bc

• Diagnostic bacteriology aims to identify the bacterial species involved in the infection resulting in the patient’s signs and symptoms
• Direct laboratory methods such as microscopy provide preliminary information about the bacteria involved in an infection
• Data provided in direct examination of the specimen are not enough to identify the causative agent of the infection, further characterization is needed
• Cultivation is the process of growing microorganisms in culture by taking bacteria from the infection site and growing them in the laboratory
• Nutrients and environmental requirements must be provided in vitro to support the growth of bacteria
• Culture media are anything (solid or liquid) that possess nutritional and environmental requirements for bacterial growth
• Complex medium:

  • Exact composition and amounts of individual components are not exactly known
  • Useful for meeting the nutritional requirements of many different microorganisms
  • Often needed for bacteria with complex nutritional or cultural requirements
• Defined medium:

  • Specific chemical composition is known and components are weighed out exactly
  • Used for culturing specific microorganisms with known nutritional needs
  • Often used in research to understand what the experimental microorganism is metabolizing
• Broth (liquid) phase:

  • Nutrients are dissolved in water
  • Bacterial growth indicated by a change in appearance from clear to turbid
  • Thioglycolate broth supports the growth of both aerobic and anaerobic bacteria
• Solidified media:

  • Solidifying agent (1.5%) added to the nutrient, agar is commonly used
  • Agar is a sulfated polymer extracted from red algae, well-suited for solidifying media
  • Agar melts at about 90°C and resolidifies at about 45°C
• Biphasic media contain both liquid and solid phases
• Types of Culture Media:

  • Supportive/Nutritive: Supports the growth of most non-fastidious bacteria
  • Enrichment: Contains added growth factors like blood, vitamins, and yeast extract
  • Selective: Contains additives to inhibit the growth of certain bacteria, e.g., gram-positives
• Sterilization:

  • Delicate media components that cannot withstand autoclaving can be sterilized by membrane filtration (0.2 to 0.45 μm pore)
• Different types of culture media used in microbiology include Columbia colistin-nalidixic acid agar (CNA), eosin methylene blue (EMB), MacConkey, Hektoen enteric (HE), and xylose lysine deoxycholate (XLD)
• Culture media are formulated to inhibit the growth of certain bacteria, such as gram-positives, and provide distinct colonial appearances based on biochemical reactions
• Phases of culture media preparation:

  • Measurement of the desired quantity of dehydrated culture medium
  • Reconstitution of the dehydrated culture medium
  • Sterilization by autoclaving
  • Distribution of the reconstituted culture medium into tubes
  • Laying the tubes down the slant rack
• Principles of Bacterial Cultivation:

  • Grow and isolate all bacteria present in a clinical specimen
  • Determine which bacteria are causing infection and which are contaminants
  • Obtain sufficient growth for identification, characterization, and susceptibility testing
• Primary culture media selection:

  • Agar plates are commonly used
  • Blood agar medium is common for primary isolation media
  • MacConkey agar or Eosin methylene blue is used to inhibit gram-positive organisms
  • Broth media limited to certain specimens
  • Enrichment broth used to recover pathogenic organisms from specimens with a heavy concentration of commensal organisms
• Inoculation process:

  • Inoculation is the process of introducing microorganisms into a culture medium
  • Quantitative inoculation involves plate count or quantitative inoculating loop
  • Semiquantitative inoculation involves quadrant streaking
  • Considerations for inoculating primary culture media vary based on specimen type
• Inoculation directly from a plate results in the most numerous and largest aggregates in liquid batch cultures
• Large aggregates impact cultures' tolerance toward tobramycin, showing the profound impact of inoculation method on antibiotic tolerance
• Preformed aggregates recruit single cells in a "snowball effect," building up aggregated biomass in the culture, relying heavily on the exopolysaccharide Psl
• Both Escherichia coli and Staphylococcus aureus produce aggregates in liquid batch cultures
• Pure liquid cultures are normally homogeneous mixtures of single-cell bacteria, but aggregation can significantly change the outcomes of experiments
• Aggregates in liquid cultures may be a challenge for microbiologists
• Incubation process involves incubating inoculated media under specific temperatures and environmental conditions depending on the organisms being sought
• Incubators should be carefully controlled for temperature with minimal fluctuations and humidity control is important for optimal growth
• Aerobic incubation provides ambient air with 21% oxygen and a small amount of carbon dioxide, while capnophilic incubation provides increased CO2 concentration
• Anaerobic incubation chamber contains specific percentages of hydrogen, carbon dioxide, nitrogen, and no oxygen
• Anaerobic jar principle involves the removal of oxygen by reaction with hydrogen in the presence of a catalyst
• Anaerobic glove box is a self-contained system for processing specimens without exposure to air
• Roll-streak system uses PRAS media and L-cysteine hydrochloride to maintain low oxidation-reduction potential
• Anaerobic disposable plastic bag system generates an atmosphere when water is added to the H2-CO2 gas generator
• Incubators are based on the principle that microorganisms require specific parameters for growth and development
• An incubator is based on the principle that microorganisms require a particular set of parameters for their growth and development
• All incubators are based on the concept that when organisms are provided with the optimal condition of temperature, humidity, oxygen, and carbon dioxide levels, they grow and divide to form more organisms
• In an incubator, the thermostat maintains a constant temperature that can be read from the outside via the thermometer
• The temperature is maintained by utilizing the heating and no-heating cycles
• During the heating cycle, the thermostat heats the incubator, and during the no-heating period, the heating is stopped, and the incubator is cooled by radiating heat to the surrounding
• Insulation from the outside creates an isolated condition inside the cabinet, which allows the microbes to grow effectively