Unit 5: Control of Microorganisms

I. Orientation: Principles of Microbial Control

Microbial control uses physical or chemical methods to destroy microorganisms, remove them, or inhibit their growth. The appropriate method depends on the organism, its number, the presence of organic matter, the environment, and whether complete sterility is required.

• Definitions:
  • Sterilization: Destruction or removal of all forms of microbial life, including bacterial endospores.
  • Disinfection: Destruction of many or all pathogenic microorganisms on inanimate objects, but not necessarily endospores.
  • Antisepsis: Chemical control of microorganisms on living tissue.
  • Sanitization: Reduction of microbial numbers to public-health standards.
  • Degerming: Mechanical removal of microorganisms from a limited area, such as handwashing.
• Microbial death: A population usually declines logarithmically; each treatment interval removes a constant proportion rather than a constant number.
• Resistance: Endospores, mycobacteria, cysts, and some non-enveloped viruses are generally more resistant than vegetative bacterial cells and enveloped viruses.
• Treatment variables: Temperature, exposure time, concentration, pH, organic material, and surface penetration all affect effectiveness.
• Control goal: Sterility is required for surgical instruments, whereas reduced microbial numbers may be sufficient for food, skin, or environmental surfaces.

II. Physical Agents: Heat, Filtration, Radiation, Desiccation, and Osmotic Pressure

Physical methods control microorganisms without relying primarily on reactive chemical molecules. They are widely used because they can be predictable, economical, and suitable for large-scale processing.

A. Heat

Heat kills mainly by denaturing proteins, disrupting membranes, and damaging nucleic acids; moist heat is generally more effective than dry heat at the same temperature.

• Moist heat: Boiling at (100^\circ\text{C}) for several minutes kills vegetative cells and many viruses, but may not destroy endospores.
• Autoclaving: Steam under pressure commonly operates at (121^\circ\text{C}), 15 psi, for 15–20 minutes. It sterilizes by coagulating cellular proteins and destroys endospores when the load is properly penetrated.
• Pasteurization: Reduces pathogens and spoilage organisms in liquids without sterilizing them. High-temperature short-time treatment commonly uses (72^\circ\text{C}) for 15 seconds in milk.
• Dry heat: Hot-air ovens require higher temperatures and longer exposure, such as (160^\circ\text{C}) for about 2 hours, because dry air transfers heat less efficiently.
• Thermal resistance:
  • D-value: Time required at a specific temperature to reduce a population by 90%.
  • Z-value: Temperature increase required to reduce the D-value tenfold.

B. Filtration

Filtration removes microorganisms from liquids or gases by physically trapping them rather than killing them.

• Membrane filters: A (0.22\ \mu\text{m}) pore-size filter commonly removes most bacterial cells from heat-sensitive solutions such as antibiotic preparations.
• Limitations: Some small bacteria, mycoplasmas, viruses, and dissolved toxins may pass through ordinary bacterial filters.
• Air filtration: HEPA filters remove at least 99.97% of particles approximately (0.3\ \mu\text{m}) in diameter and are used in operating rooms, biological safety cabinets, and clean rooms.
• Advantages: Filtration is useful for serum, vitamins, and culture media that would be damaged by heat.
• Concrete principle: A filter removes organisms only from the material that passes through it; it does not disinfect the container or the filtrate after contamination.

C. Radiation

Radiation damages DNA and other cellular components either directly or through formation of reactive chemical species.

• Ionizing radiation: Gamma rays and X-rays have enough energy to produce ions and free radicals. Gamma irradiation can sterilize disposable syringes, plastic medical supplies, and some foods.
• Non-ionizing radiation: Ultraviolet light, especially around 260 nm, forms thymine dimers in DNA and is useful for exposed air and surfaces.
• Penetration: UV has poor penetration and is blocked by dust, glass, and organic material; therefore, it cannot reliably sterilize shaded or irregular surfaces.
• Cellular repair: Some organisms repair UV damage using photoreactivation or dark-repair enzymes, reducing the effectiveness of marginal exposure.
• Safety: Ionizing radiation requires shielding; UV can injure skin and eyes.

D. Desiccation

Desiccation inhibits growth by removing water required for enzyme activity, transport, and metabolism, but it does not reliably kill all microorganisms.

• Susceptibility: Many Gram-negative bacteria die rapidly when dried, whereas bacterial endospores, fungal spores, and some viruses survive for long periods.
• Mechanism: Water loss concentrates solutes and disrupts membranes and proteins.
• Applications: Drying helps preserve grains, powdered foods, and laboratory specimens; lyophilization, or freeze-drying, preserves microbial cultures.
• Limitation: When water returns, surviving cells may resume growth. Desiccation is therefore usually bacteriostatic rather than sterilizing.

E. Osmotic Pressure

High concentrations of salt or sugar inhibit microorganisms by drawing water out of cells through osmosis.

• Plasmolysis: In a hypertonic environment, water leaves the cytoplasm, the membrane pulls away from the cell wall, and metabolism slows.
• Applications: Salting meats and curing fish use sodium chloride; jams and syrups use high sugar concentrations.
• Resistance: Halophiles require high salt, while osmophilic yeasts tolerate high sugar and can spoil syrups or concentrated fruit products.
• Limitation: Osmotic pressure usually prevents multiplication rather than killing every organism, and salt-tolerant or sugar-tolerant species may survive.

III. Chemical Agents: Disinfectants, Antiseptics, and Preservatives

Chemical control agents are selected according to their target surface, concentration, contact time, toxicity, and spectrum of activity. The same chemical may be safe on equipment but harmful to living tissue.

A. Disinfectants

Disinfectants are chemicals applied to nonliving surfaces to reduce or destroy microorganisms.

• Phenolics: Damage membranes and denature proteins; they remain active in some organic material but can be irritating.
• Halogens: Chlorine forms hypochlorous acid in water and is widely used in drinking-water treatment; iodine preparations damage proteins and membranes.
• Alcohols: Ethanol or isopropanol at approximately 60–90% denatures proteins and disrupts membranes, but alcohols do not destroy endospores and evaporate quickly.
• Aldehydes: Glutaraldehyde and formaldehyde cross-link proteins and nucleic acids; they can achieve high-level disinfection but require careful handling.
• Quaternary ammonium compounds: Disrupt membranes and are useful for environmental surfaces, but are less effective against endospores, mycobacteria, and some non-enveloped viruses.

B. Antiseptics

Antiseptics are antimicrobial chemicals safe enough for application to skin or other living tissue when used at appropriate concentrations.

• Examples: Chlorhexidine is used in surgical skin preparation; iodine compounds and alcohol-based preparations reduce skin flora before injections.
• Selective toxicity: Effective antiseptics damage microbial membranes or proteins more than host tissue, but they are not completely harmless.
• Factors reducing activity: Blood, mucus, dirt, and organic debris can consume or shield the active chemical.
• Contact time: A quick wipe may reduce organisms, but adequate wet exposure is needed for the stated antimicrobial effect.
• Limitation: Antisepsis is not sterilization; resident skin microbiota and some resistant structures may remain.

C. Preservatives

Preservatives inhibit microbial growth in food, cosmetics, pharmaceuticals, and other products during storage.

• Organic acids: Benzoic, sorbic, and propionic acids are more effective at acidic pH because the undissociated molecules enter microbial cells.
• Nitrates and nitrites: Used in some cured meats to inhibit Clostridium botulinum and help maintain color.
• Sulfites: Used in selected foods and beverages; they interfere with microbial enzymes.
• Required balance: A preservative must control organisms without causing unacceptable toxicity, taste, odor, or instability.
• Limitation: Preservatives slow growth but may not eliminate organisms already present; correct processing and hygienic packaging remain necessary.

IV. Chemotherapeutic Agents: Antibiotics, Antifungal Agents, and Antiviral Agents

Chemotherapeutic agents are antimicrobial drugs administered to a host. Their usefulness depends on selective toxicity: the drug should harm the microorganism more than the patient.

A. Antibiotics

Antibiotics are naturally produced or chemically modified substances that inhibit or kill bacteria; antibacterial drugs may also be fully synthetic.

• Cell-wall inhibitors: Penicillins and cephalosporins bind penicillin-binding proteins and block peptidoglycan cross-linking, especially affecting growing bacteria.
• Protein-synthesis inhibitors: Tetracyclines act on the 30S ribosomal subunit; macrolides act on the 50S subunit.
• Nucleic-acid inhibitors: Fluoroquinolones interfere with DNA gyrase or topoisomerase; rifampin inhibits bacterial RNA polymerase.
• Metabolic inhibitors: Sulfonamides resemble para-aminobenzoic acid and disrupt folate synthesis.
• Resistance mechanisms:
  • Enzymatic inactivation, such as beta-lactamase destruction of penicillins.
  • Altered targets, reduced permeability, and active efflux pumps.
• Therapeutic index: Selective toxicity is greatest when a microbial structure, such as peptidoglycan, is absent from human cells.

B. Antifungal Agents

Antifungal drugs target features of fungi that differ from human cells, especially ergosterol-containing membranes and chitin-containing walls.

• Polyenes: Amphotericin B binds ergosterol and forms membrane pores; toxicity occurs because humans also possess membrane sterols.
• Azoles: Fluconazole and related drugs inhibit ergosterol synthesis by blocking fungal cytochrome-dependent enzymes.
• Echinocandins: Inhibit synthesis of (\beta)-glucan in the fungal cell wall, providing a selective target.
• Limitations: Fungal cells are eukaryotic and therefore share more biochemical pathways with humans than bacteria do, narrowing the therapeutic window.
• Treatment principle: Therapy may need to continue for weeks because fungi grow slowly and infections can occur in keratinized tissues or deep organs.

C. Antiviral Agents

Antiviral drugs inhibit specific stages of viral replication because viruses use host-cell machinery and have few independent metabolic targets.

• Attachment and entry inhibitors: Prevent a virus from binding or entering its host cell.
• Polymerase inhibitors: Nucleoside analogues can terminate viral DNA or RNA synthesis after incorporation.
• Protease inhibitors: Prevent cleavage of viral polyproteins into functional components.
• Neuraminidase inhibitors: In influenza, drugs such as oseltamivir reduce release of newly formed virions.
• Limitations: Viruses replicate inside host cells, so drug toxicity and viral resistance are major concerns; combination therapy can reduce the chance of resistant mutants dominating.

V. Endospores: Types and Bacterial Sporulation

Endospores are dormant, highly resistant structures formed inside certain bacterial cells, mainly species of Bacillus and Clostridium. They are survival structures, not reproductive cells: one vegetative cell forms one endospore.

A. Types of Endospores

Endospores are classified by their position within the bacterial cell and their effect on cell shape.

• Central spores: Located near the middle of the cell; an example is the central endospore of some Bacillus species.
• Subterminal spores: Located near one end but not at the extreme pole.
• Terminal spores: Located at the end of the cell; Clostridium tetani forms a terminal spore that gives a drumstick-like appearance.
• Structural features:
  • Core: Contains DNA, ribosomes, enzymes, calcium dipicolinate, and very little water.
  • Cortex: Specialized peptidoglycan layer that supports dehydration.
  • Coat: Protein layers resist chemicals and enzymes.
  • Exosporium: Outer covering present in some species.
• Resistance basis: Dehydration, DNA-protective proteins, calcium dipicolinate, and thick protective layers help endospores withstand heat, drying, radiation, and chemicals.

B. Stages of Sporulation in Bacteria

Sporulation begins when nutrients, especially carbon or nitrogen sources, become limited and proceeds through coordinated cellular changes.

• Stage I—chromosome replication: The bacterial chromosome replicates, and the cell prepares for asymmetric division.
• Stage II—forespore septum: An asymmetric septum forms near one pole, producing a smaller forespore and a larger mother cell compartment.
• Stage III—engulfment: The mother-cell membrane surrounds the forespore, leaving it enclosed by two membranes.
• Stage IV—cortex formation: A thick, specialized peptidoglycan cortex develops between the forespore membranes.
• Stage V—coat formation: Protein layers assemble around the developing spore; calcium dipicolinate accumulates and the core dehydrates.
• Stage VI—maturation: The endospore becomes metabolically dormant and highly resistant.
• Stage VII—release: The mother cell undergoes lysis, releasing the mature endospore into the environment.
• Germination: When nutrients and favorable conditions return, activation is followed by germination, water uptake, cortex breakdown, and outgrowth into a vegetative bacterial cell.