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Microbial Assessment of Air Quality.pptx
- 2. Enumeration of Microorganisms in Air • A number of methods have been devised for enumerating bacterial population in air. Methods can be grouped on two aspects. The rate at which bacteria carrying particles are settling by gravity from the air onto exposed surfaces. Large particles will settle due to gravitational force. Settle plate method. Counting the number of bacteria carrying particles in a given volume of air. Slit sampler.
- 3. In air sampling the separation of particles from air can be achieved by 1. Settling under gravity 2. Centrifugal action 3. Filtration 4. Impingement 5. Electrostatic Precipitation
- 4. • This method makes use of the settling of bacteria containing particles under gravity. Hesse's Tube Settle Plate Method
- 5. a) Hesse's Tube • This sampler is made of a horizontal glass tube • Contains a layer of solid medium at the bottom • As air is drawn in to the tube through the inlet end, the particles settle onto the medium. • Upon incubation colonies develop on the medium. • If the tube is long enough or the flow is sufficiently slow, almost all particles will settle out before reaching the outlet end.
- 6. b) Settle Plate Method Principle • The bacteria carrying particles are allowed to settle onto the medium for a given period of time and incubated at the required temperature. • Number of colonies formed indicates the number of settled bacteria containing particles.
- 7. • Petridishes containing an agar medium of known surface area are selected. • Agar surface should be dry without any moisture. • Choice of the medium depends upon the kind of microorganisms to be enumerated. • Blood agar - For an overall count of pathogenic, commensal and saprophytic bacteria in air. • Selective media - For detecting a particular pathogen • Malt extract agar - for molds. • Labelling of plates - Note the place and time of sampling, duration of exposure etc. • Then the plates are uncovered in the selected position for the required period of time.
- 8. • During sampling it is better to keep the plates about 1 metre above the ground. • Immediately after exposure for the given period of time, close the plates and incubate for o 24 hrs at 37°C for aerobic bacteria o 3 days at 22°C for saprophytic bacteria. o 1-2 weeks at 10-50°C for molds. • After incubation the colonies on each plate are counted and recorded as the number of bacteria carrying particles settling on a given area in a given period of time. • readily countable number of well isolated colonies, 30-100 colonies.
- 9. Air Centrifuge Reuter centrifugal air sampler
- 10. a) Air Centrifuge • Developed by Wells (1993). Principle • The particles from air are centrifuged onto the culture medium. • Sampled air was passed along a tube which was rotated rapidly on its long axis • The inner surface of the tube was lined with culture medium and any bacteria containing particle deposited on it • Produce colony up on incubation.
- 11. b) Reuter centrifugal air sampler • A modern version of air centrifuge • It resembles a large cylindrical torch with an open ended drum at one end. • The drum encloses impeller blades which can be rotated by battery power when switched on. • A plastic strip coated with culture medium can be inserted along the inner side of the drum. • Air is drawn into the drum and subjected to centrifugal acceleration. This causes the suspended particles to settle on the culture medium. • After sampling the strip is removed from the instrument and incubated at 37°C for 48 hours. • Later the colonies can be counted.
- 12. Advantages • Portable hence very convenient for transportation and use • Battery operated Disadvantage • Less efficient than the slit sampler in detecting particle below 5mm in diameter. • The size of the air being sampled cannot be accurately controlled.
- 13. • This is a simple method for collecting particles from air. • The filter can be made of any fibrous or granular material like sand, glass fibre and alginate wool (in phosphate buffer). o Tube sampler Millipore filter
- 14. a) Tube sampler • Oldest device for collecting and enumerating microorganisms in the air. • It consists of a tube with an inlet at the top and an outlet at the bottom which is narrower than the top end. • Near the bottom there is a filter of wet sand which is supported by a cotton plug below. • The entire device can be sterilized. • After sterilization the air to be sampled is allowed to pass through the sand and cotton. • Microorganisms as well as dust particles containing microorganisms in the air are deposited in the sand filter as the air passes through it. • Later the sand is washed with broth and a plate count is made from the broth by taking aliquotes of the broth.
- 15. b) Millipore filter • Made up of pure and biologically inert cellulose ethers. • They are prepared as thin porous, circular membranes of about 150 μm thickness. • The filters have different porosity grades (10nm to 8 μm). • The assemblage contains a funnel shaped inlet and a tube like outlet. • In between these two the filter is fitted. • Outlet - connected to a vacuum pump to suck known amount of air. • Filter is directly placed onto the surface of a solid medium. • After incubation colonies formed can be counted.
- 16. Impingement into liquids Raised impinger Bead - bubbler device Lemon Sampler Impingement onto solids. Hollaender & Dalla Valle Sampler Slit Sampler Sieve Sampler
- 17. Impingement into liquids a. Raised Impinger • It’s a type of sampler impingement is made within bulk fluid by a jet of air.
- 18. b. Bead-Bubbler device • Oldest device for sampling air. • consists of 250ml suction flask with side outlet connected to suction pump. • Glass bubbler - glass tube with minute openings at the bottom. Placed inside the flask by a rubber stopper. • Glass beads of size about 5nm in diameter are kept around the glass bubbler. • In addition, the flask contains known volume of broth.
- 19. Working mechanism • Air is drawn into the flask through the glass bubbler. • Incoming air escapes into the broth in the form of bubbles through the holes at the bottom of glass bubbler. • The shaking action of the flask and glass beads facilitate the formation of bubbles. • Microorganisms in the air are dispersed in the broth. • After sampling, an aliquot from the broth is plated for count.
- 20. c. Lemon Sampler • Consists of a glass Folin aeration tube with a perforated bulb with six holes at one end. • Bulb end of aeration tube is kept inside a test tube containing 20ml broth with 2 to 3 drops of olive oil to prevent foaming. • Stoppered with two-hole rubber stopper. • Kjeldahl trap is a square glass baffle with slightly bent end inserted into the test tube and other end is connected to suction pump. • Flow meter measures the rate of airflow entering through the air inlet. • The entire bubbler should be sterilized by autoclaving or by rinsing with 70% alcohol and dried. • Air is drawn at the rate of 25-30 litres per minute and dispersed through the broth.
- 21. Impingements onto solids. a. Hollaender & Dalla Valle Sampler • Consists of brass container with removable bottom. • Petridish containing medium is placed at the bottom of the container. • Just above the petridish an inverted glass funnel (swabbed with alcohol before use) is fitted without touching it. • Container is screwed tightly against the gasket during sampling. • The air sample passes through the funnel stem and the airborne microorganisms are impinged upon the agar medium. • The air is drawn by using suction pump • Effective sampling rate is found to be 28 liters per minute. • Advantage: simple, portable and efficient.
- 22. b. Slit Sampler • Slit sampler is an efficient and convenient device for the enumeration of bacteria carrying particles in a unit volume of air. • It was introduced by Bourdillon et al. in 1941. Principle • Air is drawn from the environment at a fixed rate and the suspended particles are allowed to impinge on the surface of an agar plate, on incubation each particle will form a colony.
- 23. • Consists of an enclosed chamber. • There is a slit of 0.33 mm width and 27.5 mm length with 3mm depth. • The box is connected to a suction pump which maintains it at a negative pressure of -22.6 mm mercury. • At the correct negative pressure air will enter through a slit at the rate of one cubic foot (28.3 lit.) per minute. • At the bottom of the chamber there is a rotating circular platform which holds the agar plate (Distance between the slit and the agar plate - 2mm). • When the plate is rotated particles suspended in air will be deposited over the entire surface of the plate.
- 24. Size-grading slit sampler • Set of plates are used to collect particles of different sizes. • This is achieved by using slits of different sizes in series. Advantages • Highly efficient device and can collect • 95% of the water droplet particles • respiratory secretion droplet nuclei of 0.2um diameter Disadvantage • Produce noise Modification of slit sampler
- 25. c. Sieve Sampler • Simple device, similar to slit sampler • Particles containing microorganisms are distributed over the plate through air jets with several holes called Sieve plates. • Upon incubation these particles form colonies that can be enumerated.
- 26. Modification of sieve sampler Multistage sieve sampler • Developed by Anderson • It consist of a series of sieve plates with different porosity.
- 27. • Air is allowed to pass through the electrodes. • The charged particles fall on to a rotating disc which is fed with a collecting fluid at a rate of 10ml per minute. • Air is sucked into the chamber by a fan fitted at the bottom. Advantages • Efficient method • Suitable for large volume of air.