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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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