ISOLATION AND CULTIVATION OF MICROORGANISMS (Module 6-7)

• Binary fission


  Method by which prokaryotes produce new individuals that are genetically identical to the parent organism

  
• Prokaryotes, such as bacteria, propagate by binary fission

  
• For unicellular organisms, cell division is the only method used to produce new individuals
• In both prokaryotic and eukaryotic cells, the outcome of cell reproduction is a pair of daughter cells that are genetically identical to the parent cell
• In unicellular organisms, daughter cells are individuals
• Bacterial chromosome


  • Attached to the plasma membrane at about the midpoint of the cell
  • Replication is bidirectional, moving away from the origin on both strands of the loop simultaneously
• Binary fission


  1. Chromosome replication

  2. Chromosome movement to opposite ends of the cell

  3. Cytoplasmic separation

  4. Formation of septum

  5. Separation of daughter cells

  
• FtsZ


  First protein to move to the division site, essential for recruiting other proteins that produce a new cell wall (septum) between the dividing cells
• FtsZ proteins can form filaments, rings, and other three-dimensional structures that resemble the way tubulin forms microtubules, centrioles, and various cytoskeletal components
• FtsZ uses GTP (guanosine triphosphate), to rapidly assemble and disassemble complex structures
• Generation time


  Time it takes for a population of bacteria to double in number
• For many common bacteria, the generation time is quite short, 20-60 minutes under optimum conditions
• For most common pathogens in the body, the generation time is probably closer to 5-10 hours
• Bacterial growth curve


  • Lag phase
  • Exponential or log phase
  • Stationary phase
  • Death or decline phase

  
• Lag phase


  Adaptation period where bacteria are adjusting to new conditions

  
• Exponential or log phase


  Marked by predictable doublings of the population
• Stationary phase


  Bacterial population runs out of essential nutrient/chemical or growth is inhibited by waste products or lack of space
• Death or decline phase


  Number of viable cells decreases in a predictable (or exponential) fashion
• Viable but nonculturable (VBNC) state might be of importance for pathogens, where they enter a state of very low metabolism and lack of cellular division, only to resume growth at a later time, when conditions improve
• 100% cell death is unlikely, for any cell population, as the cells mutate to adapt to their environmental conditions, however harsh
• Often there is a tailing effect observed, where a small population of the cells cannot be killed off
• Direct cell count


  Counting cells in a liquid culture or colonies on a plate
• Viable count


  Count of viable or live cells based on colonies formed on plates
• Plate count


  1. Pour plate method

  2. Spread plate method
• Pour plates allow the identification of bacteria as aerobes, anaerobes or facultative aerobes

  
• Spread plates allow the isolation of specific clonal colonies
• Colony-forming units per milliliter (CFU/mL)


  Measure of viable cells, as more than one cell may have landed on the same spot to give rise to a single colony
• Inoculating plates for viable counts

  1. Pour plate method

  2. Spread plate method
• Pour plate and spread plate


  • Techniques that quantify bacterial samples
  • Require Petri dishes and nutrient agar
• Pour plate


  • Method for counting the number of colony-forming bacteria present in a liquid specimen
  • Allows identification of bacteria as aerobes, anaerobes or facultative aerobes

  
• Spread plate


  Allows the isolation of specific clonal colonies
• Difference between pour plate and spread plate


  • Pour plate - molten agar is poured on to the inoculum
  • Spread plate - inoculum is spread on the surface of the solidified agar
• Inoculum


  Microorganisms, bacteria or fungi that grow in or on the nutrient agar
• Serial dilution


  1. Important first step before proceeding to either the pour plate or spread plate method

  2. Goal is to obtain plates with CFUs in the range of 30–300

  3. Usually involves several dilutions in multiples of 10 to simplify calculation

  4. Number of dilutions chosen according to preliminary estimate of culture density
• Membrane filtration technique


  • Effective, accepted technique for testing fluid samples for microbiological contamination
  • Involves less preparation than many traditional methods
  • Allows the isolation and enumeration of microorganisms
  • Membrane filters used extensively in the laboratory and industry to sterilize fluid materials

  
• Most probable number (MPN)


  • Statistical method used to estimate the viable numbers of bacteria in a sample by inoculating broth in 10-fold dilutions
  • Based on the principle of extinction dilution
  • Often used in estimating bacterial cells in water and food

  
• Dry weight technique


  1. Microorganisms removed from medium by filtration

  2. Microorganisms on filters washed to remove all extraneous matter

  3. Dried in desiccator by putting in weighing bottle (previously weighed)

  4. Dried microbial content then weighed accurately
• Dry weight technique


  • Especially useful for measuring the growth of micro fungi
  • Time consuming and not very sensitive as bacteria weigh so little, requiring centrifugation of several hundred millions of culture to find sufficient quantity to weigh
• Measurement of nitrogen content


  1. As microbes (bacteria) grow, there is an increase in the protein concentration (i.e. nitrogen concentration) in the cell

  2. Cell mass can be subjected to quantitative chemical analysis methods to determine total nitrogen that can be correlated with growth
• Turbidimetric estimation (turbidometry)


  • Rapid cell mass determination method
  • Based on the fact that microbial cells scatter light striking them
  • Amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number
  • Increase in cloudiness (turbidity) of the medium as bacterial concentration increases
  • Turbidity can be measured using instruments like spectrophotometer and nephelometer