VPE 511: Environment and Environmental Hygiene (2+1)

SAMPLING-SEDIMENTATION METHOD

Direct Sampling

• The air source to be tested is decided and a sterile agar plate is kept open for atleast 15 minutes. Potato dextrose agar is commonly used. For fungi sabouraud dextrose agar is used. The plates are incubated or 5 days at 25^oC for fungi and 2 days at 35-37^oC for bacteria.

 SABOURAUD DEXTROSE AGAR

Ingredients	gm/lt
Mycological peptone	10
Dextrose	40
Agar	15
Final pH (at 25oc)	5.6±2

• For examination of fungi, place a drop of lactophenol cotton blue stain on a slide. Place a cellophane tape over the fungal growth and stick it over the stain. Remove excess lactophenol, dry and observe under microscope.
• For examination of bacteria, a loopful of colony is used to make a smear. This smear is stained appropriately to identify bacteria.

Air sampler

Principle

• The Air sampler operates on the impaction principle. The air under examination is sucked by the impeller centrifugally and impacted against the agar medium strip. The impeller speed of 4100 rpm is so adjusted that 280 litres of air is sampled every minute.
• The sampling volume corresponds to 40 liters / minute of separation volume. (i.e.) 1/7th of the sampling volume. The separation volume per unit of time forms the basis for calculating the number of organisms per volume of air being sampled.

Materials required

• Air sampler system and sterile agar medium strips.

Components of air sampler

• The air sampler system consists of a controller unit with a sling and a hand held metal centrifugal air sampling probe.

Controller unit

• This has a crystal controlled timer with time setting from 1-9 minutes. The controller unit has an inbuilt battery and it should be charged for 14 hours. An AC mains adapter is also provided to supply power to the handset unit.

Hand held probe

• This consists of the following
  • Impeller (fan)
  • Impeller SS cup.
  • Impeller handle.
  • Socket for supply of power from control unit.

Procedure

• The impeller and the impeller cup are sterilized 121^oC for 20 minutes.
•  The impeller handle is disinfected using alcohol.
• The Agar medium is pulled out holding the edges only from the wrapper and inserted into the slot in the metal cup without touching the medium so that only 2 cms stays out. The agar surface should face the impeller.
• The impeller handset is held in the area where the air sampling is to be carried out.
• Press N button. Red LED glows indicating starting time of the process.
• Set time 1-9 minutes by pressing the buttons
• Press 'RESET' button, Green LED glows indicating starting time of the process.
• After SET time is elapsed the buzzer beeps, indicating the completion of the time cycle.
• Holding the strip by its tab, the strip is pulled out an replaced in its original wrapper so that the agar faces away from the sliding lid.
• Then, the wrappr is sealed, marked for identification and incubated at 37^oC.

Calculation

CFU/litre = Colonies on agar strip  / (40 x Sampling time in minutes)

• 1 minute corresponds to 40 litres of air separation volume.

CFU/m³ = (Colonies on agar strip x 25) / Sampling time in minutes

Application

• Monitoring the quality of ambient air for the persence of microorganisms is necessary to avoid severe health hazards in hospitals etc. The GMP requirements could be fulfilled in pharmaceutical manufacture.
•  Similarly, in blood banks an surgical theatres as well as in tissue culture labs specified monitoring of air facilitates the maintenance of necessary asceptic conditions.
• The other environments that need monitoring are pathological labs, cosmetic manufacture, fermentation industry, food processing plants, dairy industry, abattoirs and fisheries, medical research, public health centres, dental clinics, electronic industry, vaccine manufacture, biotechnology industry.