MCB 150 Methods Used in the Study of Microorganisms

• To understand the structure and functioning of ecosystems


  • Identities of organisms
  • Requires quantitative information about:

  1. Number of organisms
  2. Biomass of population
  3. Rate of activity
  4. Cycling and transfer rates of materials
• It is rarely feasible to subject the whole ecosystem to enumeration and measurement procedures!
• Microbial Sampling Methods


  1. Undisturbed environment

  2. Subsamples

  3. Pooled

  4. Mixed sample

  5. Dilution or Concentration

  • Studies of metabolic activity (additional step: Addition of substrates)
  • Cultural studies
  • Direct Examination
  • Molecular studies
• Each determination consists of three phases

  • Sample collection
  • Sample processing
  • Actual measurements
• Samples


  • Soil samples
  • Water samples
  • Air samples
  • Biological samples
• All procedures must be taken into consideration when results are interpreted for measurements are profoundly influenced by the manner of sample collection and processing.
• Sample Collection


  • Different approaches are being used for sampling microorganisms from different environments
  • The method to use is determined by:

  1. Physical and chemical properties of the ecosystem
  2. The expected abundance of microorganisms
  3. The enumeration or measurement procedures to be performed
• Microbial Sampling Methodsfor AIR


  • Environment - Air
  • Access - Direct
  • Numbers - Low
  • Sampling Devices - Filters, Andersen samplers
• Planning/Construction of Sampling Design


  • What to take (air, soil, water)
  • What to analyze
  • Where to take samples
  • Who will take samples (sample custody)
  • When to take samples
  • How often samples will be taken
  • How to take samples
  • How many samples to take
  • How much sample is needed
  • How to preserve samples
  • How long the sample will be stable
• • Sampling procedures to be used must ensure that:

  1. The number or activity of microorganisms are not altered in a non-quantifiable manner
  2. Samples collected are not contaminated with foreign microorganisms
  3. Samples collected are representative of the whole ecosystem being sampled
• In many environments, the distribution of microorganisms is not homogenous but patchy.
• Composite sampling - individual samples are obtained, bulked together and then mixed

  • Valid on certain conditions:

  1. equal number and amount of individual samples are used
  2. no interactions exist among individual sampling units
  3. only objective of the study is to obtain an unbiased estimate of the mean
• Soil Sampling


  • As source of microorganisms, aseptic technique is not important
  • Study the natural state of soil population, materials to use must be sterile
• Devices commonly used for soil sampling

  • Soil probe or augers
  • Shovels
  • Grabs
• Buried Slide Technique or Bulb Planter Technique

  • Uses glass slides, E.M. grids or flattened capillary tubes
  • Cellutape/scotch tape
• Suggested Steps in Soil Sampling

  • Establish experimental objectives or hypothesis to be tested
  • Develop a sampling plan
• Develop a Sampling Plan

  • Evaluate the environment where samples will be obtained

  1. Determine the history of the experimental area
  2. Determine the salient physical and biological features of the experimental area (slope, elevation, vegetation)
  3. Describe the soil
• Amount of soil sample to be collected


  • Small sample size (up to 100 g)
  • Medium sample size (100g to several kilograms)
  • Large sample size (over several kilograms)
• Soil Sampling Depth


  • Plough layer (0 to 25 cm deep)
  • Densely rooted layer (0 to 10 cm deep)
• Containers to use for soil samples

  • Plastic bags (0.025 mm)
  • Sealable glass or rigid plastic container for anaerobes
• Samples should be kept in the same physical, chemical and biological state as they were in situ.
• Sample Transport


  • Samples should be brought to the laboratory as soon as possible
  • Transport at ambient temperature is preferable if analysis takes place shortly after transport
• Samples used for characterization of numbers or activities of organisms should be used as quickly as possible after collection!
• Sample Storage


  • Air-drying prior to storage leads to reduction of microbes proportional to the length of storage; increased surface acidity due to reduced Mn and increased solubility and oxidizability of soil organic matter; has a differential effect on the composition of microbial communities
• Water Sampling


  • Involves both direct and remote sampling
  • Choice of apparatus is determined by: sampling location and conditions, information sought
  • Requirements of a good sampling device: robust, capable of being sterilized, constructed of an inert material, capable of collecting sufficient volume of sample
• Types of Water


  • Groundwater - freshwater beneath the earth’s crust; in layers called aquifers; pumped to the surface by building wells
  • Surface water - open to the atmosphere and results from overland flow; freshwater like lakes and rivers •marine
• Sampling Devices for Water

  • Van Dorn water sampler, Plankton nets (for enumeration of algae and protozoa)
  • Niskin water bottle, J-Z water bottles, rat-trap bottles, glass slides (for collection of bacterial populations)
  • Corers, grabs (for sediment samples)
• Parameters Measured During Water Sampling

  • Temperature
  • Transparency
  • Organic matter content
  • Dissolved oxygen concentration
  • pH
  • Salinity
  • Ca, N, and P content
  • Current flow rate
• Water Sample Storage and Transport

  • Samples should be brought to the laboratory as soon as possible
  • Transport on ice
  • Process samples as soon as you get to the lab
  • Store at 4°C, dark
  • Max storage time: 6 hours for bacteria from surface sources, 24-30 hours for drinking water, 4 days for Giardia and other pathogenic protists
• Air Sampling


  • Commonly referred to as bio aerosol sampling
  • Sampling is usually coupled with processing
  • Objective: Efficient removal and collection of biological particles from the air in a manner which does not affect the ability to detect the organism
• Methods of Air Sampling

  • Passive sampling - by gravity or downward molecular diffusion (Gravity slide method, Gravity plate method)
  • Inertial Sampling (Impaction methods, Centrifugal methods, Filtration methods)
• Impaction


  • Particles collide and stick onto the surface
  • Air jet is directed over the impaction plate
• Types of samplersfor IMPACTION METHOD


  • Slit samplers
  • Sieve samplers
• Centrifugal methods


  • Aerosol is spun in a circular path at high velocity such that particles impact onto a collecting surface with a force proportional to the particle's velocity and mass (e.g. Reuters Centrifugal Sampler)
• Filtration methods


  • Use membrane filters to trap suspended particles
• Commonly used filtersfor FILTRATION METHOD of AIR


  • Sodium alginate
  • Glass fiber
  • Gelatin membrane filters
  • Synthetic membrane filters
• Impingement methods


  • Use of liquid to collect particles from the air
  • Quantitation is accomplished by dilution plating or membrane filtration
• Liquid media commonly usedfor IMPINGEMENT METHODS of AIR

  • Buffered gelatin
  • Peptone water
  • Tryptose saline
  • Nutrient broth
• Thermal precipitation


  1. Dust-laden air stream flows past a heated wire (100 deg C)

  2. Particles collected on glass slides for examination
• Electrostatic precipitation


  A uniform electrostatic charge is imparted to incoming airborne particles which are then collected on an oppositely charged collecting surface