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Microbiological Analysis
EXPERIMENT 9 MICROBIOLOCAL of Air and Water
ANALYSIS OF AIR
AND WATER
Structure
9.1 Introduction
9.2 Objectives
9.3 Experiment
Requirements
Procedure
Observation
9.4 Interpretation
9.1 INTRODUCTION
The air can contaminate foods including dairy products during processing with
pathogenic and spoilage microorganisms resulting in deterioration of finished product.
The common aerosol sources in a dairy plant are personnel, dust, insects, rodents,
floor drains, and ventilation system and water especially when used under pressure
in cleaning and sanitization procedures. Food contact surfaces can harbour
microorganisms and can be potential source for the aerosol formation in processing
areas. Hence it is important to regularly monitor the microbiological quality of air
in dairy environment. The entrance of any airborne microorganism into pasteurized
and sterilized dairy products is highly undesirable. To make dry milk products free
of pathogens e.g. Salmonella, it is crucial to ensure low count of air to be used
for cooling and instantizing of dry milks. Air monitoring is a part of Good
manufacturing practices (GMP) and Hazard analysis and critical control points
(HACCP) programmes. There are two basic methods of air monitoring: passive
and active. Active sampling method is an air sampler technique based on the
number of microorganisms in a given volume of air sucked by a sampler that
allows the recovery of viable particulate matter on a solid medium. Another
method, considered, as Passive method of sampling comprises sedimentation
technique based on settling of viable particles on the surface of a solid agar
medium. Passive monitoring can only detect microorganisms that fall onto the
surface of the medium while active air sampling systems provide a more
representative sample. Large quantity of water is needed for many operations in
dairy plant such as steam generation, cooling, healing in heat exchangers and for
cleaning and washing hence it is important to monitor the microbiological quality
of water supply for examination of water SPC and coliform test are carried out
on regular intervals.
9.2 OBJECTIVES
After going through this unit we should be able to
l Recall the significance of monitoring of microbiological quality of air and water in
a dairy environment; 44
l Carry out sampling and microbiological analysis of air and water; and Microbiological Analysis
of Air and Water
l Interpret the results
9.3 EXPERIMENT
i. Requirement
i) Examination of Air
a) Air sampler
b) Sterile petriplates
c) Culture Medium: Plate count Agar (PCA), Potato Dextrose
Agar(PDA),Violet Red Bile Agar(VRBA), Baird Parker Agar(BPA)
d) Autoclave
ii) Examination of Water
i) Sterilized ground glass stoppered bottle (8 oz capacity)
ii) Tryptone glucose agar
iii) Dilution blanks (9 ml)
iv) Sterilized petridishes
v) 1 ml and 10 ml bacteriological pipettes
vi) Ethyl alcohol
vii) MacConkeys broth tubes (containing Andrade’s indicator and Durham
tubes) single and double strength
viii) Sample of water
ix) EMB of Endo agar
ii. Procedure
i) Microbiological analysis of Air
a) Sampling of air
Samples may be taken as follows:
l At opening in equipment,
l At selected point for testing the quality of air in the room, for example
where products are filled into containers,
l In areas where employees are concentrated.
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b) Settling plate/ sedimentation technique (Passive Method ) Microbiological Analysis
of Air and Water
l Prepare petriplates with 20 ml of with melted culture medium (PCA,
PDA, VRBA or BPA). Allow the media to set and harden,
l Remove the tops from the plates and expose them for 5, 10, 15 and 30
minutes.
o
l Replace the tops and incubate plates at 35 C/48h for aerobic plate
o o
count, 25 C/3-5 d for yeasts and moulds, 37 C/48 h for total
coliforms and Staphylococcus aureus.
l Count the number of colonies in each plate at the end of incubation
period.
3 3
l Express the results as cfu/plate or cfu/cm /min or cfu/Cm /week.
l Examine mold like colonies using the low power objective of a
microscope.
c) Impaction Method (Active Method)
l Use an air sampler for suction of a volume of 100L, 500L or 1000L
of air from the given area followed by impaction on culture media laid
in petriplates.
o
l First sterilize the air sampler’s lid by autoclaving at 121 C for 15
minutes and then sanitize with 70 % ethyl alcohol before and after
each sampling.
l Incubate the plates in the same conditions as in settling plate technique
3
and express the results as cfu/cm .
i) Microbiological Analysis of Water
a) Sampling of water
l Allow the water to run for 3 to 4 minutes from the outlet of water say a
tap.
l Clean the inside and outside of the opening of the outlet.
l Sterile the opening (if metallic) by heating it with a blow lamp or a piece
of ignited cotton – wool soaked in methylated spirit or alcohol.
l Again allow the water to run slowly for about a minute.
l Hold the sample bottle in one hand near the tap, remove the stopper
with the other hand, flame the mouth of bottle, quickly bring the bottle
below the running stream of water and when the bottle is nearly full,
take it out and quickly replace the stopper.
l Carry out the sampling quickly to prevent undue exposure of the bottle
to environment.
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l Transfer and store the sample bottle in refrigerator till examination. Microbiological Analysis
of Air and Water
b) Microbiological analysis
ii) Plate count
l Shake the water sample thoroughly by moving the bottle up and down
25 times,
l Prepare 2 serial dilutions, 1 in 10 and 1 in 100 using the dilution blanks.
o o
l Set twelve petridishes a set of 6 each for incubation at 37 C and 22 C.
l Transfer 1 ml of the sample directly from the bottle (zero dilution), 1 ml
from dilution 1 (1/10) and 1 ml from dilution 2 (1/100) into 3 separate
petridishes (in duplicate). Mark the dilutions (0, 1, and 2) on the
respective plates.
l Add agar into the plates, mix with the inoculum and allow it to set.
o
l Invert the plates and incubate one series at 37 C for 48 h and the other
o
at 22 C for 72 h.
l At the end of the incubation, count the plates containing from 30 to 300
colonies. In the case of the zero dilution plates, the number of colonies
may be counted even if it is less than 30.
l Calculate the number of organisms per ml of water.
iii) Coliform test
l Transfer 1 ml and 1/10 dilution portions of the sample into 5 tubes each
of single strength MacConkey’s broth.
l Transfer 10 ml portions of the sample in to 5 tubes containing 10 ml of
double strength medium.
l Thus there will be 3 sets of 5 tubes each containing 0.1 ml, 1.0 ml and
10 ml of the sample.
o
l Incubate the tubes at 37 C for 25 h and tubes showing no change should
be incubated for another 24 h.
l Observe for the production of acid (red colour) and gas in Durham
tubes. Presence of acid and gas in 3 out of 5 tubes in any dilution is
considered as a positive presumptive test for presence of coliforms in
water sample.
l Record your observations and calculate the number of bacteria per 100
ml by consulting the Most Probable Number (MPN) table.
l Confirmation test on positive tubes can be carried out as in case of milk.
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