Methods used in the study of microorganisms:

1. Know identity of organisms

2. Require quantitative information about
a. Number of organisms
b. Biomass
c. Rate of activity (O2 uptake and CO2 evolution)
d. Role in cycling and transfer rates of materials

Undisturbed environment can be directly examined. It can be studied for metabolic activity if enriched.
Subsamples are pooled from undisturbed environment to get mixed samples which are either diluted or
concentrated. These diluted or concentrated samples are either studied by direct examination,
molecular, cultural or metabolic activity.

Each determination consists of 3 phases:

1. Sample collection (soil, water, air, bio)

2. Sample processing
3. Actual measurements

All procedures must be taken into account when results are interpreted for measurement. This is
influenced by sample collection and processing.

Sample collection:

1. Physical and chemical properties of the ecosystem

2. Expected abundance of microorganisms
3. Enumeration or measurement procedure to use

Environment Access Number Sampling device

Air Direct Low Filters/ Andersen
Water Direct/remote High or low Net, filters, cont?
Sediment Remote High Grab, corer
Soil Direct High Shovel, corer

Sampling devices or procedures must ensure:

1. The number or activity of microbes are not altered in a non-quantifiable manner

2. Sample collected are not contaminated with foreign microbes
3. Samples collected are representative of the whole ecosystem being sampled

Sterilized devices are important in counting studies to ensure that the organism is not a contaminant but
are from the ecosystem.

Types of Sampling:

1. Probability sampling – mathematical approach and commonly used to get representative

samples
 a. Simple random sampling – equal chance of selection
b. Systematic sampling – stipulations made to further make the procedure highly selective
c. Stratified sampling – if it is a big area, must get equal distribution
2. Non probability sampling
a. Judgement sampling – deliberately avoid areas not representative of the entire area
b. Convenience sampling – use samples which are available
c. Quota sampling – a given number of sample is obtained
3. Composite sampling
a. Varying ph etc.
b. Individual samples are obtained, bulked together and mixed

Composite sampling is valid on certain conditions:

1. Equal number and amount of individual samples are used

2. No interactions exist among individual samples
3. Objective is to obtain an unbiased estimate of the mean

Sampling from different environments:

1. Soil sampling
2. Water sampling
3. Air sampling

Soil sampling

 Source of microorganisms (aseptic technique not important)

 Study the natural state of soil population (materials to use must be sterile)
 Devices
o Shovel
o Grabs
o Soil probe
 Use glass slide to obtain direct samples

Rossi-cholodny buried slide technique

 Motile organisms can enter the flattened capillary tube (pedoscope)

 Cellutape / scotch tape

Steps:

1. Establish experimental objectives or hypothesis

2. Develop a sampling plan
a. Evaluate environment where samples will be obtained (ocular inspection
b. Determine the following
i. History of the experimental data
ii. Salient physical and biological features of the experimental area
1. Slope
2. Elevation
3. Vegetation
 iii. Description of the soil

Amount of sample to be collected?

 Small sample size (100grams)

 Medium sample size (100g to several kilograms)
 Large sample size (several kilograms)

Sampling depth

 Plough layer
 Densely rooted layer

Container

 Plastic bags used

 Sealable glass/ rigid plastic container (anaerobic)

Sample transport

 Samples brought to the lab as soon as possible

 If immediate processing, ice box not needed

Sample storage

 Samples for characterization of count / activity (used ASAP)

 Samples should be kept as in their in situ environment
 Do not air dry
o Causes reduction of microbes
o Increases surface acidity
 Lowers Mn, increases solubility and oxiditability of soil
 Differential effect on the composition of the microbial community

Temperature

 4 degrees celcius moist storage

 4 degrees ceclius dark. 3 weeks is acceptable to do research on:
o Total microbial biomass
o Counts of specific microbial group
o Available N
o Enzymatic activities

Water sampling

 Various methods are used to collect water samples

 Choice of apparatus determined by:
o Sampling location and conditions
o Information sought
 Requirements of a good sampling device
o Should be robust ( strong)
 o Capable of being sterilized
o Constructed of an inert material ( unreactive)
o Capable of collecting a sufficient volume of sample
o Does not affect samples adversely

Sampling devices for enumeration of algae or protozoa

1. Van Dorn water sampler (fiber glass)

a. Preservative used is formalin acetic acid
b. Lower at particular depth
c. Sampling of particular strata
2. Schindler plankton trap (quite heavy and used at varying depth)
3. Plankton nets

Sampling devices for bacterial populations

1. Niskin water bottle : get at a particular depth

2. JZ water bottles
3. Rat-kap bottles?
4. Glass slides

Sampling devices for sediment

1. Corers
a. Parameters during collection
i. Temperature
ii. Transparency
1. phototrophic organisms
2. Up to what level of light penetrates
iii. Dissolved oxygen concentration
iv. Organic matter content
v. pH
vi. salinity
vii. Ca, N and P conent
viii. Current flow rate
b. Samples must not be refrigerated until processed

Air sampling

 Bio aerosol sampling

 Objectives: efficient removal and collection of bio particles without affecting ability to detect
organism

Methods:

 Passive sampling
o Organisms collected by gravity
o Gravity slide method
 Not precise
  Not affected by aerodynamics
o Gravity plate method
 Affected by aerodynamic
 Precise
 Internal sampling
o Impaction methods
 Air jet directed over impaction plate
 Particles collide and stick onto surface
 Types
 SIH samplers
 Sieve samplers
o Centrifugal method
 Spin at high velocity
 Particles impact onto collecting surface
o Filtration method
 Use membrane filters to trap suspended particles
 Commonly used filters
 Glass
 Sodium alginate
 Gelatin membrane
o Thermal precipitation
 Dust-laded air stream flows past heated wire
 Particles collected on glass slide
o Electrostatic precipitation
 Uniform electrostatic charge is imparted to airborne particles

Choice of cm for bio aerosol sampling

1. Nutritional requirements of organism

2. Type of into desired
3. Sampling method
4. Sampling conditions

Biological samples (collection of vital fluids)

 Vital fluids
o Cerebrospinal fluid – use of lumbar puncture or spinal tap
o Total volume of 10ml
 Blood
o Use of venipuncture technique
o 10ml blood
o 1:10 (blood: cm) for inoculation
o Anticoagulants incorporated to cm
o Sap : drill holes into the trunk
 Excreted products
o Fecal material/stool
  Obtained using wide-mouthed
 Preservative for stool sample is 10% formalin and low viscosity polyvinyl alcohol
 Rectal swabs
 Processed immediately
o Urine sample
 Use midstream clean catch method
 Use of suprapubic aspiration of the urinary bladder
 Container must be dry
o Sputum sample
 10 ml preferred; pooled if not enough
 Translaryngeal/ transtracheal aspiration
 Fail to observe causative organism
 Aerobic pulmonary infection
 Exudates
o Wound exudates – needle/ swab
o Aspiration of pusulent? material from depth of wound
 Tissues
o Surface tissues: rinse method
o Biopsy

Specimen transport

 Maintain sample near original state

 Transport media (not rich media)
o Salt
o Buffer
o Reducing agent
 Remove oxygen
 For anaerobic organisms
o Solidifying agent
 Prevent spilling
 Packaging that withstand pressure changes

Transport media

1. Carry and blair medium – medium for feces that may contain salmonella, shigella, vibrio
2. Arnies medium with charcoal – isolate fastidious organisms; help eliminate metabolic products
of bacterial growth
3. Stuart’s medium- used for transport of specimens suspected of gonococci; throat, vaginal,
wound and skin swabs with fastidious organisms
4. Anaerobic transport medium – maintain viability of anaerobic bacteria; viability without
significant multiplication (trioglycolate broth)
5. Venkat ramakrishunan for vibrio cholera