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picture1_Processing Pdf 90853 | Experiment 9


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File: Processing Pdf 90853 | Experiment 9
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 ...

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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.
                                                                                                                                                                                                            45
                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.
                                                                                                              46
                  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.
                                                                                                                           47
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