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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 1
Dept. Environmental Science, IT, Sligo
READING LIST FOR AIR MICROBIOLOGY
See website. http://www.itsligo.ie/staff/mabroaders 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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 2
Dept. Environmental Science, IT, Sligo
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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 3
Dept. Environmental Science, IT, Sligo
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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 4
Dept. Environmental Science, IT, Sligo
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:
i. the total number of microorganisms per m3 of air i.e. the total
number of bacteria plus the total number of yeasts and moulds,
ii. The percentage of bacteria and yeasts and moulds at each location,
iii. 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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 5
Dept. Environmental Science, IT, Sligo
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 be supplemented 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).
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 6
Dept. Environmental Science, IT, Sligo
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'.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 7
Dept. Environmental Science, IT, Sligo
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
2
350
3
525
4
700
0.5
2
5
0.5
2
5
0.5
2
5
0.5
2
5
87.5
350
875
175
700
1750
262.5
1050
2625
350
1400
3500
Fig. 1 Slits from Casella Sampler
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 8
Dept. Environmental Science, IT, Sligo
Figure 2. Front panel of Casella Sampler.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 9
Dept. Environmental Science, IT, Sligo
(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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 10
Dept. Environmental Science, IT, Sligo
(ii) Millipore Impingement Apparatus, 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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 11
Dept. Environmental Science, IT, Sligo
(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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 12
Dept. Environmental Science, IT, Sligo
(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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 13
Dept. Environmental Science, IT, Sligo
(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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 14
Dept. Environmental Science, IT, Sligo
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 the volume
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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 15
Dept. Environmental Science, IT, Sligo
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
4. Press ENTER
5. HEAD LEFT is displayed.
6. Press up arrow  and select HEAD LEFT + RIGHT
7. Press enter
8. START FOR 500 may be displayed.
9. If so then press START otherwise
10.Press down  arrow
11.Select Standard Mode
12.Press ENTER
13.Std Prog 500 may be displayed
14.Select Volume of air using up/down buttons for 500l
15.Press ENTER
16.START.
Remove agar plates, cover and incubate at the appropriate temperature.
Report CFU’s per m3 air sampled in your location.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 16
Dept. Environmental Science, IT, Sligo
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;
total fungi, (yeasts and moulds)
Staphylococci
Oral streptococci
TSA
Sabaroud Dextrose Agar or
Malt Agar acidified, (2ml lactic
acid (10%) per 100ml agar
a) Mannitol salt
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.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 17
Dept. Environmental Science, IT, Sligo
Presentation Of Results From Sampling Devices.
For your Location , for each sampler present the following in Table format:
Total microorganisms collected, i.e. total bacteria plus total yeasts/moulds
in (CFU/m3). For this you need to know the volume of air sampled.
Percentage of the total made up of bacteria and yeasts/moulds.
What proportion of the total are respirable and non respirable.
Of the bacteria, what proportion are Gram positive/Gram negative.
Present your results on the isolation and identification of suspect
Staphylococcus aureus.
Need a diagram of a yeast with the diameter of the yeast cell measured
Need a diagram of a filamentous fungus recovered from the agar plates.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 18
Dept. Environmental Science, IT, Sligo
Further Analysis Of The Bacteria Recovered from the air.
Confirmation of Staphylococcus aureus and Streptococcus spp.
Pick suspect Staph. colonies from the Mannitol Salt agar plates and transfer
onto TSA, Blood Agar, Dnase, and phosphatase agar, using spot inoculation.
Incubate @ 37C for 48 hrs. Likewise, spot Blood agar and TSA with
suspect colonies from Mitis Salivarius agar and incubate @ 37C for 72 hrs.
Staphylococci and micrococci are frequently isolated from pathological material and
foods. Distinguishing between the two groups is important. Some Staphylococci are
known to be pathogens; some are doubtful or opportunist pathogens; others, and
micrococci, appear to be harmless but are useful indicators of pollution. Staphylococci are
fermentative capable of producing acid from glucose anaerobically; micrococci are
oxidative and produce acid from glucose only in the presence of oxygen.
Identification
They are Gram-positive, oxidase negative, catalase positive, fermentative cocci arranged in
clusters.
Colonies of staphylococci and micrococci are golden brown, white, yellow or pink,
opaque, domed 1-3 mm in diameter after 24 hr. on blood agar and are usually easily
emulsified. There may be -haemolysis on blood agar.
On Baird-Parker medium after 24 hr., Staphylococcus aureus gives black, shiny,
convex colonies, 1-1.5 mm in diameter; there is a narrow white margin and the colonies
are surrounded by a zone of clearing 2-5 mm in diameter. This clearing may be evident
only at 36 h.
Other staphylococci, micrococci, some enterococci, coryneforms and enterobacteria may
grow and may produce black colonies but do not produce the clear zone.
Some strains of S. epidermidis have a wide opaque zone surrounded by a narrow clear
zone. Any grey or white colonies can be ignored. Most other organisms are inhibited.
Examine Gram-stained films. Do coagulase and DNase tests on Gram-positive cocci
growing in clusters. This is a short cut: strains positive by both tests are probably S.
aureus.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 19
Dept. Environmental Science, IT, Sligo
Coagulase test
Possession of the enzyme coagulase which coagulates plasma is an almost exclusive
property of S. aureus. There are two ways of performing this test:
(l) Slide coagulase test Emulsify one or two colonies in a drop of water on a slide. If no
clumping occurs in 10-20 s dip a straight wire into human or rabbit plasma (EDTA)
and stir the bacterial suspension with it. S. aureus agglutinates, causing visible
clumping in 10 s.
Use water instead of saline because some staphylococci are salt sensitive,
particularly if they have been cultured in salt media. Avoid excess (e.g. a loopful) of
plasma as this may give false positives. Check the plasma with a known coagulase
positive staphylococcus.
(2) Tube test Do this (a) to confirm the slide test, (b) if the slide test is negative. Add 0.2
ml of plasma to 0.8 ml of nutrient (not glucose) broth in a small tube. Inoculate
with the suspected staphylococcus and incubate at 37°C in a water-bath. Examine
at 3 h and if negative leave overnight at room temperature and examine again.
Include known positive and negative controls. It is advisable to use EDTA plasma
(available commercially) or oxalate or heparin plasma. Check Gram films of all
tube coagulase positive organisms.
S. aureus produces a clot, gelling either the whole contents of the tube or forming a loose
web of fibrin. Longer incubation may result in disappearance of the clot due to digestion
(fibrinolysis).
The slide test detects 'bound' coagulase ('clumping factor'), which acts on fibrinogen
directly; the tube test detects 'free' coagulase, which acts on fibrinogen in conjunction with
other factors in the plasma.
Either or both coagulases may be present .
DNase test
Inoculate DNase agar plates with a loop so that the growth is in plaques about 1 cm in
diameter. Incubate at 37°C overnight. Flood the plate with 1 M hydrochloric acid. Clearing
around the colonies indicates DNase activity. The hydrochloric acid reacts with unchanged
deoxyribonucleic acid to give a cloudy precipitate.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 20
Dept. Environmental Science, IT, Sligo
Phosphatase test
Inoculate phenolphthalein phosphate agar and incubate overnight. Expose to ammonia
vapour. Colonies of phosphatase positive staphylococci will turn pink.
S. aureus gives a positive test (but negative strains have been reported). Coagulasenegative staphylococci and micrococci are usually phosphatase negative
The API Staph system is useful if identification to species is required.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 21
Dept. Environmental Science, IT, Sligo
Staphylococcus aureus
This species is coagulase and DNase positive, forms acid from lactose, maltose and
mannitol, reduces nitrate, hydrolyses urea and reduces methylene blue. It is usually
phosphatase positive but does not grow on ammonium phosphate agar.
Some strains are haemolytic on horse blood agar but the zone of haemolysis is relatively
small compared with the diameter of the colony (differing from the haemolytic
streptococcus).
S. aureus is usually identified by either the coagulase or the DNase test. False-positive
coagulase tests are possible with enterococci.
The fermentation of mannitol is not reliable. Mannitol-fermenting, coagulase negative,
strains occur.
S. aureus is a common cause of pyogenic infections and food poisoning. Staphylococci are
disseminated by common domestic and ward activities such as bedmaking, dressing or
undressing. They are present in the nose, on the skin and in the hair of a large proportion of
the population.
Micrococcus
These are Gram-positive, oxidase negative, catalase positive cocci that differ from the
staphylococci in that they utilise glucose oxidatively or do not produce enough acid to
change the colour of the indicator in the medium. They are common saprophytes of air,
water and soil and are often found in foods.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 22
Dept. Environmental Science, IT, Sligo
Streptococcus
Gram-positive cocci that always divide in the same plane, forming pairs or chains; the
individual cells may be oval or lanceolate. They are Gram-positive, nonsporing, non-motile
and some are capsulated. Most strains are aerobic. The catalase test is negative.
Isolation
Plate on blood agar and trypticase yeast extract cystine agar or mitis salivarius agar.
Air
For -haemolytic streptococci, use crystal violet agar containing 1:500 000 crystal violet with
slit samplers. For evidence of vitiation use mitis salivarius agar.
Identification of streptococci
Colonies on blood agar are usually small, 1-2 mm in diameter and convex with an entire edge.
The whole colony can sometimes be pushed along the surface of the medium. Colonies may
be 'glossy', 'matt' or 'mucoid'. Growth in broth is often granular, with a deposit at the bottom
of the tube.
The primary classification is made on the basis of alteration of haemolysis on horse blood
agar.
-Haemolytic or 'viridans' streptococci produce a small, greenish zone around the colonies.
This is best observed on chocolate blood agar.
' (Alpha prime)-haemolytic streptococci are surrounded by an area of haemolysis which
superficially resembles that of -haemolytic streptococci (below) but with a hazy outline and
unaltered red blood cells within the haemolysed area.
-Haemolytic streptococci give small colonies surrounded by a much larger, clear
haemolysed zone in which all the red cells have been destroyed.
Some streptococci show no haemolysis.
Haemolysis on blood agar is only a rough guide to pathogenicity. The -haemolytic
streptococci include those strains which are pathogenic for humans and animals but the type
of haemolysis may depend on conditions of incubation and the medium used as a base for the
blood agar.
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Dept. Environmental Science, IT, Sligo
Streptococcal antigens
Species and strains of streptococci are usually identified by their serological group and type.
There are 15 Lancefield groups characterised by a series of carbohydrate antigens contained
in the cell wall.
The API 20 Strep and Rapid ID Strep systems are useful for identifying streptococci.
Group A
These are -haemolytic, are the so-called haemolytic streptococci of scarlet fever, tonsillitis,
puerperal sepsis and other infections of humans, and are known as S. pyogenes. Some strains
are capsulated and form large (3-mm) colonies like water drops on the surface of the medium.
S. salivarius
These are commensals in the human upper respiratory tract and are therefore useful indicators
in air hygiene and ventilation investigations. The colonies are large and mucoid on media
containing 5% sucrose.
Aerccoccus
These are Gram-positive, oxidase negative, fermentative cocci that are usually in clusters,
pairs, tetrads or short chains.
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Dept. Environmental Science, IT, Sligo
Results:
Total CFU/m3 (Total Bacteria +total Yeasts/moulds)
Devices
Microlab
Sci.
Corridor
Front Hall
Eng.
Corridor
Back Hall
Casella
Anderson
Biotest
Hawksley
Impinger
For each Location
Total CFU/m3
% bacteria
% Yeasts/moulds
CFU/m3
% Respirable
% non Respirable
Total CFU/m3
% Respirable
% non Respirable
Casella
Anderson
Biotest
Impinger
Anderson Sampler
At each Location
Total Bacteria
Total
Yeasts/moulds
Mannitol Salts
Mitis Salivarius
Anderson Results
Microlab
Sci Corridor
Front Hall
Eng Corridor
Back Hall
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Dr. M.A. Broaders 25
Dept. Environmental Science, IT, Sligo
A SELECTION OF AIR SAMPLING DEVICES
Diagram showing relationship between particle size and spore deposition.
MULTISTAGE LIQUID IMPINGER.
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Dept. Environmental Science, IT, Sligo
CYCLONE SEPARATOR
Two Stage Anderson Sampler, with non respirables 8  on the upper
stage and respirable 4 diam on the lower stage.
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Dept. Environmental Science, IT, Sligo
ES&T 3& H&S 3
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Dr. M.A. Broaders 28
Dept. Environmental Science, IT, Sligo
Immunity to Fungi
Fungal infections are normally only a superficial nuisance (e.g. Ringworm),
but a few fungi can cause serious systemic disease, usually entering via the
lung in the form of spores, the outcome depends on the degree and type of
immune response, and may range from an unnoticed respiratory event to
rapid fatal dissemination or a violent hypersensitivity reaction. (Type 3:Extrinsic Allergic Alveolitis)
In general, the survival mechanism of successful fungi are similar to those
of bacteria: anti-phagocytic capsules (e.g. cryptococcus),
resistance to digestion within macrophage (e.g. histoplasma) and
destruction of polymorphs (e.g. coccidiodes).
The severe respiratory difficulties associated with Farmer's Lung occur
within 6-8 hours of exposure to the dust from mouldy hay. People are found
to be sensitized to thermophilic actinomycetes which grow in the mould hay.
Inhalation of the spores into the lungs introduces antigen into the lungs and
a complex-mediated hypersensitivity reaction occurs.
Names Applied To Extrinsic Allergic Alveolitis Caused By Inhaled
Spores.
SOURCE OF THE DISEASE
DUST
Mouldy Hay
Farmer's Lung
Air-conditioning
systems
Bagasse
Redwood sawdust
Malting barley
Maple bark
Cheese
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ORGANISM
Micropolyspora faeni
Thermoactinomyces
vulgaris
Hypersensitivity
Micropolyspora faeni
pneumonitis
Thermoactinomyces
vulgaris
Bagassosis
Thermoactinomyces
sacchari
Sequoiosis
Aureobasidium
pullulans
Graphium sp.
Maltworker's Lung
Aspergillus clavatus
Aspergillus fumigatus
Maple
bark Cryptostroma
pneumonitis
corticale
Cheese washer's Lung Penicillium caesi
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 29
Dept. Environmental Science, IT, Sligo
Fungi And Actinomycetes Associated With Respiratory Infections.
Disease
Source
Cryptococcosis
Pigeon droppings
blastomycosis
blastomycosis
Coccidiodmycosis
Histoplasmosis
Sporotrichosis
Adiaspiromycosis
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Organism
Cryptococcus
neoformans
soil
Blastomyces
dermatitidis
soil
Paracoccidiodes
brasiliensis
soil
Coccidioides immitis
chicken, bat droppings Histoplasma
capsulatum
Straw, sphagnum moss Sporothrix schenckii
Nests of field mice
Emmonsia crescens
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Dr. M.A. Broaders 30
Dept. Environmental Science, IT, Sligo
Bacterial Infections Which May Be Acquired By Inhalation.
Disease
Organism
Pulmonary tuberculosis
Pulmonary anthrax
Staphylococcal respiratory
infections
Streptococcal respiratory
infections
Pneumococcal pneumonia
Nocardiosis
Q fever
Whooping cough
Diphtheria
Sinusitis, bronchitis
Primary atypical pneumonia
Pneumonic plague
Legionnaires Disease
Mycobacterium tuberculosis
Bacillus anthracis
Staphylococcus sp.
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Streptococcus pyogenes
Diploccus pneumonia
Actinomadura asteroides
Coxiella burnetii
Bordetella pertussis
Corynebacterium diphtheria
Haemophilus influenza
Mycoplasma pneumoniae
Yersinia pestis
Legionella pneumophila
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Dept. Environmental Science, IT, Sligo
Classification of fungi.
The fungi are divided into two divisions:
1. Myxomycota (slime moulds)
2. Eumycota (true fungi).
Our interest is with the true fungi or Eumycota.
Two basic growth forms:
(i) unicellular or yeast form which reproduces by simple budding. Colonies
usually moist or mucoid.
(ii) filamentous or mould form which reproduces by spores or conidia.
Colonies usually velvety or cottony.
The filaments are known as hyphae and the mass of hyphae form the mycelium.
There are two kinds of hyphae, non-septate (coenocytic) and septate. The septa
divide the hyphae into compartments but not into cells.
Subdivisions of Eumycota:
1. Mastigomycotina - one class only: oomycetes- typically aquatic fungi
containing 580 species, non-septate hyphae.
2. Zygomycotina - one class only: zygomycetes- rapidly growing,
predominantly saprophytic fungi containing 665 species, non-septate hyphae.
Medically important genera include Absidia, Basidiobolus, Conidiobolus,
Mucor, Rhizopus.
3. Ascomycotina - classes no longer recognized: mostly terrestial
saprophytes and parasites of plants containing 28,650 species, septate hyphae,
sexual spores produced within asci. Medically important genera include
Allescheria, Aspergillus, Blastomyces, Geotrichum, Microsporum, Piedraia,
Trichophyton.
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Dept. Environmental Science, IT, Sligo
4. Basidiomycotina - four classes: hymenomycetes (mushrooms)
gasteromycetes (puff balls) uredimomycetes (rusts) ustilaginomycetes
(smuts) - terrestial saprophytes and parasites of plants containing 16,000
species, septate hyphae, sexual spores produced externally on basidia.
Medically important genera include the poisonous mushrooms and
Cryptococcus.
5. Deuteromycotina - a subdivision created for the "fungi imperfecti" i.e. no
sexual forms detected. Two classes: coelomycetes (produce conidia in sac-like
structures) hyphomycetes (conidia produced without sac-like structures).
Contains 17,000 species, septate hyphae. Most of the medically important fungi
are included in the "fungi imperfecti" including Candida, Cladosporum
Coccidioides, Epidermophyton, Fonsecaea, Madurella, Malassezia,
Microsporum, Sporothrix, Trichosporon.
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Dept. Environmental Science, IT, Sligo
A "Clinical Classification" of medical fungi can be made by grouping the
organisms according to the diseases they cause in humans. The following
fungi represent only the more common genera:
1. The superficial mycoses (no living tissue invaded and no pathological
changes occur). Malassezia, Piedraia, Trichosporon.
2. The cutaneous mycoses (no living tissue invaded but pathological
changes occur). Candida, Epidermophyton, Microsporum, Trichophyton.
3. The subcutaneous mycoses (chronic infections by soil fungi after skin
injury - mycetoma). Absidia, Allescheria, Basidiobolus, Cladosporum,
Conidiobolus, Fonsecaea, Madurella, Mucor, Phialophora, Rhizopus,
Sporothrix.
4. The systemic mycoses (dimorphic fungal pathogens causing
pulmonary infection from air-borne conidia). Blastomyces,
Coccidiomyces, Histoplasma, Paracoccidioides.
5. The opportunistic systemic mycoses (fungi of low virulence which can
invade immuno-compromised hosts). Absidia, Aspergillus, Candida,
Cryptococcus, Geotrichum, Mucor, Rhodotorula, Rhizopus, Torulopsis.
Contaminating fungi:
The more common contaminating fungi are also opportunistic pathogens e.g.
Absidia, Aspergillus, Mucor, Penicillium, Rhizopus, Rhodotorula,
Scopulariopsis.
Contaminating fungi may have very small pathogenic risk, other than
allergic reactions, but some of them are of great importance to food and
agricultural mycologists.
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Dept. Environmental Science, IT, Sligo
Culture media for fungi:
Mycological media should inhibit bacterial growth so that the slower
growing fungi can develop. The traditional way to do this is to use low pH
media e.g. Sabouraud Dextrose Agar and Potato Dextrose Agar at pH
5.6, Malt Extract Agar at pH 5.4 and Wort Agar at pH 4.8.
Although these media are still widely used, it is accepted that low pH levels
can suppress the growth of stressed fungal cells. The pH can be raised to
neutrality, if antibiotics are added to suppress the growth of bacteria e.g.
Oxytetracycline-Glucose-Yeast Extract Agar (OGYE Agar) Dermasel Agar
Base with Dermasel Selective Supplement SR75. It should be noted that the
cycloheximide in the Selective Supplement will inhibit the growth of
Aspergillus, Candida, Cephalosporium, Fusarium, Penicillium and
Trichosporum species.
An alternative to selective media is elective media i.e. a formulation which
allows only those organisms to grow which can utilise the growth factors
provided. Czapek-Dox Agar Modified, a synthetic medium in which sodium
nitrate is the sole source of nitrogen, is a popular example of an elective
fungal medium.
A second problem with mycological media is the tendency of rapidly
growing fungal colonies to spread and overwhelm neighbouring colonies of
slower-growing organisms.
The incorporation of ox-bile or preferably rosebengal inhibits spreading.
Rose-Bengal Chloramphenicol Agar combines inhibition of spreading and
bacterial growth. Rose-Bengal is not quite effective enough to control
spreading of very rapidly growing fungi, such as Rhizopus and Mucor
species.
Dichloran can be added to assist rosebengal.
Dichloran-Rose Bengal-Chloramphenicol Agar (DRBC Agar with
chloramphenicol) is a good example of this type of medium.
ES&T 3& H&S 3
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Dr. M.A. Broaders 35
Dept. Environmental Science, IT, Sligo
Many fungi are marginally xerophilic and can tolerate conditions of greatly
reduced levels of free water (water activity aw. 0.95) e.g. Aspergillus and
Penicillium species. Some fungi e.g. Wallemia, Eurotium , Xeromyces and
Chryosporum species can be very exacting in their water activity
requirements and may not grow on commonly used aw 0.999 media.
Dichloran-Glycerol (DG18) Agar Base with chloramphenicol has an aw 0.95
and improves the growth of these fungi. This medium has shown good
growth with many other fungi isolated from dried foods.
The presence of toxigenic fungi in foodstuffs is a serious matter. A selective
self-indicating medium for the specific Aspergillus species which can
produce mycotoxins, is an advantage. AFPA Base with chloramphenicol will
detect A. flavus and A. parasitcus colonies in 24-48 hours at 30°C. Colonies
are recognised by their yellow/orange pigmentation on the reverse of the
colonies.
Corn Meal Agar is a nutritionally impoverished medium, which is highly
versatile. Yeasts and other fungi will form chlamydospores in this medium,
especially in the presence of 1 % v/v Tween 80. It will also enhance the
chromogenesis of fungi, especially with 0.2%., w/v glucose. It can be used to
store stock cultures of fungi for long periods of time.
ES&T 3& H&S 3
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Dr. M.A. Broaders 36
Dept. Environmental Science, IT, Sligo
Examples of some fungi which may be dispersed into the air.
A. Absidia
B. Blastomyces
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Dept. Environmental Science, IT, Sligo
C. Botrytis.
D. Fusarium.
Helminthosporium.
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Dr. M.A. Broaders 38
Dept. Environmental Science, IT, Sligo
Cladosporium.
Geotrichum
Fonsecaea
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Dr. M.A. Broaders 39
Dept. Environmental Science, IT, Sligo
Allescheria.
Paecilomyces.
Phialophora.
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Dr. M.A. Broaders 40
Dept. Environmental Science, IT, Sligo
Rhodotorula.
Piederaia
Rhizopus.
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Dept. Environmental Science, IT, Sligo
Gliocladium.
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Dr. M.A. Broaders 42
Dept. Environmental Science, IT, Sligo
Sampling for Bioaerosols.
Bioaerosols, meaning airborne particles derived from microbial, viral,
and related agents, come in a wide variety of sizes, shapes, and
classifications.
Bioaerosols can cause two basic conditions: infections and allergies.
Infections are generally the result of multiplication and growth of
microbes inside humans while allergies are the result of exposures to
antigens. Not all infectious organisms cause pathogenic diseases in
humans, but those that can are of concern. Well-known diseases
associated with occupational exposures include anthrax, Q fever, and
brucellosis. Diseases for which concerns are increasing are those
associated with health workers and include AIDS and hepatitis.
Antigens are capable of stimulating the production of antibodies that
produce allergic diseases. Allergic reactions are the result of an antigen
producing a response from the immune svstem.
Hypersensitivity disease is another term for the allergic reactions
produced by these agents. These include hypersensitivity pneumonitis,
allergic asthma, and allergic rhinitis. Sources of airborne antigens include
bacteria, fungi, pollen, insect body parts, and skin scales (dander) and
saliva of mammals. In these situations antibody assays on blood from
affected individuals may be performed in conjunction with monitoring for
bioaerosols.
Sometimes it is not the microbe itself that produces the harmful effect
but the fact that it produces a toxin. Botulism is an example wherein the
botulinum toxin is the responsible agent. When release of a toxin is
involved, the organism can produce a disease without extensive
multiplication or dissemination throughout the body.
Just as there are factors that can predispose individuals to the health
effects caused by chemicals, certain persons are also at increased risk
when exposed to bioaerosols if they are over 50 years old, drink alcohol
excessively, smoke, or have preexisting respiratory disease or other
illnesses such as diabetes or kidney disease.
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Dept. Environmental Science, IT, Sligo
Bioaerosols can exist in both viable (living) and nonviable states. Viable
microorganisms such as bacteria, fungi, yeasts, and molds originate from
sprays or splashes of media, from the agitations of dusts, and from
sneezes and coughs of which only the small particles (<10 µm) remain in
the air long enough to travel any distance. Examples of nonviable agents
that are occasionally sampled include pollens and insect parts. Grains,
clusters of cells, and skin scales are much larger-sized particles (10—50
µm) than bacteria and viruses. Spores, which can be formed by fungi and
certain bacteria, can be both viable and nonviable and are capable of
causing disease in both forms. Most techniques attempt to sample for
only viable particles as these can be cultured so that they multiply,
making identification easier.
The specialized characteristics of viable agents require specialized
sampling instruments in order to preserve the organisms for laboratory
culture, which is the primary means of identification. Their fragility and
temperature, moisture, and nutrient needs are the primary considerations
when selecting a sampling device. While passive air sampling is simple
and can be done by setting out plates containing culture media, it is not as
effective as the use of active techniques involving the use of pumps.
There are two basic methods for collecting these air samples:
(1) specialized instruments and (2) air sampling trains incorporating a
personal air sampling pump, rotameter and media, such as is used for
integrated chemical sampling.
The specialized instruments can be used to house culture media and
therefore in most cases are preferred to integrated sampling techniques.
Area air samples are more commonly collected for bioaerosols than
personal samples, regardless of the type of situation being monitored,
due to the need to house culture media inside of instruments
specialized for sampling viable bioaerosols.
ES&T 3& H&S 3
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Dr. M.A. Broaders 44
Dept. Environmental Science, IT, Sligo
Most air sampling for bioaerosols is related to occupational exposures
in hospitals, laboratories, and research facilities; certain industrial
operations, such as brewery fermentation, cotton preparation and ginning,
wool sorting, hemp handling, and sawmills; and agricultural operations,
including hay preparation and the use of biological insecticides and
wastewater and sewer treatment facilities. A current interest is
exemplified by a recent survey for a biological insecticide, Bacillus
thuringiensis, to characterize exposures to personnel during a large-scale
spraying application.
Workers in some sectors of the food industry where fruits and
vegetables are processed may also be affected. As an example, slicing
sugar beets was found to generate exposures to bacteria originating in soil
during beet growth.
Other examples of operations where bioaerosol sampling might be
done include pharmaceutical manufacturing plants, clean rooms in
semiconductor manufacturing plants, animal laboratories, and food
processing plants.
Historically most environmental monitoring has consisted of collecting
bulk samples of water, especially drinking water and wastewater. Indoor
air surveys in buildings incorporate both indoor and outdoor air samples
for various agents.
Sampling usually attempts to determine whether the agents are being
generated from a source within a building rather than from an outside
source where they naturally occur It has been noted that the majority of
fungal spores found indoors are derived from outdoor sources, such as
decaying plant and animal materials, while the primary source indoors is
human bacteria shedding.
ES&T 3& H&S 3
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Dr. M.A. Broaders 45
Dept. Environmental Science, IT, Sligo
Sources of microbes include organic materials; humidifiers; vaporizers;
heating, ventilating, and air conditioning systems (HVAC); as well as
their associated equipment, such as cooling towers.
Another situation of growing interest that incorporates both occupational
and environmental exposures is bioremediation of contaminated soils
and waters using specially engineered "super bugs." These techniques
have proved very successful for treating petroleum compounds. In
these situations, the release of volatile organic compounds (VOCs) is
also a concern, so a boundary line monitoring strategy would need to
incorporate both sets of agents. Industrial wastewater treatment has
incorporated the use of various microbes for many years.
Air sampling for bioaerosols may be combined with sampling for
chemical agents in some situations such as indoor air surveys and
exposures to wood dust or bark. In some situations, monitoring is
performed for chemicals where there are metabolic by-products of the
organism, such as endotoxin, released by certain bacteria. Biological
monitoring may also need to be performed. For example, blood and
urine samples have been collected in cases of suspected Legionnaires
disease, since Legionella pneumophila infections cause the release of
antigens to the urine. Surface contamination may also be a concern.
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Dept. Environmental Science, IT, Sligo
BACTERIA
Bacterial infections are the most commonly seen in humans, such as those
that occur in minor wounds and scratches. Diseases related to bacteria
that are found in occupational exposures include those caused by anthrax,
also called wool sorter's disease, transmitted by handling imported goat
hair, wood, and hides; Brucella canis infections (brucellosis) from the
contaminated blood of slaughtered animals; and Leptospira-induced
disease (leptospirosis). associated with farm animals, dogs, and rodents.
which is spread through contact with infected urine, animal tissue, or
water.
Other bacteria of concern in sampling include Staphylococcus and
Streptococcus, which are carried by humans and cause infections under
the right conditions; Pseudomonas, which cause pneumonia; and bacillus,
which is associated with hypersensitivity pneumonitis. Thermophilic
bacteria of concern include Thermoactinomyces, known to cause
hypersensitivity; and Micropolyspora, Thermomonospora, and
Saccharomonospora. Another exposure of concern is the Salmonella
bacteria responsible for food poisoning. In this case, ingestion rather than
inhalation is the route of exposure. While not strictly occupational in
nature, this may be a concern in indoor air investigations.
Rickettsiae are intracellular parasites in fleas, ticks, and lice that are
considered to be bacteria. The tick is the most common reservoir and tick
bites are the primary route of transmission. Rickettsiae do not appear to
produce symptoms of disease in their hosts, but if they are transmitted to
humans, a severe and often fatal infection may result. The major
rickettsial disease of humans is epidemic typhus. (other human diseases
are Rocky - Mountain spotted fever and Q fever, both transmitted by
ticks, and scrub typhus, normally transmitted by mites to field mice, but
also transmissible to humans. The chlamydias, other specialized bacteria,
are carried in birds and the primary disease they are associated with,
ornithosis, is transmitted by inhalation of dried discharges and droppings
of birds.
Most bacteria are 1 µm to 5 µm in size
In order to select the proper sampling medium, it will be necessary to
know what the specific characteristics are of the bacteria suspected of
being present. It has been suggested that Gram-positive bacteria are more
likely to survive during air sampling than gram negative.
ES&T 3& H&S 3
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Dr. M.A. Broaders 47
Dept. Environmental Science, IT, Sligo
Gram-negative bacteria produce endotoxins that are pyrogenic and
induce local inflammatory responses. It is currently thought that
endotoxins may be responsible for a number of diseases in workers. Some
bacteria, like fungi, can produce spores whose characteristics are
discussed further in the section on fungi.
Bacteria are widely present in the environment and the presence of
certain odors, slime, and foam on a surface are often an indication of
bacterial growth. Certain types live in the human body while others live
outdoors on vegetation; therefore, when sampling indoors, those bacteria
associated with humans will predominate while outdoor samples will
contain mostly the other type. Bacteria are spread primarily through
inhalation, although bacteria that are very small are often dispersed on
skin scales. It has been estimated that 7 million skin scales are shed per
minute by humans, each containing an average of 4 viable bacteria.
Bioaerosols are usually associated with water, and an undisturbed source
of water or a humid environment is highly conducive to growth. When
water is aerosolized, droplets range in size, but larger droplets can
evaporate and become smaller, thus increasing the potential for
inhalation. Taps, showers, whirlpool spas, and cooling towers are all
sources. When bacteria attach to particles, they are often protected
against the environment.
The primary tools for collecting bacteria for culture are impingers
and cascade impactors. Screening samples can be collected with a slit
or centrifugal impactor.
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Dr. M.A. Broaders 48
Dept. Environmental Science, IT, Sligo
Legionella pneumophila
Legionnaires' disease became a concern when it became apparent that
aerosol from contaminated cooling towers could enter fresh air vents and
spread this agent to the HVAC system in buildings. Legionella
pneumophila, the agent in this case, is a rod-shaped, slow-growing, gramnegative bacterium. Ideal growth conditions require a temperature of
35—45°C and a pH of 6.9—7.0. The bacterium is ubiquitous in the
environment. It can coexist with amoebae and can survive and grow on
blue-green algae. The vast majority of outbreaks have been associated
with Legionella sero group 1, but other sero groups, if detected, are also
of concern, and control measures should be initiated if they are detected.
Legionella sero group 1 has been isolated from a variety of surface and
potable aquatic habitats. It is viable in tap water for more than a year.
Hot-water tanks, and in particular their bottom sediments, are excellent
media for its survival and proliferation.
Cooling towers are especially susceptible to contamination, since
their primary function involves inducing large amounts of air into large
amounts of flowing water; thus, they act as air scrubbers, washing out
dust, debris, pollen, insects, and plant materials. These bacteria have also
been known to build up in water softeners.
Most sampling for Legionella is done by collecting bulk samples of
suspected sources. One source considers heavy contamination by
Legionella to be counts greater than 10 colony-forming units per liter in a
bulk sample. Sampling should be done in both suspected and background
areas. As a precautionary measure, bulk samples of suspect sources of
Legionella should be collected every 6 months. Chemical analyses of
makeup water and system water should be performed monthly. Samples
should not be collected following cleaning or immediately after startup. It
is best to sample in the middle or end of each 6-month period during
normal operation.
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Dept. Environmental Science, IT, Sligo
The best places are system water dead spots or slow flowing areas;
however, these should be selected so they are not near incoming fresh
water or biocide treatment points.
Testing should precede but not follow slug feed of biocide.
In the course of evaluating the potential for this microorganism to be the
source of an outbreak, the operational procedures and facilities are
considered as important as the sources of microbial contamination and
dissemination. An investigation for Legionella in cooling towers would
focus on the following areas:
Temperature : If the normal operating temperature of the water is greater
than 30°C, there is a high risk of bacterial growth.
Contamination: Nonmetallic materials such as washers, coating, gaskets,
linings, and sealants in the system can harbor bacterial growth.
Stagnation : If the water is left standing for more than 5 days at a time,
there is a high risk of bacterial growth.
Particulate matter The sump can accumulate sludge, debris, scale and
bacterial growth.
Aerosol generation : If there is a significant amount of spray the
likelihood of spread is increased, especially if there is any possibility
of aerosol escaping from the cooling tower.
Susceptible populations : If the cooling tower is associated with any
buildings or sites occupied by susceptible people, such as hospitals or
schools, the risk is increased.
Other factors of importance include whether a responsible person is in
charge of the cooling system, the type of training provided to the staff
responsible for its upkeep, and the availability of adequate record
keeping.
If a routine test for Legionella is positive, the cooling tower should be
cleaned immediately. Following startup, the system should be resampled
within seven days. As Legionella require soluble iron for growth, control
of corrosion is an important factor. Often it involves adjusting the pH of
the water. Biocides are often used to kill Legionella and other waterborne
microbes, but their effectiveness depends on controlling water chemistry
including pH, alkalinity, and cycles of concentration (i.e., addition
periods).
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 50
Dept. Environmental Science, IT, Sligo
Endotoxin
Endotoxin is a distinct lipopolysaccharide (LPS) found in the outer
membrane of gram-negative bacteria, and it varies among bacterial types.
Endotoxin can be present in several forms: the bacteria, fragments of
bacteria membranes incorporated in dust, as well as the endotoxin
molecule itself. Endotoxin is considered highly toxic and is suspected of
causing pulmonary impairment in humans. Endotoxin has been
implicated as having a significant role in the development of byssinosis
from cotton dust exposures.ls It has been found in agricultural, industrial,
and office environments.
Endotoxin in air has been sampled using filters attached to personal
sampling pumps. Bulk samples are useful when endotoxin is suspected.
These must be collected in oven-baked glassware and must be analyzed
promptly.'
The most common analytical method for endotoxin is known as the
Limulus assay. Testing results are often reported as picograms per meter
cubed (p/M3), which is an extremely small amount of material. LPS is
inactivated by filter media, including 5.0-mm PVC, l.O-mm Teflon, 0.45mm MCE and Polyflon, the result being reduced concentrations when
testing is conducted. It is of note that currently there are a number of
variations both in the Limulus assay and in the extraction technique used
to remove endotoxin from the filter media; therefore, comparison of
results from different laboratories will depend on how similar their
analytical techniques are.
Given the fact that an agreed upon method that eliminates the problem of
loss of sample on filters has not yet evolved, the best approach to
sampling is to collect background and source samples and compare the
results rather than attempt to associate the hazard with some type of
standard. The same method should be used for all samples and similar
volumes should be collected.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 51
Dept. Environmental Science, IT, Sligo
FUNGUS AND MOLDS
The fungi class includes yeasts, mold, mildew, and mushrooms. Soil is
the most common habitat of the fungi, although many of the primitive
fungal groups are aquatic. Fungi occur on the surface of decaying plant or
animal materials in ponds and streams or grow on top of aqueous
industrial fluids such as metal-working coolants. They are common in
grain-handling facilities, paper mills, fruit warehouses, and agricultural
environments as well as indoor air environments. Fungal species
commonly encountered include Aspergillus, which is ubiquitous in the
soil and air, especially in agricultural products and in standing water,
whose spores are known to cause a variety of pulmonary effects; and
Histoplasma and Cryptococcus, found in bird droppings. Penicillium is a
mold that grows on damp organic materials and standing water and is
associated with hypersensitivity pneumonitis. Candida albicans is a yeast
that is ubiquitous and known to cause Candidiasis, a disease of the skin
and mouth that occurs in dishwashers, cooks, cannery workers, and others
who frequently have their hands in food-contaminated water. In
immunosuppressed individuals it can have systemic effects. Other fungi
of concern are Alternaria, Aureobasidium, Chaetomium, Cladosporium,
and Mucor.
Fungal-related diseases can be divided into two types: mycosis and
mycotoxicosis. Mycosis represents a variety of toxic effects, including
dermatitis, hypersensitivity pneumonitis, and some systemic diseases that
result from an infection by the organisms themselves. Mycotoxicosis is
produced by metabolites of various fungi and causes diseases such as
toxic aleukia and yellow rice disease.
Occupations associated with exposure to fungi include sawmill,
sugarcane, and cork workers as well as jobs where seeds and textile fibers
are handled. Other work environments conducive for the growth and
sporulation of fungi are farming, grain handling, mushroom cultivation,
insect rearing, and pharmaceutical manufacturing.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 52
Dept. Environmental Science, IT, Sligo
Like other microbes, fungi have specific nutritional requirements that
vary among the species and produce metabolic products, a classic
example being penicillin produced from the mold penicillium. Fungi are
also dependent on having water present. The presence of a moldy odor is
suggestive that fungi are growing.
A unique stage of some fungus' life cycle is the spore stage consisting
of a wide variety of shapes in a very broad size range (<2 mm to >100
mm). In this stage they form a durable coating over the exterior and
become dormant. Spores can be classified by size, morphology, and
color, allowing them to be categorized into different taxonomic groups.
Since spores are relatively hardy structures, they can survive in dry
environments and become airborne when disturbed. Fungal spores are
released into the air either by mechanical means, such as wind or other
agitation, or biologically by specialized (active) spore discharge
mechanisms usually occurring during periods of high relative humidity.
When airborne, fungal spores tend to travel as single units.
Certain foods such as peanuts and animal feed contain fungal spores that
begin growing and producing aflatoxins when environmental conditions
(time, temperature, moisture, nutrients, and pH) are favorable. Aflatoxins
are a group of chemically similar compounds known to be acutely toxic
and carcinogenic at low doses, and are metabolites of two common fungi:
Aspergillus favus and Aspergillus parasiticus. If fungal spores are
suspected, water reservoirs should be identified and bulk samples should
be collected at all suspected sources. Suitable niches for growth and
sporulation include stored food, house plants, air conditioners,
humidifiers, cold air vaporizers, books and papers, carpets, and damp
areas.
The primary air sampling tools used for fungi spores are slit impactors
and filters. Screening samples can be collected with a centrifugal
impactor.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 53
Dept. Environmental Science, IT, Sligo
VIRUSES
Viruses represent a unique class of agent and are different from cellular
organisms. A virus alternates in its life cycle between two phases: one
extracellular and the other intracellular. In its extracellular phase, a virus
exists as an inert, infectious particle, or virion. A virion consists of one or
more molecules of nucleic acid, either DNA or RNA, contained within a
protein coat, or capsid. In its intracellular phase, a virus exists in the form
of replicating nucleic acid, either DNA or RNA. Viruses utilize the host
cell for replication (reproduction) and thus are intracellular parasites. In
the extracellular phase, some viruses are quite stable and resistant to heat
and light.
Viral infections may be acquired from vectors such as needles or from
handling of animals or animal products and from humans. Laboratory
acquired infections may result from needle sticks; animals; clinical or
autopsy specimens; or contaminated glassware. Diseases include rabies,
cat- scratch disease, and viral hepatitis (both serum and infectious).
Viruses survive best in situations where high humidity and moderate
temperatures are present. Situations where water containing a virus is
being aerosolized are especially conducive to viral multiplication.
Collection of viruses often requires very specific techniques, although
some have been collected on filters. Viruses have also been collected on
the slit sampler and the multistage cascade impactor.
Viruses are usually measured as either infectious units or total
particle numbers. While it is important to be aware that viruses may be a
cause of an outbreak of illness, it is unlikely that air sampling would be
useful in most situations, due to complicated analytical techniques
usually requiring that a live species be injected and the degree of
specialization necessary to perform these analyses. Instead, a more
common technique is to identify the symptoms associated with a suspect
virus and determine if the disease is present through a physician's clinical
evaluation.
Collection of viruses often requires very specific media, although some
have been collected on filters.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 54
Dept. Environmental Science, IT, Sligo
OTHER MICROORGANISMS
Spirochetes have a unique cell structure relative to other bacteria in that
they have very long, wormlike bodies. Thus, they can swim in liquid
media and are found in mud and water.
Mycoplasmas, the smallest known cellular organisms, are a large and
widespread group. The first member of this group to be identified was the
agent of bovine pleuropneumonia.
Factors That Affect the Survival and
Dispersion of Bacteria and Viruses in Wastewater Aerosols
Relative humidity
Bacteria and most enteric viruses survive longer at
high relative humidities, such as those occurring
during the night. High RH delays droplet evaporation and retards organism die-off.
Sunlight
Sunlight, through UV radiation, is deleterious to
microorganisms. The greatest concentration of
organisms in aerosols from wastewater occurs at
night.
Open air
It has been observed that bacteria and viruses are
inactivated more rapidly when aerosolized and
when the captive aerosols are exposed to the open
air than when held in the laboratory.
Wind speed
Low wind speeds reduce biological aerosol
transmission .
Temperature
Increased temperature can also reduce the viability
of organisms in aerosols, mainly by accentuating
the effects of RH. Pronounced temperature effects
do not appear until a temperature of 80°F (26°C)
is reached.
ES&T 3& H&S 3
Air Pollution (Microbiology) Practicals
Dr. M.A. Broaders 55