Chapter 7 — The Control of Microbial Growth

Key Terminology

• Sepsis – presence of microbial contamination in blood/tissues; associated with systemic inflammatory response.
• Asepsis – absence of significant contamination; goal of medical/surgical practice.
  • Aseptic technique in surgery: flame sterilization of instruments, disinfected field, gloved/ gowned staff → minimizes wound infection.
• Sterilization – complete removal/destruction of all life-forms (incl. endospores & viruses).
  • Commercial sterilization – heat treatment (e.g., in canning) sufficient to destroy Clostridium botulinum endospores without ruinous food damage.
• Disinfection – destruction of pathogenic (non-endospore) microbes on inert surface.
• Antisepsis – disinfection of living tissue/skin.
• Degerming – mechanical removal of microbes from limited area (e.g., alcohol wipe before injection).
• Sanitization – lowering microbial counts on utensils to safe public-health levels.
• Biocide / Germicide – agent that outright kills microbes.
• Bacteriostasis – inhibition of growth and metabolism; organisms remain viable if agent removed.

Kinetics of Microbial Death

• Exponential (logarithmic) death: during control treatment, equal percentages of the population die per equal time unit.

  • If 90 % of cells die each minute, survivors after n minutes = N_0(0.1)^n.
  • Practical implication: complete sterilization requires allowing sufficient multiples of decimal reductions (see D-value).

• Thermal constants

  • Thermal Death Point (TDP) – lowest temperature that kills all microbes in a suspension in 10 min.
  • Thermal Death Time (TDT) – minimal time required to kill all cells at a given temperature.
  • Decimal Reduction Time (D-value, DRT) – minutes to achieve 90\% reduction (1-log drop) at given temperature; critical in food industry.

Factors Influencing Antimicrobial Effectiveness

• Initial number of microbes – larger populations require longer or more intense treatment.
• Environmental conditions
  • Presence of organic matter (blood, vomitus, biofilm matrix) protects microbes & inactivates disinfectants.
  • Temperature: higher T° often increases action of chemicals; cold may protect microbes.
  • Biofilms: extracellular polymeric substance retards penetration; mixed-species synergy.
• Time of exposure – some agents (e.g., glutaraldehyde) need hours for endospore effect.
• Microbial characteristics – inherent resistance hierarchy (see comparative list).

Mechanisms of Action for Control Agents

• Alteration of membrane permeability – damage → leakage of vital components & lysis.
• Protein damage/denaturation – loss of 3-D structure → enzyme inactivation, lethal metabolism halt.
• Nucleic-acid damage – breaks, mutations, or cross-linking prevent replication & protein synthesis (e.g., UV-induced thymine dimers).

Physical Methods of Microbial Control

1. Heat

• Moist Heat – coagulates/denatures proteins.
  • Autoclave: saturated steam under pressure (standard: 121^{\circ}\text{C},\ 15\,\text{psi},\ 15\,\text{min}) → sterilizes culture media, instruments, dressings.
  • Pasteurization – reduces spoilage/pathogens in beverages.
  • Classical batch: 63^{\circ}\text{C},\ 30\,\text{min}.
  • HTST: 72^{\circ}\text{C},\ 15\,\text{s} (milk industry).
  • UHT: 140^{\circ}\text{C}<1\,\text{s} → shelf-stable products.
  • Thermoduric microbes (e.g., Lactobacillus) may survive but not pathogenic.
• Dry Heat – kills by oxidative destruction.
  • Direct flaming (loops, needles).
  • Incineration (contaminated dressings, carcasses, anthrax spores).
  • Hot-air oven: 170^{\circ}\text{C},\ 2\,\text{h} (equivalent to autoclave standard in lethality).

2. Filtration

• Physically removes microbes from heat-sensitive liquids/gases.
  • Membrane filters with 0.22\,\mu\text{m} or 0.45\,\mu\text{m} pores (viruses require nanofilters).
  • HEPA filters in biosafety hoods remove >99.97\% of particles ≥ 0.3\,\mu\text{m}.

3. Low Temperature

• Refrigeration (≈4^{\circ}\text{C}) – bacteriostatic for most pathogens.
• Deep-freezing (≈-50^{\circ}\text{C} to -95^{\circ}\text{C}) – long-term culture preservation.
• Lyophilization – freeze-drying; water removed by sublimation under vacuum.

4. High Pressure (Pascalization)

• 50$–800\,\text{MPa} disrupts protein conformation → cell death; preserves flavor (used for deli meats, juices).

5. Desiccation & Osmotic Pressure

• Removal of water (drying, freeze-drying) halts metabolism; many pathogens can survive dormant (e.g., M. tuberculosis on fomites).
• High solute (salt/sugar) causes plasmolysis; basis of jams, salted fish.

6. Radiation

• Ionizing (γ-rays, X-rays, high-energy e⁻ beams) – produces hydroxyl radicals → breaks chromosomes; used for disposable medical supplies, spices.
• Non-ionizing UV (200–300 nm) – forms thymine dimers; most germicidal at 260\,\text{nm}.
  • Limited penetration; used for surface/air sterilization, biosafety cabinets.
• Microwaves – heat produced kills indirectly; not reliable for unevenly distributed microbes.

Chemical Methods of Control

Principles of Effective Disinfection

• Concentration/strength of agent (often logarithmic relation to kill rate).
• Presence of organic matter (blood, feces) may neutralize chemicals.
• pH: certain agents (e.g., phenolics) more active at acidic pH.
• Time of exposure: spores & mycobacteria need longer.

Standard Evaluation Protocols

• Use-dilution test (regulatory reference method)
  1. Stainless-steel rings dipped in standardized bacterial suspension → dried.
  2. Rings immersed in disinfectant 10\,\text{min},\ 20^{\circ}\text{C}.
  3. Rings transferred to recovery broth; growth assessed.
• Disk-diffusion assay – chemical-soaked paper disks on agar lawn; zone of inhibition diameter indicates relative efficacy.

Major Classes of Disinfectants / Antiseptics

• Phenol (carbolic acid)
  • First surgical antiseptic (Lister); rarely used now due to irritation & odor.
• Phenolics (e.g., O-phenylphenol, Lysol®)
  • Derivatives with -\text{Cl} or -\text{CH}_3 groups; remain active in organic matter; disrupt membranes & proteins.
• Bisphenols
  • Hexachlorophene – surgical scrub; active vs staphylococci/streptococci.
  • Triclosan – consumer soaps/plastics (targets fatty-acid synthesis); resistance emerging.
• Biguanides
  • Chlorhexidine – broad-spectrum; binds skin/mucosa, low toxicity → mouthwashes, pre-op scrub.
• Halogens
  • Iodine: tincture (in alcohol) or iodophor (povidone-iodine); impairs protein synthesis, oxidizes cell components.
  • Chlorine: forms hypochlorous acid \text{HOCl} in water; strong oxidizer.
  • Household bleach ≈5\% NaOCl; swimming pools, drinking water.
• Alcohols
  • Ethanol 70\%, isopropanol 70$–90\% – protein denaturation + lipid dissolution; require water for action; not sporicidal.
• Heavy Metals (Oligodynamic action)
  • \text{Ag}^+, \text{Hg}^{2+}, \text{Cu}^{2+} bind sulfhydryl groups → protein denaturation.
  • 1\% AgNO₃ previously for neonatal ophthalmia; now replaced by antibiotics.
• Surface-Active Agents (Surfactants)
  • Soaps – anionic acids; mechanical emulsification (degerming).
  • Acid-anionic detergents – sanitizers in dairy; react with plasma membrane.
  • Quaternary ammonium compounds (quats) – cationic; disrupt membranes, denature proteins; ineffective vs spores, Pseudomonas.
• Chemical Food Preservatives
  • Organic acids (sorbic, benzoic, propionic) inhibit mold metabolism; safe at \text{pH}<5.
  • Nitrites/nitrates in cured meats stabilize red color & inhibit C. botulinum spore germination.
  • Bacteriocins (nisin) & antifungals (natamycin) for cheese.
• Aldehydes
  • Glutaraldehyde (2 % cidex) – sterilant for heat-labile endoscopes; cross-links \text{–NH}_2,\ –SH,\ –OH groups.
  • Formaldehyde – gas or 37 % formalin; tissue fixative, vaccine preparation.
• Gaseous Sterilants
  • Ethylene oxide – alkylates DNA/proteins; penetrates packaging; requires aeration (carcinogenic/explosive).
  • Propylene oxide, β-propiolactone, chlorine dioxide (line disinfection ↔ anthrax spores in US mail).
• Peroxygens (Oxidizing Agents)
  • Hydrogen peroxide \text{H}2\text{O}2 (antiseptic, vapor sterilization in hospitals).
  • Ozone \text{O}_3 – water disinfection.
  • Peracetic acid – effective on endospores & viruses; food processing equipment.

Comparative Resistance of Microorganisms

(Decreasing order of resistance to chemical & physical controls)

1. Prions – infectious proteins; require incineration or >1\,\text{N}\,\text{NaOH}+\text{autoclave}.
2. Bacterial endospores – thick keratin cortex; survive boiling, many disinfectants.
3. Mycobacteria – waxy mycolic acid cell wall; resist drying & weak chemicals.
4. Protozoan cysts – wall-protected dormant form.
5. Vegetative protozoa
6. Gram-negative bacteria – outer membrane limits drug entry; porins selective.
7. Fungi & most fungal spores
8. Non-enveloped (naked) viruses – capsid resists detergents.
9. Gram-positive bacteria – thick peptidoglycan but no outer membrane.
10. Enveloped viruses – lipid envelope easily disrupted by soaps & alcohol.

Snapshot: Chemical Agent vs Hard-to-Kill Forms

• Phenolics – poor vs spores; good vs mycobacteria.
• Quats – none vs spores & mycobacteria.
• Chlorines – fair vs both.
• Alcohols – poor vs spores; good vs mycobacteria.
• Glutaraldehyde – fair vs spores; good vs mycobacteria (with prolonged exposure).

Practical & Ethical Considerations

• Over-use of triclosan/quats in consumer goods → selective pressure for resistant strains; environmental persistence.
• Food irradiation raises consumer perception issues despite demonstrated safety & nutrient retention.
• Ethylene oxide is a potent sterilant but toxic/carcinogenic → facilities must balance efficacy with worker safety.
• Global health: inexpensive bleach, filtration & chlorination drastically lower diarrheal disease in resource-limited settings.

Quick Reference Equivalencies & Formulas

• Moist vs dry heat equivalence:
  • 121^{\circ}\text{C},\ 15\,\text{min} (autoclave steam) ≈ 170^{\circ}\text{C},\ 120\,\text{min} (dry hot-air).
• D-value equation: D = \frac{t2 - t1}{\log{10}N1 - \log{10}N2} where N = survivors.
• Relationship of decimal reductions to sterilization assurance:
  • Probability of survivor after n D-values: 10^{-n}.