Microbiology 1

History of Microbiology

• Lucretius and Girolamo Fracastoro suggested that diseases were caused by "invisible living creatures" (1478-1553)
• Anton van Leeuwenhoek, the "first true microbiologist" and "Father of Bacteriology and Protozoology", used the term "animalcules" or "beasties" (1632-1723)
• John Needham suggested that organic matter possessed a "vital force" that could give rise to life (1731-1781)
• Francesco Redi invalidated abiogenesis, proving that maggots cannot rise spontaneously from decaying meat (1626-1697)
• Lazzaro Spallanzani improved on Needham's experiments by heating the broth in a sealed jar, observing no growth (1729-1799)
• Rudolf Virchow challenged the doctrine of spontaneous generation with the concept of biogenesis (1821-1902)
• Louis Pasteur disproved the doctrine of spontaneous generation, developed vaccines for anthrax and rabies, and improved the wine-making process (1822-1895)

Germ Theory of Disease

• Ignaz Semmelweis advocated for handwashing to prevent the spread of puerperal fever (1818-1865)
• Joseph Lister used phenol to prevent surgical wound infections (1827-1912)
• Robert Koch discovered Bacillus anthracis and Mycobacterium tuberculosis, showing that bacteria indeed cause diseases (1843-1910)

Exceptions to Koch's Postulate

• Many healthy people carry pathogens but do not exhibit symptoms of the disease
• Some microbes are difficult or impossible to grow in vitro in artificial media
• Introducing a pure culture to the experimental animal, the animal must be susceptible to the pathogen
• Use of human volunteers is difficult to find and has ethical considerations

Vaccination

• Buddhist monks drank snake venom to confer immunity to snake bite
• Variolation was practiced in 17th century China
• Edward Jenner inoculated a person with cowpox virus, resulting in protection against smallpox in 1796
• Louis Pasteur developed the first vaccine against smallpox in 1798
• Paul Ehrlich developed a synthetic arsenic drug, Salvarsan, to treat syphilis in 1910
• Sulphonamides were synthesized in the 1930s
• Alexander Fleming discovered the first antibiotic, Penicillin, in 1928

Cell Envelope Structures

• Contains plasma membrane (PM)
• Contains cell wall and some do not
• Surface polymers: capsule, slime layer, and other surface structures

Division of Microbiology

• Parasitology: study of parasites, their hosts, and the relationship between them
• Mycology: study of fungi, including their genetic and biochemical properties
• Phycology: study of algae
• Virology: study of viruses
• Bacteriology: study of bacteria

Bacterial Genetics

• Mechanisms of gene transfer: transformation, transduction, and conjugation
• Mutation: a change in the original nucleotide sequence of a gene or genes within an organism's genome
• Recombination: exchange of genes between homologous regions on two DNA molecules

Bacterial Metabolism

• Respiration: efficient ATP-generating process in which molecules are oxidized to generate energy
• Fermentation: anaerobic process in which glucose is converted into ATP and other products
• Types of fermentation: alcoholic, homolactic, heterolactic, mixed acid, and butanediol

Microbial Growth and Nutrition

• Major nutritional needs: carbon, nitrogen, and energy
• Oxygen requirements: aerobic, anaerobic, facultative, and microaerophilic
• Temperature requirements: psychrophiles, mesophiles, and thermophiles
• pH requirements: neutrophiles, acidophiles, and alkaliphiles
• Other requirements: halophiles, capnophiles, and osmophiles

Common Stains for Microscopic Visualization

• Gram stain
• Acid-fast stains
• Acridine orange
• Calcofluor white
• Methylene blue
• Lactophenol cotton blue (LPCB)
• India ink
• Endospore stain

Methods of Studying Bacteria

• Goal of the specimen collector: maintain the viability of the organisms with minimal contamination
• Basic principles: select the correct anatomic site, collect the specimen using the proper technique and supplies, and package the specimen in a container or transport medium
• Collection procedures: stool, sputum, and other specimens
• Transportation of specimens: notify the laboratory in advance, and store the specimen in an environment that will not degrade the suspected organism/s### Laboratory Procedures
• Specimen collection:
  • Upper respiratory tract, external ear, eye, and genital tract: swab collection systems with transport media to prevent drying and maintain specimen integrity
  • Lesions, wounds, and abscesses: clean area, collect from advancing margin, and use needle aspiration instead of swab
• Urine specimen collection:
  • Clean-catch midstream urine specimen
  • First morning urine preferred
  • Sterile container with tight-fitting lid

Preservatives

• Urine and stool specimens can be preserved with:
  • Boric acid: maintains accurate urine colony counts at room temperature for 24 hours
  • Cary-Blair transport media: for delayed transportation (up to 2 hours) and refrigeration

Anticoagulants

• Used to prevent clotting of specimens (blood, bone marrow, and synovial fluid)
• Sodium polyanethol sulfonate (SPS): most common anticoagulant used for microbiologic specimens (concentration not exceeding 0.025% wt/vol)
• Heparin: often used for viral cultures and Mycobacterium spp. isolation from blood

Transport and Holding Media

• Stuart's or Amie's transport media: commonly used for preserving microorganisms during transportation
• JEMBEC system: contains selective agar and a CO2-generating tablet for N.gonorrhoeae specimens

Unacceptable Specimens and Specimen Rejection

• Reasons for rejection:
  • Information mismatch on requisition and specimen label
  • Inadequate quantity or specimen transport time exceeding 2 hours without preservation
  • Specimen in formalin or dried up
  • Leaking or incorrect transport container

Microscopic Examination

• Gram staining: developed by Hans Christian Gram
• Errors in Gram staining: Gram (+) becomes Gram (-) and vice versa, due to acidic Gram's iodine or inadequate decolorization

Culture Media

• Categories: simple, differential, and probe-mediated stains
• Classification according to physical state, composition, and use
• Types: liquid, semisolid, and solid (agar, BAP, TSI)

Isolation Techniques

• General purpose isolation streak: used for initial isolation of microorganisms
• Quantitative isolation technique: used to determine the number of microorganisms in a sample

Culture Incubation

• Most bacteria cultures incubated at 35°C-37°C
• Some bacteria require specific conditions: capnophiles (Candle jar, CO2 incubator), microaerophiles (jars or bags)

Incubation Time

• Most bacterial cultures held for 48-72 hours
• Culture for anaerobes and broth cultures may be held for 5-7 days

Gross Colony Characteristics

• Hemolysis
• Size
• Form/margin
• Elevation
• Color
• Density
• Consistency
• Pigment
• Odor

Microbial Control

• Early civilizations: practiced salting, smoking, pickling, drying, and use of spices to control microbial growth
• Sterilization: killing or removing all forms of microbial life
• Disinfection: reduction of pathogenic microorganisms to prevent disease
• Antiseptics: applied to living tissue to prevent infection
• Degerming: mechanical removal of microbes in a limited area
• Sanitization: use of chemical agents to minimize disease transmission
• Physical methods: heat, radiation, and filtration
• Heat: kills microorganisms by denaturing enzymes and proteins
• Thermal Death Point (TDP): lowest temperature at which all microorganisms in a liquid suspension are killed in 10 minutes
• Thermal Death Time (TDT): minimal length of time in which all bacteria are killed at a given temperature
• Decimal Reduction Time (DRT): time in minutes at which 90% of bacteria are killed at a given temperature### Sterilization Methods
• Ultra High Temperature Pasteurization (UHT): Milk treated at 140°C for 3 seconds and then cooled quickly in a vacuum chamber, allowing storage at room temperature for several months.
• Tyndallization: Intermittent sterilization using free-flowing steam for 30 minutes on each of 3 successive days to kill all vegetative microorganisms and spores.

Radiation Methods

• Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy rays with short wavelengths (< 1 nanometer), high energy, and penetrating power, used to sterilize pharmaceuticals and medical supplies, and to retard spoilage in food.
• Nonionizing Radiation: Ultraviolet light with wavelengths (> 1 nanometer), used to disinfect operating rooms, nurseries, and cafeterias.
• Microwave Radiation: Wavelengths (1 millimeter to 1 meter), absorbed by water molecules, killing vegetative cells in moist foods, but not bacterial endospores.

Heat Methods

• Dry Heat: Kills by protein oxidation, used to sterilize inoculating loops and needles by direct flaming, and disposable items by incineration.
• Hot Air Sterilization: used to sterilize objects, requiring 2 hours at 170°C, with damages to skin, eyes, and some materials.

Filtration Methods

• Membrane Filters: Uniform pore size, used in industry and research, retaining all viruses and some large proteins.
• High Efficiency Particulate Air (HEPA) Filters: Used in operating rooms and burn units to remove bacteria from air.

Chemical Methods

• Phenols and Phenolics: Damage cell membranes and inactivate enzymes, used as antiseptics, disinfectants, and in mouthwashes and throat lozenges.
• Halogens: Effective against microorganisms, but may cause skin irritation, used to disinfect drinking water, pools, and sewage.

Osmotic Pressure and Desiccation

• Osmotic Pressure: Uses high concentrations of salts and sugars in foods to create a hypertonic environment, inhibiting microbial growth.
• Desiccation: Removes water from microorganisms, creating a static effect, not effective against bacterial endospores.

Gaseous Sterilizers

• Ethylene Oxide: Kills all microorganisms and endospores, requiring 4 to 18 hours, used to sterilize hospital instruments and mattresses.
• Peroxygens (Oxidizing Agents): Oxidize cellular components, disrupting membranes and proteins, used as antiseptics and disinfectants.