Dept. Environmental Science, IT, Sligo

Microbiological Analysis of the Air.

Degree: Environmental Science & Technology,

Year 3. (Module: Air Pollution E301)

Diploma: Occupational Health & Safety, Year 3

(Module: Occupational Hygiene 1 S302)

Lecturer: Dr. Michael Broaders.

READING LIST FOR AIR MICROBIOLOGY

See website. for more reference material

CH.Collins & AJ. Beale. Safety in Industrial Microbiology and Biotechnology 1992. Butterworth Heinemann. Isbn 0 7506 1105 7

660.6

CH. Collins & JM. Grange. The Microbiological Hazards of Occupations. 1990

Occupational Hygiene Monograph No. 17. Series ed. Dr D. Hughes. Isbn 0 905927-23-0. H&H Scientific Consultants Ltd. In assoc with Science Reviews Ltd.

Harriet A. Burge Bioaerosols..1995. CRC Press Inc. 0-87371-724-4...... 613.5

Christopher S.Cox & Christopher M. Wathes 1995. Bioaerosols Handbook.. CRC Press Inc. 1-87371-615-9 ...... 576.190961

Gregory,P.H.(1973). MICROBIOLOGY OF THE ATMOSPHERE. 2nd ed. Leonard Hill...... 576.190691

Dart,R.K. & Stretton,R,J.(1980). MICROBIOLOGICAL ASPECTS OF POLLUTION CONTROL. ( Chapter on Microorganisms as Air pollutants.) Elsevier Publishing Co.....628.536.

RULES, REGULATIONS AND CODE OF CONDUCT FOR SAFETY IN THE MICROBIOLOGY LABORATORY

  • APROPRIATE PROTECTIVE CLOTHING MUST BE WORN IN THE LABORATORY AT ALL TIMES.
  • SAFETY GLASSES TO BE WORN AT ALL TIMES.
  • (LABORATORY COATS MUST BE WORN AT ALL TIMES AND MUST BE CLEAN AND FREE OF GRAFFITTI.)
  • BEHAVIOUR IN THE LABORATORY MUST BE APPROPRIATE TO REFLECT SAFETY STANDARDS. (Performance and behaviour in the laboratory are taken into account for CA marks.)
  • EATING, DRINKING AND SMOKING ARE NOT PERMITTED IN THE LABORATORY.
  • HANDS MUST BE WASHED WITH SOAP ON ENTERING THE LABORATORY AND AT ALL TIMES LEAVING THE LABORATORY.
  • BENCH TOPS MUST BE SWABBED WITH DISINFECTANT AT THE START AND END OF EACH CLASS. (ETHANOL IS PROVIDED)
  • WASTE DISPOSAL BAGS ARE PROVIDED FOR PETRI DISHES AND OTHER DISPOSABLES WHICH REQUIRE AUTOCLAVING.
  • WASTE DISPOSAL BINS ARE PROVIDED FOR WASTE PAPER .
  • DISCARD JARS ON THE BENCH TOPS CONTAINING DISINFECTANT ARE PROVIDED FOR DISPOSAL OF GLASS SLIDES AND USED PIPETTES AND PIPETTE TIPS
  • SINKS MUST NOT BE USED FOR WASTE DISPOSAL.
  • HANDLE ALL CULTURES AS IF POTENTIALLY PATHOGENIC (i.e DANGEROUS DISEASE CAUSING ORGANISMS).
  • HANDLE ALL MATERIAL I.E, WATER FROM RIVERS/LAKES etc., SOIL, SLUDGES AND MATERIALS FROM OTHER SOURCES AS CONTAINING POTENTIAL PATHOGENS.
  • DO NOT LICK LABELS, PENCILS, FINGERS etc.
  • TRY TO PREVENT RUBBING YOUR EYES AND LIPS, BE AWARE OF THE POSSIBILITY OF CONTAMINATION AT ALL TIMES.
  • THINK ASEPTIC TECHNIQUE AT ALL TIMES
  • IN CASE OF ACCIDENT (BREAKAGES, SPILLAGES etc.) INFORM THE LECTURER IMMEDIATELY.
  • ALWAYS LEAVE THE LABORATORY CLEAN AND TIDY FOR YOUR NEXT CLASS. Clean bench top of stains and put away microscopes, hot plates etc.

Objectives

You should be able to:

  • prepare instruments for recovery of viable bioaerosol from the air in occupied and other habitats;
  • determine the materials required and prepare and sterilise all materials for use with the instruments;
  • operate the instruments, incubate the plates and record and report the results;
  • present the results in an acceptable format and be able to analyse and manipulate the data to intrepert the result i.e. convert CFU’s on the agar plates to CFU’s per m3 air;
  • draw conclusions about the extent and nature of the contamination and;
  • compare and contrast the use of the instruments in air analysis.

Microbiological Analysis of the Air.

In this series of practicals you will sample the air at a number of locations in the college for its microbial content.

You will:-

a)compare the microbial loading of the atmosphere at each location in terms of:

  1. the total number of microorganisms per m3 of air i.e. the total number of bacteria plus the total number of yeasts and moulds,
  2. The percentage of bacteria and yeasts and moulds at each location,
  3. The proportion of microorganisms that are respirable and non respirable.

Also of the bacterial population, what proportion are Gram positive or Gram negative or rods or cocci. Recognise the difference between bacteria and yeasts, Identify some bacteria as far as possible and examine some filamentous fungi.

b) compare the sampling efficiency of each device at each location. i.e. compare the amount of bacteria and fungi collected at each location by each device.

The devices and methods used in the analysis are as follows:-

  1. Casella Slit–to–agar sampler;
  2. Anderson Two Stage Sampler
  3. Millipore Liquid Impingement (as a demonstration only);
  4. All Glass Liquid Impingement;
  5. Hawksley Air Sampler;
  6. Biotest Centrifugal Air Sampler;
  7. Surface Air Sampler
  8. Settle Plates.

Present the results in proper manner using Tables, Graphs, Bar charts.

Materials and Procedures.

Casella Slit–to–Agar Sampler
Large Petri dishes (14.5 cm) are filled with agar medium to within 5 mm. of the top of the dish, (approx. 200ml).

TSA is used to collect total bacteria. (near neutral pH)

Sabaroud Dextrose Agar or Malt Agar is used to select for yeasts and moulds. (more acidic pH)

This medium may besupplemented with either Triton N101 (500 mg/l) or Rose Bengal (50 mg/l) and the antibiotics penicillin (20 units/ml) and streptomycin (40 units/ml) to make the medium even more selective for yeasts and moulds.

Agar plates are incubated at the appropriate temperatures to allow the microorganisms to develop. (37ºC for bacteria and 22-25ºC for yeasts and moulds.

Before using the instrument follow the procedure carefully as in the notes below.

The sampler works by drawing air through one or more of the narrow slits (1mm width) positioned 0.2 cm above the surface of the agar plate. While the air is being drawn through the machine, the plate is rotated through 360° so that the microorganisms are distributed over the surface of the agar.

Both the volume of air per minute drawn through the machine and the total time for the rotation of the plate, are variable and this data is used to determine the volume of air passed through the sampler.

The total volume of air per plate can be varied from 87.5 litres to 3,500 litres. (Table 1).

For routine use, 175 l/min for a 2 min. sampling time should be used. (Total 350 l air sampled)

The volume of air passing through the sampler can be altered by blanking off one or more slits and by adjusting the vacuum reading accordingly.

If only one slit is used, the needle on the dial should be adjusted to the first thin line. If two slits are used the needle should be adjusted to the second thin line. If three slits are used the needle should be adjusted to the third thin line. (Fig.2).

Preliminary checks.

Before commencing sampling, the slits and tube above should be cleaned by swabbing over the slit faces and around the tube with 70% iso-propyl alcohol. Do not steam sterilise.

Sampling procedure

1. After checking that switch 'A' is off, connect the vacuum pump to the sampler and plug in the mains.

2. Check that you are using the correct number of slits, according to the volume of air to be sampled (Table 1)

3. Turn on switch 'A', put switch 'B' into the "down" position. Adjust the vacuum to the correct mark on the gauge. Turn off switch 'A', and put switch 'B' up.

4. Unclamp the slit box and lower the turn-table with the control knob.

5. Turn the turn-table so that that the indicator is at zero. Place the agar plate centrally on the table.

6. Replace the slit box.

7. Turn on mains switch 'A'.

8. Raise the turn-table with the control knob until the neon light glows.

9. Select speed with switch 'C'.

10. Put switch 'B' in "down" position until the turn-table is past 30° and then return to "up" position.

11. When the cycle has finished, turn off mains switch 'A', lower turn-table, remove slit box and plate.

12. Incubate plate at the appropriate temperature

13. Repeat the above procedure for each sample location.

Table 1.

No. of slits / Flow/Min (litres) / Time of one cycle in Min. / Volume sampled
(litres)
1 / 175 / 0.5
2
5 / 87.5
350
875
2 / 350 / 0.5
2
5 / 175
700
1750
3 / 525 / 0.5
2
5 / 262.5
1050
2625
4 / 700 / 0.5
2
5 / 350
1400
3500

Fig. 1 Slits from Casella Sampler

Figure 2. Front panel of Casella Sampler.

(ii) Anderson Two stage Viable Sampler.

Collects 95% of particles above 0.8 µm.

The sampler separates viable particles into two size ranges, with a 50% cut off diameter of Stage 1 at 8.0 µm.

The pump maintains a flow rate of 28.3 liters/min.

Use regular agar plates of TSA, Malt Agar as before,

Mannitol salt (for suspect Staphylococcus aureus)and Mitis salivarius (for oral Streptococci).

Plates should contain 25 ml of agar.

The sampler requires two plates, one for each stage.

Each stage contains 200 tapered orifices.

The diameter of the stage 1 orifices is 1.5 mm and 0.4 mm on the second stage.

Sample for four minutes.

Incubate plates at the appropriate temperature, i.e 25C for yeasts and moulds and 35C for all bacteria.

Count all colonies after incubation from both plates to determine number of microorganisms in the air sampled.

Calculate number of cfu's per m3 of air.

Calculate the percentage of particles on each stage and represent as respirable particles from stage 2 and nonrespirable particles from stage 1.


(ii) Millipore ImpingementApparatus, vacuum pump, limiting orifice and impingement fluid.

IMPINGEMENT FLUID.

In 1l of distilled water dissolve:

2g powdered gelatin,

4g Na2HPO4,

37g Brain Heart Infusion broth,

0.1ml octyl alcohol.

Mix the ingredients in a flask and boil for 15 min. Use 20 ml of impingement fluid in the filter funnel.

Prepare the apparatus according to the instructions from demonstrator.

After sampling the air, remove the filter and place onto an absorbent pad, moisten the pad with yeast and mould recovery medium or bacterial recovery medium.

Incubate at the appropriate temperature.

Note the volume of air sampled from the duration and rate of sampling.(l/min)

Report on the number of microorganisms collected from the various locations as CFU/m3 air.

(iv) All Glass Liquid Impingement.

Airborne Microorganisms may be collected without significant loss of viability by impingement in a sterile buffered broth.

The impingement fluid is then drawn off and filtered through a membrane for culturing and counting or diluted serially and plate counted.

Air is drawn through the impinger at 12.5 litres per minute with a vacuum pump.

30 ml sterile impingement fluid is aseptically added to the lower section of the impinger.

Be sure the tip of the impinger is covered by liquid.

After the sampling period (note the sampling time), turn off the vacuum pump.

Collect the impingement fluid in a sterile universal, wash the walls of the impinger with a small volume of sterile diluent and make the volume up to 10 mls. Carry out serial dilution and recover total bacteria and yeasts and moulds. Other selective media can be used to recover staphs, streps or other specialized microorganisms.

(v) Hawksley Air Sampler.

This sampler collects particles in the air directly onto the surface of a membrane held in a membrane holder attached to a vacuum pump.

The vacuum pump is set to collect between 10-30 litres of air per minute. The actual rate of sampling is largly determined by the level of contamination of the air to be sampled. Heavily contaminated air can only be sampled for a short duration, otherwise the membrane becomes overcrowded, however prolonged sampling tends to desiccate the delicate microorganisms on the membrane.

Set up the sampling device as described. Note the rate of air sampling and the duration of the sampling and record the total volume of air sampled.

Recover the cells on the membranes using suitable recovery media i.e. total bacteria, staphs, yeasts and moulds. Incubate at the appropriate temperature.

Report on the number of microorganisms collected from the various locations.
(vi) Biotest Centrifugal Air Sampler.

Calibrate and sterilise the impeller head. Use the instrument as directed. Sampling time is normally 4 mins.

Sampling Volume.

Because of the design of the machine not all the particles in the air sampled are impacted onto the agar strips. The volume of air sampled is 280 l/min but the separation volume for particles 4 µm diameter is 40 l/min. Therefore for a 4 min sampling period the amount of air sampled is 160 liters.

Use TSA strip for total bacteria, Rose Bengal agar medium for the fungi. Note the volume of air sampled, incubate the strips at the appropriate temperature to recover the microbes.

Report on the number of microorganisms collected from the various locations.

The detected number of organisms per unit of air volume can be calculated as follows:-

CFU/m3 = Colonies on the agar strip x 25

Sampling time (mins)

Principle of operation

The Biotest RCS Air Sampler works on the impaction principle. The function of the Air Sampler is to collect airborne microorganisms quantitatively onto a culture medium. The air under examination is sucked into the sampler from a distance of at least 40 cm by means of the impeller.

The air enters the impeller drum concentrically and in a conical form, as the blades are set in rotation, and the particles contained in the air are impacted by centrifugal force onto a plastic strip containing a culture medium. The air then leaves the drum in a spiral form around the outside of the cone of air entering the sampler. After the sample has been taken, the agar strips are removed carefully, placed in the carrier tray and are incubated at the appropriate temperature after which the colonies counted.

The sampler has an average rotational speed of 4096 rpm with an accuracy of + 2%. The separation volume is 40 litres per minute.

Volume characteristics

Due to its principle of operation and the geometric properties of the impeller drum the RCS Air Sampler has special volume characteristics. It is therefore necessary to differentiate between the total volume sampled (= Sampling Volume) and thevolume relevant for separating the particles ( = Separation Volume). The Separation Volume per time unit is the basis for calculating the number of organisms per air volume.

1 Sampling Volume The air which is to be examined enters the instrument head concentrically with a diameter of 2a and at velocity Cax. Here it is picked up by the impeller blade, deflected through 180° and routed to flow past a strip filled with a nutrient medium. The air is expelled via an annular gap with width b. The total sampling volume (V) can be determined by point-by-point measuring of the velocity and angle of flow over the radius r and subsequent mathematical evaluation. This sampling volume is 280 I/min at a speed of rotation of 4096 rpm. This sampling volume is a parameter for calculating the volume of air that is relevant to separation of the particles.

2. Separation Volume By virtue of the high centrifugal force, the particles in the rotating ring of air are forced outwards and impacted onto the surface of the nutrient medium. However, this separation takes place only from one part of the sampling volume. It is possible to determine the separation volume mathematically. In doing so, a major parameter for separation is the height of the instrument head. This height (Imin) can be calculated for the separation of all particles contained in the total sampling volume. The basis for this is the resolution of a differential equation which describes the spiral flight path of the particles under the influence of the air flow velocity, the direction of flow and the centrifugal force that arises.

For a relevant particle diameter of 4 µm, this produces a height of 14 cm.

However, since instead of 14 cm, only 2 cm are available in the instrument head as the separating height, separation is not effected from the whole sampling volume but from only 1/7 of this.

Thus the separation volume for the instrument is 40 l per minute.

7. SAS Surface Air Sampler.

This instrument is designed for use with the regular Contact (RODAC) plates, containing agar suitable for recovery of various microorganisms.

The sampler has two sampling heads which can be used similtaneously.

It is possible to use the same agar to give two replicate samples or you may use two different agars.

In this case we will use duplicate contact plates containing TSA for total bacteria, Mannitol Salt for presumptive Staphylococcus and Sab Dex for yeasts and moulds.

Instructions for SAS

Open covers and place Contact plates into holders without lids.

  1. Replace perforated cover.
  2. Switch ON button

3.Allow display to reach SELECT HEAD & DATE

  1. Press ENTER
  2. HEAD LEFT is displayed.
  3. Press up arrow  and select HEAD LEFT + RIGHT
  4. Press enter
  5. START FOR 500 may be displayed.
  6. If so then press START otherwise
  7. Press down  arrow
  8. Select Standard Mode
  9. Press ENTER
  10. Std Prog 500 may be displayed
  11. Select Volume of air using up/down buttons for 500l
  12. Press ENTER
  13. START.

Remove agar plates, cover and incubate at the appropriate temperature.

Report CFU’s per m3 air sampled in your location.

8. Settle Plates.

A variety of agar media can be used to sample the air for the microbial load using this technique. You will prepare plates of agar medium suitable for the growth of the following microorganisms:

total bacteria; / TSA
total fungi, (yeasts and moulds) / Sabaroud Dextrose Agar or Malt Agar acidified, (2ml lactic acid (10%) per 100ml agar

Staphylococci

/ a)Mannitol salt
Oral streptococci / b)Mitis salivarius

check with the manufacturers manual on the expected characteristics of the organisms appearing on the plates.

The agar dishes are left open on the benches in each location and the lids closed after 10,20,40 and 80 mins.

The plates are incubated at appropriate temperatures and total colonies counted.

The results are presented in table or graph form to show the number of colonies deposited per settlement area per unit of time.

Presentation Of Results From Sampling Devices.

For your Location , for each sampler present the following in Table format: