Aeromicrobiology

1. Assessment of intra and extramural Air quality

a. Sampling air-borne microbes
b. Cultivation of air microbes
c. Microbiological air standards
Water microbiology
1. Municipal water quality
a. Bacteriological examination of water (Coliform MPN test)
b. Membrane filter technique
2. Waste water management
a. Biological oxygen demand
b. Chemical oxygen demand
3. Detection of other microorganisms in water environment
a. Detection of water-borne enteric viruses
b. Detection of water-borne parasites
4. Use of Bacteriophages in water quality assessment
Soil microbiology (Similar to the one contained in SOIL MICROBIOLOGY)
 ASSESSMENT OF THE INTRAMURAL AIR QUALITY BY SEDIMENTATION

LEARNING OBJECTIVES:
At the end of this experiment, student should be able to:
Collect air sample in a controlled environment e.g. Laboratory, Room, Operation theaters
Incubate the sampled air
Determine microbial concentration in the sampled air

INTRODUCTION
Microorganisms present in air are believed to be transient and they originate from soil, water,
plants and animals. A collection of airborne biological particles is called a bioaerosol.
Bioaerosols are generated by a wide variety of natural and human-made processes including
coughing, sneezing, wave action, splashes, wind, cooling towers, ventilation systems, etc.

PRINCIPLE
Sedimentation method of air sampling is a non-quantitative method used in which agar medium
is exposed to the environment and airborne microorganisms are collected primarily by settling.
This method is often used because it is inexpensive and easily performed.

EQUIPMENT AND REAGENTS

Nutrient agar plate
Sabouraud dextrose agar (SDA) plate
Incubator

PROCEDURE
1. Place open Petri dishes (containing agar) to different locations in a laboratory and expose
for 5, 10 and 15 minutes.
2. Close the Petri dishes and label them.
3. Put the Petri dishes into an incubator and incubate them for 24 hrs
4. For Sabouraud dextrose agar (SDA) plate, incubate for one week at room temperature.
OBSERVATION
Observe the colonies formed on the surface of the agar plates and compare media with difference
in exposure times and sampling locations.

RESULTS AND INTERPRETATION

Different types of bacterial and fungal colonies will be seen all over the nutrient agar and
Sabouraud dextrose agar plates. The number of colonies will increase with longer time of
exposure.
 MICROBIOLOGICAL AIR QUALITY STANDARDS
In the field of aeromicrobiology, there is lack of any official reference limit for microbiological
quality of both indoor and outdoor; hence interpretations of results are difficult. However,
Residential Limit Values (RLV) is one of the valuable proposals of reference data presented by
Górny and Dutkiewicz on the WHO Expert Meeting in Berlin, 2002. This can serve as a
provisional air quality standard in intramural settings¶¶.

Occupational Exposure Limits (OEL) and Residential Limit Values (RLV)

¶¶
GÓRNY, R.L. and DU TKIEWICZ, J. Bacterial and fungal aerosols in indoor environment in Central and Eastern European Countries. Annals
of Agricultural & Environmental Medicine 9(1), 17-23, 2002.
 BACTERIOLOGICAL EXAMINATION OF WATER (COLIFORM MPN TEST)

LEARNING OBJECTIVES
At the end of this experiment, you will be able to:
Detect coliform bacteria in water by the most probable number (MPN) method
Inoculate water sample into lactose broth
Observe tubes for gas production
Confirm coliform detection by streaking of EMB and Endo agar
Calculate coliform concentrations in the water via most probable number (MPN) procedure

INTRODUCTION
Water can serve as vehicle for transmission of infectious pathogen including bacteria, viruses,
and protozoa. Most of the microorganisms transmitted by water usually grow in the intestinal
tract of man and leave the body through feces. Contamination of water with feces renders it unfit
for drinking. Coliform bacteria occur normally in the intestines of humans and other warm-
blooded animals and are discharged in great numbers in human and animal waste. In polluted
water, coliform bacteria are found in densities roughly proportional to the degree of fecal
pollution. When members of the coliform group are present, other kinds of microorganisms
capable of causing disease also may be present. Hence coliforms have been shown to be useful
indicators of the presence of fecal contamination.

PRINCIPLE
Most Probable Number (MPN) test has been the method most commonly used for the detection
of coliforms in water. It consists of three steps: a presumptive test, a confirmation test, and a
completed test.
Water to be tested is diluted serially and inoculated in lactose broth, coliforms if present in water
utilize the lactose present in the medium to produce acid and gas. The presence of acid is
indicated by color change of the medium and the presence of gas is detected as gas bubbles
collected in the inverted Durham tube present in the medium. The number of total coliforms is
determined by counting the number of tubes giving positive reaction (i.e both color change and
gas production) and comparing the pattern of positive results (the number of tubes showing
growth at each dilution) with standard statistical tables.

EQUIPMENT AND REAGENTS

Water sample
3 test tubes containing Durham tubes and double strength lactose broth (DSLB)
6 test tubes containing Durham tubes and single strength lactose broth (SSLB)
1 10-ml pipette
1 1-ml pipette
Incubator at 35°C

PROCEDURE
Step one: Presumptive Test
1. Set up a one set of three DSLB and two sets of three SSLB tubes and label each tube
according to the amount of water that is to be dispensed to it: 10 ml, 1.0 ml, and 0.1 ml,
respectively.
2. Mix the bottle of water to be tested by shaking 25 times.
3. With a 10 ml pipette, transfer 10 ml of water to the DSLB tubes.
4. With a 1.0 ml pipette, transfer 1 ml of water to each of the middle set of SSLB tubes and 0.1
ml to each of the last three SSLB tubes.
5. Incubate the tubes at 35°C for 48 h.

OBSERVATIONS
Examine the tubes and record the number of tubes in each set that have a gas bubble(s) in the
inverted Durham tube. Determine the MPN by referring to the Table below.
Record this data for your next experiment and also for report.
Step two: Confirmatory test
Equipment and reagents
Incubated tubes from the previous experiment
1 Petri plate of EMB agar or any of Endo agar, Brilliant green lactose bile broth (BGLB),
Eijkman’s medium
Inoculation loop
Bunsen burner
Procedure
1. Select one positive lactose broth tube from the presumptive test
2. Using inoculating loop, streak a plate of EMB agar
3. Incubate the plate for 24 h at 35°C.
Observations
Observe for nucleated colonies with dark centers on EMB (pinkish - red colonies on Endo agar)
media.
Step three: Completed test
Equipment and reagents
Incubated petri dishes from the previous experiment
Lactose broth
Nutrient agar slant
Inoculation loop
Procedure
Using an inoculating loop, pick a small colony with dark centers and a green metallic sheen in
the previous petri dish
Inoculate it into lactose broth tube and a nutrient agar slant
Incubate the broth tube and agar slant at 37°C for 24 hours.
Observation
1. Check for gas production in the lactose broth tube.
2. Make a Gram stain from the organisms on the nutrient agar slant and observe for Gram
Negative, non-spore forming.
3. Record results
RESULTS AND INTERPRETATION
Presumptive test: Any test tube in which gas production was observed is considered positive.
The number of tubes with gas production is counted based on the strength of the LB media. For
example, if there is formation of gas in all the 3 tubes in DSLB, 1 tube in SSLB (containing 1m
water sample) and none in the last SSLB (containing 0.1m water sample) tube, then the MPN is
read on the Table above as 3-1-0. The MPN index for this figure (as shown on the Table) is 43.
Therefore, it can be said that: in 100ml of the water sample, there are probably 43 coliforms,
and thus the water is PRESUMED to be contaminated and unsafe for drinking.
Confirmatory test: Gram-negative lactose fermenters (coliforms) that grow on EMB medium will
produce “nucleated colonies” (dark centers) and Escherichia coli and Enterobacter aerogenes
are the only organisms expected to grow. The two organisms can be differentiated on the basis of
size and the presence of a greenish metallic sheen. Escherichia coli colonies are small and have
this metallic sheen, whereas E. aerogenes colonies usually lack the sheen and are larger.
If only E. coli or if both E. coli and E. aerogenes appear on the EMB plate, the test is considered
positive. If only E. aerogenes appears on the EMB plate, the test is considered negative.
Completed test: If the organism is a Gram-negative, nonspore-forming rod and produces gas in
the lactose tube, then it is positive that coliforms are present in the water sample.
NB: While conducting the completed test, you may proceed with IMViC test for proper
identification of the organism in positive EMB plates.
 MEMBRANE FILTER TECHNIQUE

LEARNING OBJECTIVES
At the end of this experiment, student should be able to:
Filter water samples through membrane filters
Incubate membrane filters on media agar
Observe and count colonies of coliform and fecal coliform bacteria
Calculate coliform and fecal coliform bacteria in water

INTRODUCTION
The Membrane Filtration (MF) method is used to estimate bacterial populations in water that is
low in turbidity. This method is especially useful for large sample volumes or for many daily
tests.

PRINCIPLE
Membrane filters have a known uniform porosity of predetermined size (generally 0.45 µm)
sufficiently small to trap microorganisms. Using the membrane filter technique, sample is passed
through the membrane using a filter funnel and vacuum system. All organisms in the sample are
concentrated on the surface of the membrane. The membrane, with its trapped bacteria, is then
placed in a special plate containing a pad saturated with the appropriate medium. The passage of
nutrients through the filter during incubation facilitates the growth of organisms in the form of
colonies, on the upper surface of the membrane. Discrete colonies thus formed can be easily
transferred to confirmation media.

EQUIPMENT AND REAGENTS

Water sample
Forceps
Ethyl alcohol for flame sterilization
Bunsen burner
6 sterile 50 × 12 mm Petri dishes with tight cover
6 sterile 0.45m pore, 47 mm diameter membrane filters with pads
1 non sterile 1 liter filter flask
1 filter unit,
Nutrient broth
Nutrient agar plate
1 sterile 10 ml pipette

PROCEDURE
1. Collect the sample and make dilutions (if necessary).
2. Select the appropriate nutrient broth. Dispense the broth into a sterile Petri dish, evenly
saturating the absorbent pad.
3. Flame the forceps, and remove the membrane from the sterile package.
4. Place the membrane filter into the funnel assembly.
5. Flame the pouring lip of the sample container and pour the sample into the funnel.
6. Turn on the vacuum and allow the sample to draw completely through the filter.
7. Rinse funnel with sterile buffered water. Turn on vacuum and allow the liquid to draw
completely through the filter.
8. Flame the forceps and remove the membrane filter from the funnel.
9. Place the membrane filter into the nutrient agar (or Endo agar) Petri dish.
10. Incubate at the 30° C and for 24hrs period.
11. Count the colonies under microscope.
12. Confirm the colonies and report the results.
OBSERVATIONS
Examine the incubated plates using a low power (10–15¥ magnification) dissection microscope.
Coliform colonies are red or pink showing a bright green metallic sheen. Colonies without the
green sheen are non-coliforms. Count the coliform colonies and record the results in the form
provided.

RESULTS AND INTERPRETATION

The bacterial count per 100 ml is calculated as follows:
Count on filter
Number of coliforms in 100ml of water sample = Volume of water filtered
× 100
 BIOCHEMICAL OXYGEN DEMAND (BOD)

LEARNING OBJECTIVES
At the end of this experiment, student should be able to:
Measure the amount of biodegradable organic matter in wastewater.
Prepare dilution water
Make dilutions of wastewater to be tested (where necessary)
Measure amount of oxygen in the dilutions
Incubate the samples for 5 days at 20°C and measure amount of oxygen remaining
Calculate the amount of BOD5

INTRODUCTION
Biochemical Oxygen Demand is the amount of dissolved oxygen needed (i.e. demanded) by
aerobic microorganisms to break down organic material present in a given water sample at
certain temperature over a specific time period. The BOD5 value is most commonly expressed in
milligrams of oxygen consumed per litre of sample during 5 days of incubation at 20 °C and is
used as an indicator of the degree of organic pollution of water.

PRINCIPLE
BOD is the amount of dissolved oxygen in water consumed by microorganisms for the
biochemical oxidation of organic and inorganic matter. The amount of oxygen being consumed
can give a rough idea of how much organic matter is left in the water. If the amount of oxygen is
used by organisms for the oxidation of organic matter decreases after wastewater treatment, it is
assumed that the organic content has decreased also. Therefore, the waste water can be
discharged to the environment without causing serious contamination.

EQUIPMENT AND REAGENTS

Water sample
Dissolved Oxygen Sensor
3 BOD bottle, 300 mL, with glass stoppers and plastic caps
Buffer dilution
3 plastic bottles
Distilled water
Measuring cylinder
Incubator

PROCEDURE
1. Saturate the test water with air by pouring 1 L of it into a 2-L bottle, capping, and shaking
vigorously.
2. Allow this water to sit undisturbed with the cap off until the air bubbles dissipate and the
water temperature is the same as the room temperature.
3. Using a Dissolved Oxygen Sensor that has just been calibrated, measure the DO in the test
sample and record this value.
4. Pour about 50 mL of buffer dilution solution into each of the BOD bottles that will contain
diluted test samples.
5. Prepare a range of 3 sample : distilled water dilutions (300ml : 0ml, 100ml : 200ml and 50ml :
250ml) by measuring each specified volume of test water and pour it into the BOD bottle using a
graduated cylinder.
6. Fill the BOD bottles to the brim with the distilled water, and cap the bottle so it is air-tight.
7. Wrap bottles in aluminum foil to block out light.
8. Incubate the BOD bottles at room temperature for 5 days in a dark place (especially if the
BOD bottles are not completely opaque).
Note: If the BOD bottles are not completely opaque, incubate in a dark room or box.
Observations
After 5 days, open each bottle and measure the dissolved oxygen with the sensor.

RESULTS AND INTERPRETATION

Use the following formula to calculate the final BOD5 value:
BOD5 (mg/L) = (D1 – D2)/P
Where:
D1 is the initial DO of the sample
D2 is the final DO of the sample after 5 days, and
P is the decimal volumetric fraction of sample used.
For example, P value of the sample with 100 ml : 200ml dilution is P = 100/300 = 0.33
Note: If no dilution was necessary, then P = 1.0 and the BOD5 is determined by D1 – D2.
NB: this experiment is for water that is not disinfected by addition of chlorine or other
disinfectants. In the case where disinfection is suspected or confirmed, addition of bacterial
cells is required.
 DETECTION OF WATER-BORNE ENTERIC VIRUSES

LEARNING OBJECTIVES:
To detect coliphages in water/sewage
Incubate water or sewage sample with host bacteria
Incubate 24–48 hrs
Count viral plaques in the bacterial lawn
Calculate the number of coliphage in the sample

INTRODUCTION
The use of bacteriophages as indicators of the presence and behavior of enteric bacteria and
animal viruses has always been attractive because of the ease of detection and low cost
associated with phage assays. In addition, they can be quantified in environmental samples
within 24 hours as compared to days or weeks in some other methods. Bacteriophages are also
used as indicators of sewage contamination, efficiency of water and wastewater treatment, and
survival of enteric viruses and bacteria in the environment.

PRINCIPLE
Bacteriophages in water are assayed by addition of a sample to soft or overlay agar along with a
culture of bacteria (eg. E. coli, if coliphage is suspected) that is in the log phase of growth. The
phage attaches to the bacterial cell and lyse the bacteria. The bacteria produce a confluent lawn
of growth except for areas where the phage has grown and lysed the bacteria. These resulting
clear areas are known as plaques. A soft agar overlay is used to enhance the physical spread of
the viruses between bacterial cells.

EQUIPMENT AND REAGENTS

Sewage or waste water sample
3–4 h nutrient broth culture of E. coli
2 tubes containing 9ml of saline buffer
4 test tubes of 3 ml each of semisolid agar (0.7% of nutrient agar or trypticase soy)
4 Petri dishes with solidified agar (nutrient agar or trypticase soy)
6 1-ml pipettes
Water bath at 45–48°C
Paper towels
Incubator at 37°C
PROCEDURE
1. Bring the sample of sewage or waste water to your bench.
2. Dilute the sample 1 : 10 and 1 : 100 by making 10-fold dilutions in saline buffer by
transferring 1.0 ml to 9ml of the buffer.
3. Melt four tubes of semisolid agar by placing in a water bath at 45–48°C and allow 15 min for
the temperature of the agar to adjust to 45°C.
4. To the first tube add 1 ml of a log phase broth culture of E. coli 1 and 1ml of undiluted sample
while the tube is still in the water bath.
5. Remove the tube from the water bath and gently rock between your hands to mix the
suspension for 2–3 seconds.
6. Wipe the water from the tube with a paper towel.
7. Pour the agar over the Petri dish containing solid agar.
8. Quickly rotate the plate to spread the molten agar. Be sure the agar covers the entire surface.
9. Repeat Step 4 using 1 ml of bacteria and 1 ml of each phage dilution.
10. Allow the molten agar to solidify
11. After the agar has solidified, invert the Petri dishes and incubate at 37°C for 24 h.

OBSERVATIONS
After the incubation period, observe the surface of the agar for any small hole (s) or clear zone or
plaques.
Count the number of plaques on each dilution and calculate the concentration of phage in your
original sample.

RESULTS AND INTERPRETATIONS

Presence of clear zones or plaques indicates the presence of bacteriophages in the water sample.
The number of plaques determines the concentration of the phages in water. It is expressed as
plaque forming units PFU/ml.