, Hepatitis B virus.
2. RNA viruses: contain RNA as genetic material, e.g. Influenza virus, HIV, Poliovirus, TMV. Each group is further divided into double-stranded and single-stranded, and into (+) sense and (−) sense RNA viruses.

C. On the basis of symmetry and shape:

1. Helical (rod-shaped): capsomeres arranged in a spiral, e.g. TMV, influenza virus.
2. Icosahedral (spherical/polyhedral): capsomeres arranged in 20 triangular faces, e.g. adenovirus, polio virus.
3. Complex or binal: neither purely helical nor icosahedral, e.g. poxvirus, bacteriophage T4.
4. Pleomorphic: variable shape, e.g. Mycoplasma-like viruses.

D. On the basis of presence or absence of envelope:

1. Enveloped (complex) viruses: have an outer lipoprotein envelope, e.g. HIV, influenza, herpes.
2. Naked (simple) viruses: only nucleocapsid, no envelope, e.g. adenovirus, TMV.

E. On the basis of replication site and mode:

1. Viruses replicating in the nucleus (e.g. herpes, adenovirus).
2. Viruses replicating in the cytoplasm (e.g. poxvirus, most RNA viruses).

F. Baltimore classification (modern molecular scheme): This is the most widely accepted modern classification, proposed by David Baltimore (1971). It is based on the mode of viral mRNA synthesis and the relationship between the viral genome and mRNA. It divides all viruses into seven groups:

G. ICTV classification: The International Committee on Taxonomy of Viruses (ICTV) classifies viruses into a hierarchical system of Order → Family → Subfamily → Genus → Species, using genome type, replication strategy, host range and serological relationships as criteria. Orders end in -virales, families in -viridae and genera in -virus (e.g. order Mononegavirales, family Rhabdoviridae, genus Lyssavirus — rabies virus).

In short, viruses are structurally simple — a nucleic acid core enclosed in a protein capsid, sometimes with an envelope — but they are classified by multiple schemes based on host, nucleic acid, symmetry, envelope and replication strategy, with the Baltimore and ICTV systems being the most important today.

Detection Techniques of Helminthic Parasites

Helminths (parasitic worms) are detected in the laboratory mainly by microscopic examination of stool, supported by blood tests, serology, and molecular methods. The choice of technique depends on the parasite and its stage present in the specimen.

1. Direct Microscopic Examination of Stool

The simplest and most widely used method. A small amount of fresh stool is mixed with normal saline on a slide, covered with a coverslip, and examined under low and high power.

• Saline wet mount — detects motile ova, larvae, and adult worms; also shows motility of Strongyloides larvae.
• Iodine wet mount — stains nuclei and glycogen, aiding identification of cysts and eggs (e.g., Entamoeba, Giardia).
• Limitation — low sensitivity when parasite load is scanty; repeated samples on different days improve yield.

2. Concentration Techniques

These separate parasites from fecal debris and increase sensitivity.

A. Flotation methods (use solutions of higher specific gravity so eggs float):

• Saturated salt (brine) flotation — good for nematode eggs (Ascaris, Trichuris, hookworm) and cestode eggs.
• Zinc sulfate flotation — preferred for protozoan cysts and light helminth eggs (e.g., Ascaris, hookworm); heavy eggs such as Schistosoma do not float.
• Limitation — operculated eggs (Diphyllobothrium, Fasciola) and heavy eggs do not float.

B. Sedimentation methods (eggs settle at the bottom):

• Formol-ether (formalin-ethyl acetate) concentration — the method of choice for most helminth eggs and larvae; also detects cysts.
• Simple gravity sedimentation — useful for heavy eggs like Fasciola and Schistosoma.

3. Kato–Katz Thick Smear Technique

A standardized quantitative method using a cellophane tape strip soaked in glycerine-malachite green over a measured amount of sieved stool. It is the WHO-recommended method for field surveys of soil-transmitted helminths (Ascaris, Trichuris, hookworm) and gives egg counts (EPG) to assess intensity of infection. It is less useful for Strongyloides and hookworm larvae.

4. Cellophane Tape (Perianal Swab) Method

A piece of clear adhesive tape is pressed against the perianal skin in the morning before bathing or defecation, then stuck on a slide and examined. It is the method of choice for Enterobius vermicularis (pinworm) eggs, which are deposited outside the anus and are rarely found in stool.

5. Stool Culture and Larval Recovery

• Harada–Mori filter paper culture — stool on filter paper strips is cultured for 7–10 days; hookworm and Strongyloides larvae migrate to the water at the bottom and are collected and identified.
• Baermann funnel technique — warm water drives larvae out of feces into a collecting tube; used for Strongyloides and hookworm.
• Koga agar plate culture — detects Strongyloides larvae with high sensitivity.

6. Blood and Tissue Examination

• Thick and thin blood films (Giemsa stain) — detect microfilariae (Wuchereria bancrofti, Brugia spp.), which show nocturnal periodicity.
• Knott's concentration — lyses RBCs to concentrate microfilariae in blood.
• Tissue biopsy / aspirate — muscle biopsy for Trichinella larvae; rectal snip or urine examination for Schistosoma eggs; skin snip for Onchocerca microfilariae.

7. Serological and Immunological Tests

Used when parasites are not easily demonstrated in stool or blood:

• ELISA — for Strongyloides, Trichinella, Fasciola, Echinococcus.
• Indirect hemagglutination and immunoblot — for cysticercosis and hydatid disease.
• Circulating antigen detection — for filariasis and schistosomiasis.

8. Molecular Techniques

• PCR and real-time PCR — highly sensitive and specific; detect DNA of Strongyloides, hookworm, Schistosoma, and Taenia even at very low parasite loads.
• Multiplex PCR — identifies several helminths in a single stool sample.
• LAMP (loop-mediated isothermal amplification) — field-friendly, low-cost molecular method.

9. Imaging and Other Aids

• Ultrasound — for Schistosoma bladder fibrosis and hydatid cysts.
• CT / MRI — for neurocysticercosis and hydatid disease.
• Eosinophil count — supportive evidence of helminthic infection.

Conclusion

No single technique detects all helminths. A rational laboratory approach combines direct wet mount for motile stages, formol-ether concentration for most eggs and larvae, Kato–Katz for quantitative field surveys, and cellophane tape for Enterobius. Serology and PCR are reserved for cases where microscopy is negative but clinical suspicion remains high.

EMP Pathway (Glycolysis)

The EMP pathway is named after Gustav Embden, Otto Meyerhof and Jakub Parnas, who worked out its steps. It is the sequence of reactions by which one molecule of glucose is broken down to two molecules of pyruvate in the cytoplasm of the cell. It is the first and common stage of carbohydrate catabolism, needs no oxygen, and occurs in almost all living cells.

$\text{Glucose} + 2\text{NAD}^+ + 2\text{ADP} + 2\text{P}_i \rightarrow 2\text{Pyruvate} + 2\text{NADH} + 2\text{H}^+ + 2\text{ATP} + 2\text{H}_2\text{O}$

The pathway has 10 steps divided into two phases.

Phase I — Preparatory (investment) phase

In this phase glucose is split into two 3-carbon molecules, and 2 ATP are used.

Phase II — Payoff phase

Each of the two G3P molecules now passes through steps 6–10, so every reaction occurs twice per glucose.

Steps 7 and 10 are substrate-level phosphorylations, where ATP is formed directly without the electron transport chain.

Calculation of ATP production from one molecule of glucose

ATP consumed (Phase I):

• Step 1 (hexokinase) = 1 ATP
• Step 3 (phosphofructokinase) = 1 ATP
• Total used = 2 ATP

ATP produced (Phase II, ×2):

• Step 7 (phosphoglycerate kinase) = 2 ATP
• Step 10 (pyruvate kinase) = 2 ATP
• Total produced = 4 ATP

Net ATP from substrate-level phosphorylation: $4 - 2 = \mathbf{2\ ATP}$

NADH produced: 2 NADH (step 6), which yield ATP only through oxidative phosphorylation:

Conclusion: The EMP pathway gives a net gain of 2 ATP directly, and 6 or 8 ATP in total when the NADH is oxidised aerobically. In the absence of oxygen only 2 ATP per glucose are obtained.

Plasmid

A plasmid is a small, circular, double-stranded, extrachromosomal DNA molecule found mainly in bacteria (and some yeasts) that replicates independently of the chromosomal DNA. It is not essential for the normal survival of the cell but often carries genes that give the host a selective advantage, such as antibiotic resistance, virulence factors, or metabolic capabilities. Plasmids can be transferred from one bacterium to another by conjugation, transformation, or transduction.

R-plasmid (Resistance plasmid)

An R-plasmid is a conjugative plasmid that carries genes conferring resistance to one or more antibiotics (and sometimes to heavy metals or other drugs). It typically consists of two parts: the RTF (resistance transfer factor), which carries the genes for transfer (tra genes) and replication, and one or more r-determinants, which carry the actual antibiotic-resistance genes. Many r-determinants are transposons, so resistance genes can jump between plasmids and chromosomes.

Why R-plasmids are increasingly important in medical sectors

1. Spread of antibiotic resistance (AMR): R-plasmids transfer resistance genes rapidly between unrelated bacteria by conjugation, converting a susceptible strain into a resistant one within hours. This is a major driver of multidrug-resistant (MDR) and extensively drug-resistant (XDR) pathogens such as E. coli, Klebsiella, Salmonella, Shigella, Staphylococcus aureus (MRSA), and Pseudomonas.

2. Failure of standard therapy: Infections caused by R-plasmid-bearing organisms no longer respond to first-line and even second-line antibiotics. This forces the use of expensive, toxic, last-resort drugs (e.g., colistin, carbapenems) and increases treatment failure, complications, and mortality.

3. Horizontal gene transfer across species: Unlike chromosomal mutation, which is vertical and slow, plasmid-mediated resistance spreads horizontally — even from harmless gut flora to pathogens. This makes resistance a shared, community-wide problem rather than an individual one.

4. Nosocomial (hospital-acquired) infections: Hospitals concentrate sick, immunocompromised patients and heavy antibiotic use, creating ideal conditions for R-plasmid exchange. Outbreaks of MDR organisms in ICUs, surgical wards, and neonatal units are frequently plasmid-mediated.

5. Transfer of multiple resistances at once: A single R-plasmid may carry resistance to 5–10 different antibiotics. Treating such an infection with any one of those drugs selects for the whole plasmid, so one drug can indirectly promote resistance to many others.

6. Rise of carbapenemase and ESBL genes: Genes such as bla_NDM-1, bla_KPC, and bla_CTX-M are commonly plasmid-borne. Their global dissemination through plasmids has made carbapenems and cephalosporins unreliable in many regions.

7. Impact on public health and economy: AMR lengthens hospital stays, raises healthcare costs, and threatens the success of surgery, chemotherapy, and organ transplantation. WHO has declared AMR one of the top ten global public health threats.

8. Diagnostic and surveillance value: Detecting R-plasmids in clinical isolates helps identify resistance mechanisms, guide antibiotic choice, and track the spread of resistance determinants in a population.

Because of these reasons, R-plasmids are of central importance in medical microbiology — they are the principal vehicle for the global rise of antibiotic resistance, making infection control, rational antibiotic use, and plasmid surveillance essential in modern medicine.

Principle of Phase Contrast Microscope

The phase contrast microscope was developed by Frits Zernike (1934) to observe unstained, transparent living specimens (cells, tissues, microorganisms) that differ from their surroundings only in refractive index and thickness, not in colour or absorption.

Basic principle:

1. Phase difference instead of amplitude difference — Light passing through a transparent specimen is not absorbed, so no contrast in intensity (amplitude) is produced. However, the specimen has a slightly different refractive index and thickness than the surrounding medium, so light passing through it is retarded (phase-shifted) relative to light passing through the background. This is called the phase difference, usually about ¼ wavelength (λ/4).

2. Conversion of phase difference into amplitude (brightness) difference — The human eye cannot detect phase differences, only intensity differences. The microscope therefore converts the invisible phase shift into a visible light–dark contrast using two special optical elements:

   • Annular diaphragm (annulus) in the condenser — makes the illuminating light pass as a hollow cone (ring) through the specimen.
   • Phase plate in the objective — a transparent disc with a phase-retarding ring (usually retarding light by λ/4) coated with a light-absorbing film.

3. Interference at the image plane — Light emerging from the specimen consists of:

   • Direct (surrounding) light — passes through the phase ring of the phase plate and is retarded by λ/4.
   • Diffracted (scattered) light from the specimen — falls outside the ring and is not retarded.

   The two sets of light waves interfere at the image plane. Because of the extra λ/4 retardation, the total phase difference becomes λ/2 (half wavelength), causing destructive interference in regions of higher refractive index (denser parts) and constructive interference in less dense parts. Thus:

   • Denser structures (nucleus, organelles) appear dark against a bright background — positive phase contrast.
   • If the phase plate is made to advance instead of retard, the contrast is reversed — negative phase contrast (specimen appears bright on dark background).

Result: The invisible differences in refractive index and thickness of the specimen are converted into visible differences in brightness (contrast), allowing clear observation of living, unstained cells and their internal structures without fixation or staining.

Germ Theory of Disease

The germ theory states that many diseases are caused by the invasion and multiplication of microorganisms (germs) such as bacteria, viruses, fungi and protozoa in the body. Its main points are:

1. Microorganisms cause disease — Specific microscopic organisms (pathogens) are responsible for specific infectious diseases.
2. Pathogens enter the host — Germs enter the body through air, water, food, contact, vectors or wounds, and multiply there.
3. Specificity of pathogens — Each disease is caused by a particular type of germ (e.g. Mycobacterium tuberculosis causes tuberculosis, Vibrio cholerae causes cholera).
4. Disease results from growth and toxin production — The damage is caused by the multiplication of germs and/or the toxins they release.
5. Koch's postulates — The germ must be found in the diseased host, isolated and grown in pure culture, and must reproduce the same disease when introduced into a healthy host.
6. Prevention and control — Since germs cause disease, diseases can be prevented by sanitation, disinfection, sterilization, vaccination and treated by antibiotics/antimicrobial drugs.
7. Contagiousness — Infectious diseases can spread from one person to another through germs, explaining epidemics.

This theory replaced the earlier "spontaneous generation" idea and formed the basis of modern microbiology, hygiene and public health.

Role of microorganisms in the food sector (5 marks)

Microorganisms are used deliberately in the food sector both to produce foods and to preserve them, while some also act as spoilage agents that must be controlled.

1. Fermentation (production of foods and beverages)

• Lactic acid bacteria (Lactobacillus, Streptococcus) ferment milk sugar to lactic acid, curdling milk protein — used to make curd (dahi), yoghurt, cheese, ghee (from cream), butter and fermented vegetables like gundruk and sinki.
• Yeasts (Saccharomyces cerevisiae) ferment sugars to ethanol and CO₂ — used in bakery (bread raising), brewing (beer, wine), and distillation (spirit).
• Moulds are used in soy sauce (Aspergillus oryzae), tempeh (Rhizopus) and cheese ripening (e.g. Penicillium roqueforti for blue cheese).

2. Food preservation

• Fermentation produces organic acids, alcohol and bacteriocins that lower pH and inhibit spoilage and pathogenic organisms, so foods keep longer without refrigeration.
• Pickling, sauerkraut and ensiling of fodder are microbial preservation methods.

3. Food enrichment and quality improvement

• Microbes improve flavour, aroma, texture and digestibility (e.g. fermentation breaks down antinutrients like phytate in cereals).
• Single-cell protein (SCP) from Spirulina, Chlorella and yeasts is used as protein-rich food and animal feed.
• Probiotics (Lactobacillus, Bifidobacterium) in curd and supplements improve gut health.

4. Food safety and quality control

• Indicator organisms (coliforms, E. coli) and pathogen testing (Salmonella, Staphylococcus aureus) are used to check hygiene of water, milk and processed foods.
• Microbial toxins (aflatoxin from Aspergillus flavus, botulinum toxin) are monitored to prevent food poisoning.

5. Food spoilage (negative role)

• Bacteria, yeasts and moulds cause souring of milk, rotting of meat and vegetables, and moulding of bread, leading to economic loss — controlled by refrigeration, canning, drying, irradiation and preservatives.

Conclusion: Microorganisms are indispensable in the food sector — as fermenting agents, preservatives, protein sources and quality-control tools — but must be carefully controlled to prevent spoilage and food-borne disease.

MacConkey agar contains bile salts and crystal violet, which inhibit the growth of Gram-positive bacteria, so only Gram-negative bacteria (mainly enteric bacilli) can grow on it — this makes it selective.

It also contains lactose and the pH indicator neutral red. Gram-negative bacteria that ferment lactose (e.g. Escherichia coli) produce acid, which lowers the pH and turns the colonies pink/red; non-lactose fermenters (e.g. Salmonella, Shigella) do not produce acid and form colourless/pale colonies — this visible colour difference between colonies makes it differential.

Thus, by inhibiting Gram-positive organisms and by distinguishing lactose fermenters from non-fermenters through colony colour, MacConkey agar acts as both a selective and a differential medium.

Functions of different types of RNA

RNA (ribonucleic acid) is a single-stranded polynucleotide made of ribose sugar, phosphate and the bases adenine, guanine, cytosine and uracil. In a cell it occurs mainly in three types, each with a distinct function.

1. Messenger RNA (mRNA)

• Formed in the nucleus by transcription of DNA, carrying the genetic message in the sequence of its bases.
• It is the carrier of genetic information from DNA to the ribosome in the cytoplasm.
• Its base sequence is read in triplets (codons), each codon specifying one amino acid, so mRNA acts as the template for protein synthesis.
• It is the most heterogeneous and least abundant RNA, and is short-lived.

2. Ribosomal RNA (rRNA)

• The most abundant RNA (about 80% of total cellular RNA), found in the ribosome.
• Together with ribosomal proteins it forms the structural framework of the ribosome (large and small subunits).
• It provides the site for protein synthesis and helps in the binding of mRNA and tRNA to the ribosome.
• Some rRNA molecules have catalytic (peptidyl transferase) activity, catalysing peptide bond formation between amino acids.

3. Transfer RNA (tRNA)

• The smallest RNA (about 10–15% of total RNA), soluble in the cytoplasm.
• It acts as an adaptor molecule, carrying a specific amino acid from the cytoplasm to the ribosome.
• It has an anticodon loop whose triplet base sequence is complementary to the codon of mRNA, so it recognises and binds the correct codon.
• It has an amino acid attachment site (CCA end) at the 3′ end, and thus helps in placing amino acids in the correct sequence during translation.

Other RNAs

• hnRNA (heterogeneous nuclear RNA): precursor of mRNA, processed into mature mRNA.
• snRNA (small nuclear RNA): takes part in splicing of hnRNA to form mRNA.
• miRNA / siRNA: regulate gene expression by silencing or degrading specific mRNA.

Conclusion: mRNA carries the coded message, rRNA forms the machinery and catalytic site of the ribosome, and tRNA brings the amino acids and reads the codon — together they carry out the translation of genetic information into proteins.

MR-VP test

The MR-VP test is a composite biochemical test performed on a single glucose phosphate broth culture (MR-VP medium) to detect two different metabolic pathways by which bacteria ferment glucose. It actually consists of two separate tests — the methyl red (MR) test and the Voges–Proskauer (VP) test — read from the same culture after incubation.

Principle of the MR test

The methyl red test detects organisms that carry out mixed acid fermentation of glucose. Such bacteria (e.g. Escherichia coli) convert glucose mainly into stable acidic end products — lactic acid, acetic acid, formic acid and succinic acid — which lower the pH of the medium to about 4.0–4.5. Methyl red is a pH indicator with a transition range of pH 4.4 (red) to pH 6.2 (yellow). When a few drops of methyl red are added to the culture, a red colour at pH ≤ 4.4 indicates a positive MR test (acid production), while a yellow colour indicates a negative test (pH above 6.2, i.e. no strong acid production).

Principle of the VP test

The Voges–Proskauer test detects organisms that carry out the butanediol fermentation pathway. Such bacteria (e.g. Enterobacter aerogenes, Klebsiella pneumoniae) first produce acetoin (acetylmethylcarbinol) as an intermediate, which is subsequently reduced to 2,3-butanediol. The medium therefore remains near neutral (pH about 6.0–6.5) and gives a negative MR reaction. In the VP test, Barritt's reagent (α-naphthol and 40% KOH) is added to the culture. In the presence of atmospheric oxygen, acetoin is oxidised by KOH to diacetyl, which then condenses with the guanidine group of peptone (creatine/creatinine) in the medium to form a cherry-red (rose-red) complex. Development of a red colour within 15–30 minutes is a positive VP test; absence of red colour is negative.

Interpretation

Thus the MR-VP test distinguishes between the two major types of glucose-fermenting enteric bacteria on the basis of the end products of fermentation — the MR test detects acid production (low pH), while the VP test detects the neutral end product acetoin.

Phases of the Bacterial Growth Curve

When bacteria are grown in a closed (batch) culture, the population does not increase at a constant rate. A plot of the logarithm of cell number against time gives a characteristic growth curve with four phases:

1. Lag phase

• No increase in cell number; the curve is flat.
• Bacteria adapt to the new medium: they synthesise enzymes, ribosomes, ATP and other macromolecules.
• Cells are metabolically very active and increase in size, but do not divide.
• Duration varies with the age of the inoculum and the composition of the medium.

2. Log (exponential) phase

• Cells divide at a constant, maximum rate; the curve rises steeply and linearly.
• Growth is balanced — every cell component increases in proportion.
• Generation time is shortest and constant, so the population doubles at regular intervals.
• This is the phase used for biochemical and genetic studies, and for preparing inocula.

3. Stationary phase

• Growth rate equals death rate, so the total viable count remains constant; the curve is horizontal.
• Caused by depletion of an essential nutrient, accumulation of toxic metabolites (acids, alcohols), and overcrowding.
• Cells become smaller, synthesize storage granules, and produce secondary metabolites such as antibiotics and endospores.
• The total count may still rise while the viable count stays flat.

4. Death (decline) phase

• Death rate exceeds growth rate; the curve falls.
• Nutrients are exhausted and toxic products build up.
• Cells lose viability, lyse, and the viable count falls exponentially.
• Some cells may survive for long periods, and spore-formers persist as endospores.

Conclusion: The growth curve shows that bacterial growth is not uniform but passes through lag, log, stationary and death phases, each with distinct physiological and metabolic features.

Identification of Fungi in the Laboratory

Fungi are identified by studying their cultural characteristics (appearance on media) and morphological characteristics (microscopic structures), supported where needed by biochemical and molecular tests. The main steps are:

1. Specimen collection The specimen (pus, sputum, skin scraping, hair, nail clipping, CSF, tissue, or soil) is collected aseptically in a sterile container and transported to the laboratory without delay.

2. Direct microscopic examination The specimen is examined directly to see fungal elements before culture:

• KOH mount (10–20% KOH): clears keratin and debris, revealing budding yeast cells, pseudohyphae, true hyphae, or fungal spores. Used for skin, hair, and nail specimens.
• Gram stain: fungi appear as gram-positive, purple-stained budding yeast cells and hyphae.
• India ink preparation: demonstrates the capsule of Cryptococcus neoformans as a clear halo around the cell.
• Lactophenol cotton blue (LPCB) mount: stains fungal structures blue and kills the organism; used for examining culture growth.
• Calcofluor white stain with fluorescence microscopy: binds chitin and gives bright fluorescence.

3. Culture (isolation) The specimen is inoculated onto suitable media:

• Sabouraud dextrose agar (SDA), pH 5.6 — the standard medium; low pH inhibits bacteria. Antibiotics (chloramphenicol, gentamicin) and cycloheximide may be added.
• Brain heart infusion agar / blood agar for dimorphic and systemic fungi.
• Incubation at 25–30 °C for 4–6 weeks for dermatophytes and moulds; 37 °C for dimorphic fungi and yeasts.

4. Study of cultural characteristics The growing colonies are examined for:

• Rate of growth (rapid, moderate, slow)
• Colony morphology — texture (cottony, woolly, velvety, powdery, yeast-like), colour of surface and reverse, topography (flat, raised, folded), and pigment production. For example, Aspergillus gives powdery green colonies, Trichophyton rubrum gives white cottony colonies with a deep red reverse, and Candida albicans gives smooth, creamy, pasty colonies.

5. Microscopic examination of culture A small portion of the colony is teased out and mounted in LPCB to study:

• Hyphae: septate or aseptate, branching pattern
• Conidia: shape, size, arrangement (chains, clusters, or single), and type (macroconidia, microconidia, arthroconidia, sporangiospores)
• Fruiting bodies: conidiophores, phialides, vesicles, sporangia, cleistothecia
• Yeasts: budding cells, pseudohyphae, true hyphae, chlamydospores (as in Candida albicans on cornmeal agar with Tween 80)

6. Special and confirmatory tests

• Germ tube test: Candida albicans forms germ tubes in human serum at 37 °C within 2 hours.
• Chlamydospore production on cornmeal agar.
• Sugar fermentation and assimilation tests for identifying yeasts.
• Urease test: positive for Cryptococcus.
• Slide culture technique: for detailed microscopic study of moulds without disturbing the arrangement of spores.
• Hair perforation test and urease test for differentiating dermatophyte species.
• Molecular methods (PCR) and serological tests (latex agglutination for cryptococcal antigen) for rapid and specific identification.

Conclusion Thus, fungi are identified by combining direct microscopy, culture on SDA, study of colony and microscopic morphology, and biochemical or molecular confirmatory tests, which together give the genus and species of the fungus.

Non-specific immunity (also called innate or natural immunity) is the body's inborn, first-line defence system that protects against all foreign agents in the same general way, without recognising any particular pathogen. It is present from birth, acts immediately or within hours, and has no memory. It is broadly of two types: external (surface) defence and internal defence.

1. External (surface) defence — barriers

• Physical barriers: intact skin (keratin) and mucous membranes lining the respiratory, digestive and urogenital tracts stop the entry of microbes. Mucus traps particles, and cilia in the respiratory tract sweep them out (ciliary escalator). Coughing, sneezing, vomiting, diarrhoea, tears and urine flow also wash microbes away.
• Chemical barriers: sebum and sweat (lactic acid) keep skin pH acidic (pH ~3–5); lysozyme in tears, saliva and nasal secretions digests bacterial cell walls; HCl in the stomach kills swallowed microbes; bile and digestive enzymes in the intestine are antimicrobial; vaginal acidity and spermine in semen inhibit microbes.
• Microbiological barriers: the normal flora of the skin, mouth, gut and vagina compete with pathogens for nutrients and attachment sites and release inhibitory substances.

2. Internal defence

• Phagocytosis: neutrophils and macrophages (and monocytes) engulf and digest microbes by phagocytosis. Macrophages in the liver, spleen, lungs and lymph nodes act as fixed scavengers.
• Natural killer (NK) cells: lymphocytes that destroy virus-infected cells and tumour cells without prior sensitisation.
• Inflammation: injury or infection causes redness, heat, swelling and pain. Vasodilation brings more blood; increased capillary permeability lets phagocytes and plasma proteins enter the tissue; histamine, kinins and prostaglandins mediate the response. It localises the infection and speeds repair.
• Fever: pyrogens (e.g. interleukin-1) raise body temperature, which inhibits microbial growth and speeds up the immune reactions.
• Antimicrobial substances: complement proteins (lyse microbes, promote phagocytosis), interferons (from virus-infected cells, block viral replication in neighbouring cells), lysozyme, and acute-phase proteins such as C-reactive protein.
• Protective reflexes: coughing, sneezing and the vomiting reflex expel pathogens.

Characteristics: present from birth; non-specific (same response to all invaders); no memory; no antigen recognition; acts rapidly; same strength on repeated exposure.

Conclusion: Non-specific immunity provides immediate, broad protection and holds the infection in check until the slower, antigen-specific acquired (specific) immunity develops.

a. Nutritional types of bacteria

On the basis of source of carbon and energy, bacteria are classified into:

1. Photoautotrophs – use light as energy source and CO₂ as carbon source (e.g., Chlorobium, cyanobacteria).
2. Photoheterotrophs – use light as energy source and organic compounds as carbon source (e.g., Rhodospirillum).
3. Chemoautotrophs (chemolithotrophs) – use inorganic substances as energy source and CO₂ as carbon source (e.g., Nitrosomonas, Nitrobacter).
4. Chemoheterotrophs (chemoorganotrophs) – use organic compounds as both energy and carbon source (e.g., E. coli, Staphylococcus).

b. Applications of SEM and TEM

SEM (Scanning Electron Microscope):

• Study of surface topography and three-dimensional structure of cells, tissues and microorganisms.
• Examination of biofilms, spores, insect surfaces and forensic samples.
• Quality control in semiconductor and material industries.

TEM (Transmission Electron Microscope):

• Study of internal ultrastructure of cells — organelles, membranes, ribosomes, viruses.
• Observation of virus particles, bacteriophages and protein molecules.
• Diagnosis of viral infections and study of cell pathology.

c. Thermophilic and psychrophilic bacteria

Thermophilic bacteria:

1. Thermus aquaticus
2. Bacillus stearothermophilus

Psychrophilic bacteria:

1. Pseudomonas fluorescens
2. Flavobacterium species

d. AFB staining (Acid-Fast Bacilli staining)

AFB staining is a differential staining technique used to detect acid-fast organisms, mainly Mycobacterium tuberculosis. It is based on the high lipid (mycolic acid) content of the cell wall.

Method (Ziehl–Neelsen staining):

1. Prepare a smear, air-dry and heat-fix it.
2. Flood with carbol fuchsin and steam gently for 5 minutes.
3. Wash with water.
4. Decolourise with 20% sulphuric acid (or acid-alcohol) until no more colour comes off.
5. Wash and counterstain with methylene blue for 1–2 minutes.
6. Wash, dry and examine under oil immersion.

Result: Acid-fast bacilli appear red/pink (retain carbol fuchsin), while non-acid-fast organisms and background appear blue.

e. Oxidase positive bacteria

1. Pseudomonas aeruginosa
2. Neisseria gonorrhoeae
3. Vibrio cholerae

f. Features of genetic code

1. Triplet – each codon consists of three nitrogenous bases.
2. Degenerate – more than one codon may code for the same amino acid.
3. Non-overlapping – each base is read only once.
4. Comma-less – no punctuation between codons.
5. Universal – the same code is used by almost all organisms.
6. Unambiguous – a particular codon always codes for the same amino acid.
7. Start and stop codons – AUG initiates and UAA, UAG, UGA terminate translation.

g. Chemoorganotrophic bacteria

Chemoorganotrophic (chemoheterotrophic) bacteria are those that obtain both energy and carbon from organic compounds such as carbohydrates, proteins and fats. They oxidise these organic substances to release energy. Most bacteria of medical importance, e.g., E. coli, Staphylococcus aureus and Salmonella typhi, belong to this group. They may be saprophytic (feeding on dead organic matter) or parasitic (feeding on living hosts).

h. Haemagglutination test

It is a serological test in which antibodies or antigens cause the clumping (agglutination) of red blood cells. When a specific antigen is coated on RBCs and mixed with its homologous antibody, the RBCs clump together, forming a visible lattice.

Applications:

• Blood grouping (ABO and Rh typing).
• Detection of viruses such as influenza and dengue.
• Diagnosis of syphilis (TPHA) and rheumatoid factor.
• Titration of antibody levels in serum.

i. Antibiotics (any three)

1. Penicillin
2. Streptomycin
3. Tetracycline

j. Differential media (any three)

1. MacConkey agar – differentiates lactose fermenters from non-fermenters.
2. Blood agar – differentiates haemolytic from non-haemolytic bacteria.
3. Eosin Methylene Blue (EMB) agar – differentiates lactose fermenters by colony colour.