EXPERIMENT 8 MICROBIOLOGICAL ANALYSIS OF MILK AND MILK PRODUCTS

Transcription

1 EXPERIMENT 8 MICROBIOLOGICAL ANALYSIS OF MILK AND MILK PRODUCTS Determination of acid vaue in ghee Structure 8. Introduction 8. Objectives 8. Experiment Principe Requirement Procedure Observation Resuts 8. Precaution 8. INTRODUCTION Microbioogica anaysis of mik and mik products provides vauabe information regarding: Market mik contro and grading, Improvement of practices of production (at farm), handing and processing, Screening of mik suppies for their suitabiity for processing or preparation of mik products, Pubic heath aspect. Some of the common microbioogica tests (excuding dye-reduction tests) carried out for testing dairy products are described in this chapter. 8. OBJECTIVES After going through this unit you wi be abe to: reca the types of microorganisms present in mik and mik products and their significance; judge and grade the microbioogica quaity of dairy products; evauate the hygienic conditions maintained during the production, handing and processing of mik; and carry out the various microbioogica methods commony used for testing of dairy products.

2 8. EXPERIMENT Microbioogica Anaysis of Mik and Mik Products i. Principe Direct Microscopic Count (DMC) This method consists of examining under a compound microscope stained fims of a measured voume of mik or mik products spread and dried on gass sides over a specified area. The major advantages are: The rapid estimation of the tota bacteria popuation of a sampe of mik/ mik products. Recognition of distinctive shapes and arrangements of bacteria and somatic ces in fims as faciitated by staining, Reveation of usefu information regarding the tracing of possibe sources of contamination. Standard Pate Count Method (SPC) This method empoys universa standardization of equipment, materias and incubation methods. It aims at determining the popuation of viabe bacteria in the sampe of mik/ mik products. A sma quantity of the sampe is mixed with the appropriate nutrient agar medium and poured into a Petri dish. The agar is aowed to set and pates are incubated at specific temperature for a definite period of time. The bacteria coonies grown on the agar surface during incubation are counted presuming each coony to have grown from one bacterium or bacteria cump present in the inocuum. The standard pate count is estimated by mutipying number of coonies with diution factor. This method is specificay suitabe for foowing purposes: Estimation of number of bacteria in pasteurized mik or mik products, In-ine testing of products at various stage of processing, Detection of the sources of contamination. Cutured dairy products or dairy products, to which a bacteria cuture has been added, however are not tested ordinariy by this method. The ratio of the standard pate count to direct microscopic count has been reported to be :. Count of Thermoduric, Thermophiic and Psychrotrophic bacteria a) Thermoduric count In the dairy industry those bacteria that survive pasteurization but do not grow at this temperature are considered as thermoduric bacteria. The major sources of contamination are poory ceaned and sanitized utensis and equipment on farms and in processing pants. Their undesirabe growth in mik and mik products resuts in spoiage of dairy products such as acids/ rennet coaguation, peptonization and off favour. The thermoduric count in dairy industry is used as

3 A test of sanitization of dairy utensis, A means of detecting sources of organisms responsibe for high counts in pasteurized products. Microbioogica Anaysis of Mik and Mik Products This test is carried out by determining the bacteria count in raw mik subjected to aboratory pasteurization (heating of mik at 6.5 o C for 0 min in a water bath) with the standard pate count technique. b) Thermophiic count The term thermophiic bacteria in dairy industry appies particuary to those bacteria which grow in mik hed at eevated temperature (55 o C or higher) incuding pasteurization. These organisms enter mik from various sources on the farm or from poory ceaned equipment in the processing pant. When mik is hed at high temperatures for onger duration, these bacteria rapidy mutipy in number and may cause favour defects or probems with respect to bacteria standards. Thermophiic count is obtained by the SPC method with incubation temperature of 55 o C. c) Psychrotrophic count - In the dairy industry the term psychrotrophic indicates organisms capabe of appreciabe growth in mik and mik products at commercia refrigeration temperature irrespective of their optimum growth temperature. The number of psychrotrophic bacteria in raw mik depends on sanitary conditions prevaiing during production and on time and temperature of mik storage before processing. These bacteria are generay non-pathogenic, but in dairy products they may be hed responsibe for. Production of off-favours, Loss of favour in cutured mik products, Discooration of mik products, Decrease in the yied of cheese, Probem in meeting bacteria standards. The SPC method with ow temperature incubation is used to enumerate psychrotrophic organisms. iv) Coiform Test The coiform group of bacteria comprises a aerobic and facutative anaerobic, gram-negative, non-spore forming rods abe to ferment actose with production of acid and gas at 7 o C within 8 h. One source of these organisms is the intestina tract of human and animas. Their presence in mik and mik products is indicative of possibe faeca contamination athough some species (e.g. Enterobacter aerogens) may be derived from feeding materias and soi. As these organisms are heat abie, their presence in pasteurized mik is considered to indicate post-pasteurization contamination. For testing presence of coiforms in mik and mik product, a sma quantity of the product (.0, 0. or 0.0 m) is added to iquid or soid media containing actose and bie sat with a suitabe indicator. Production of acid and gas in iquid media and appearance of typica coiform coonies on the pates is taken as evidence of coiform contamination. A few other bacteria, such as those beonging to the genus Costridium and Bacius and certain

4 yeasts aso produce acid and gas under these conditions giving rise to fase positive resut. Hence, the test commony empoyed to detect the presence of coiform bacteria in mik is caed presumptive coiform test and in the event of doubt the confirmed test is conducted to ascertain presence of coiforms in dairy products. Microbioogica Anaysis of Mik and Mik Products v) Yeast and Moud count Yeasts and mouds are specia cass of microorganisms beonging to group fungi. Yeasts are singe ce organisms arger than bacteria. They reproduce by budding and aso by formation of spores. They are commony found in soi, fruits, and dairy products e.g. butter & cheese. Yeasts are used as starter organisms in manufacture of fermented mik products e.g. Kefir and Koumiss. Mouds occur in fiamentous forms and are arger than bacteria. Mouds are often present in air and cause contaminations and subsequent spoiage of dairy products e.g. cream, butter, cutured mik products, indigenous mik products, and condensed mik. Their presence in dairy products indicates improper pasteurization and poor sanitary conditions. Mouds are aso known to produce mycotoxins e.g. afatoxins. Some of the mouds are used for ripening of certain varieties of cheese. ii. Requirements i) Direct microscopic count method i) Cean grease-free sides with one square centimeter area ceary marked on each of them. ii) Breed s pipettes caibrated to deiver 0.0 m of mik. iii) Neede with bent point for spreading mik. iv) Compound microscope. v) Stage micrometer side rued in mm. vi) Newman s strain. ii) Standard pate count method i) Incubator. ii) Bacterioogica deivery pipettes (.0 and. m). iii) Diution banks (9 or 99 m). iv) Tryptone gucose agar or mik agar. v) Petridishes (outside diameter 98 mm; inside diameter 9 mm; depth 5 mm). iii) Thermoduric, Thermophiic and Psychrotrophic count i) Water bath maintained at 6.5-o-C. ii) Test tubes.

5 iii) 0 m and m bacterioogica pipettes. iv) Petridishes. Microbioogica Anaysis of Mik and Mik Products v) Diution banks (9 and 99 m). vi) Tryptone gucose agar/ mik agar. vii) Thermometer; stopwatch, ice cod water. viii) Incubator iv) Coiform Count i) Bacterioogica pipettes (,., and 0 m). ii) MacConkey s broth tubes with Durham s fermentation tubes. iii) MacConkey s agar iv) Eosine Methyene bue agar v) Endo agar vi) Diution banks. vii) Test tubes viii) Petridishes. ix) Inocuation neede v) Yeast and Moud Count i) Diution banks. ii) Potato dextrose agar. iii) Pipettes (.0 and 0.0 m). iv) Petridishes v) Incubator iii. Procedure i) DMC Method a) Determination of Microscopic Factor Pace the stage micrometer on the stage the microscope and focus on the scae first with the 6 mm objective and then with the oi immersion objective. Count the number of sma divisions (0.0 mm each) in the maximum diameter of the fied and thus determine the diameter of the fied. The microscopic factor (MF) is cacuated as foows: 5

6 Area f smear (00 sqmm) 0,000 MF = = Area of microscopic fied Voume of mik (0.0m).6 r Microbioogica Anaysis of Mik and Mik Products b) Preparation of Mik Spear Mix the sampe of mik thoroughy by shaking. Draw mik into the Breed s pipette above the graduation mark, and adjust the voume of the sampe to exacty 0.0 m mark. Touch the tip of the pipette to the centre of a one square centimeter area on a side and expe the entire voume of mik. Spread the portion of mik uniformy over the centre of one square centimeter area on the side with the hep of famed bent pint neede. Dry the smears at 0 o 5 o C within 5 minutes. c) Staining the Fims Dip the sides in Newman s strain (in a jar) for ½ to minute. Remove excess strain by aowing water to run over from one end to another end. Air-dry the smear. d) Microscopic Examination Observe the smear under oi immersion objective. Count the singe organisms or we-isoated cumps of ces on a number of microscopic fieds. The fied for counting shoud be so seected to represent a parts of the fim as foows: Average number of cumps/ fied Number of fieds to be counted ii) Standard Pate Count Method a) Preparation of Diutions Prior to remova of the sampe from its container, thoroughy and vigorousy mix contents to ensure the samping of representative portion. Before opening a sampe container, wipe the top of container with a sterie coth or cotton saturated with 70% acoho. 6

7 Immediatey before transferring test portion of mik or cream, shake container, making 5 compete up-and-down/ back-and-forth movements of about one foot in 7 seconds. Seect diution(s) in a manner that the tota number of coonies on a pate wi be between 0 and 00. Remove m of the sampe of mik or mik products having viscosity simiar to mik e.g. cream with a sterie bacterioogica pipette and transfer it to the first tube of diuents (9 m). Aow about seconds for the content of the pipette to drain and genty bow out the ast drop. Rotate the test tube between pams of the hand to compete the mixing. This makes a diution of :0. Simiary, a series of diutions can be prepared by transferring m of the first diution (:0) into another 9 m diution bank to get :00 diution and so on. Where the soids content or viscosity of sampes exceeds that of whoe mik e.g. dried mik, condensed mik, ice cream, cutured dairy products, prepare the initia :00 (or :000) diution by weighing g (or g) asepticay into diution bottes containing 99 m of diution bank. Microbioogica Anaysis of Mik and Mik Products b) Preparation and Incubation of Pates Use of fresh pipette and transfer m of each required diution into sterie petridishes in dupicate. Aow - seconds for the pipette to drain, touch the top of the pipette to a dry pace in the petridish to drain out the ast drop. Add 0-5 m of standard mik agar previousy meted and cooed to 5 o C. Mix the contents of the pate thoroughy whie the medium is sti iquid by genty rotating the petridishes and aow the agar to coo and set. Invert the pates and incubate at 7 o C for 8 h. c) Counting of Coonies Remove the pates after 8 h and seect the pair of pates having coonies between 0 and 00 on each pate. Count the number of coonies with the hep of a coony counter and determine the average of the counts in the two pates and mutipy this by the diution factor and report as SPC/ m or g. iii) Count of Thermoduric, Thermophiic and Psychrotrophic Bacteria a) Thermoduric Bacteria Arrange the water bath at 6.5 o C. Perform the proper mixing of mik sampes. 7

8 Transfer 0 m of mik into test tubes asepticay. Insert a thermometer into one of the test tubes under observation. Lower the test tubes in the water bath. When the temperature of mik reaches 6.5 o C start your stopwatch. Terminate the incubation exacty after 0 minutes by taking out the tubes from the water bath and immediatey chiing the mik by immersing in ice-cod water. Prepare appropriate diutions and perform the standard pate count method with incubation at 7 o C for 8 h. Mutipy the average number of coonies with diution factor and report as Laboratory pasteurization count per m or 9 (LPC/ m or g). Microbioogica Anaysis of Mik and Mik Products b) Thermophiic Bacteria Count Perform the standard pate count method with incubation of pates at 55 o C for h. Report the resuts as thermophiic bacteria count/ m or g (TBC/ m or g). c) Psychrotrophic Bacteria Count Prepare diutions and pates as per the method of standard pate count. Incubate pate at 7 o C for 0 days. After determining the coony count, report as psychrotrophic bacteria count per m or g (PBC/ m or g). iv) Coiform Test A. Presumptive Test a) Liquid Media Prepare seria diutions of the sampe of mik or mik products. Transfer m of required diution into MacConkey s broth tubes in tripicate. Incubate the tubes for h at 7 o C and observe for the production of acid and gas. The production of acid is exhibited by change of coour of medium from purpe to yeow in the case of bromo creso purpe and orange to pink in the case of Andrade s indicator. Production of gas is observed in the Durham s tubes, which may be partiay or competey fied with gas. In case of no change, further incubate for another h and record the observation. 8

9 b) Soid Media Prepare seria diutions of the sampe. Microbioogica Anaysis of Mik and Mik Products Incubate m portions of the required diutions into sterie petripates in dupicates. Add to each pate 0-5 m of MacConkey s agar previousy meted and cooed to 5 o C. Mix the contents by rotating the pates. Aow to agar to soidify. Pour additiona ayer (- m) of the medium competey over the surface of the soidified medium. Invert and incubate the pates at 7 o C for h. Once incubation is over, examine the pates for presence of typica dark red coonies measuring at east 0.5 mm in diameter. Count such coonies and express the resuts as coiform count per m of mik. B. Confirmation Test Pour 0 to 5 m of meted Eosine Methyene Bue Agar or Endo agar into petridish and aow the media to set. Introduce the sterie inocuating needs to the depth of 0.5 cm beow the surface of the positive tube. In case of positive agar pates, transfer portion of typica coonies to the EMB/ Endo agar s pates. Pace the curved section of the neede on the agar surface and streak genty to avoid tearing of the medium. Invert the pates and incubate at for h. Observe the appearance of typica coonies of coiform on the agar surface. Such coonies wi appear pink with dark centre and metaic sheen on EMB agar. Endo agar produces red coonies. v) Yeast and Moud Count Prepare :0 diution. Transfer m of diution to dupicate petridish for pating. Adjust the ph of potato dextrose agar to.5 by adding cacuated amount of sterie tartaric acid soution at the time of pouring pates. Pour the meted agar cooed to 5 o C and mix the contents we. Aow the agar to set. 9

10 Invert and incubate the pates at 5 o C for -5 days. Count the number of coonies. Microbioogica Anaysis of Mik and Mik Products iv. Observations We shoud record the foowing observations from the foowing tabes. Tabe. Direct Microscopic Count Sampe Number of Cumps or Average DMC/ m Ces Per Microscopic Fied,,,, 0 Tabe. Standard Pate Count Sampe Diution Counts in Pate Average SPC/ m or g,,,, 0 Tabe. Thermoduric Bacteria Count Method Mik/ Mik Diution Coony Counts Average LPC/ m Product Sampe or g 0

11 Tabe Thermophiic Bacteria Count Method Mik/ Mik Diution Coony Counts Average TBC/ m Product Sampe or g Microbioogica Anaysis of Mik and Mik Products Tabe 5. Psychrotrophic Bacteria Count Method Mik/ Mik Diution Coony Counts Average PBC/ m Product Sampe or g Tabe 6. Presumptive Coiform Test (MacConkey s Broth) Sampe Diution Observation at the end of h and 8 h Tabe 7. Presumptive Coiform Test (MacConkey s agar) Sampe Presence of Coiforms Number of Coonies in Coiform Diution Coonies (±) the Pates Count/ m or g

12 Tabe 8. Confirmation test for coiform Sampe Medium Coour of Medium Coonies Typica Negative Microbioogica Anaysis of Mik and Mik Products EMB Agar Endo Agar Tabe 9. Yeast and Moud Count Sampe Diution Number of yeast and Yeast and Moud Moud Coonies in Pates Count/ m or g v. Resuts/Interpretation Interpret the resuts obtained by microbioogica anaysis of mik and mik products on the basis of Microbioogica standards (BIS & PFA) furnished in the textbook. 8. PRECAUTIONS During DMC method, avoid the rapid heating of smear for the purpose of drying before staining, Rapid heating at this stage may cause the fim to crack and pee out during atter treatments. If number of cumps per microscopic fied exceeds 0, diute the mik suitaby before determining microscopic count. Before opening a sampe container, remove from the cosure a obvious materias which may contaminate the sampe. The interva between making of sampe and removing the test aiquot shoud not exceed minutes. Do not prepare or dispense diutions or pour pates in direct sunight. Whie dispensing diutions do not bow out into a petridish. Avoid proonged exposure to unnecessariy high temperatures during and after meting of agar medium.

13 Seect the number of sampes to be pated in any one series so that not more than 0 minutes eapse between diuting the first sampe and pouring the ast pate in the series. Whie streaking the inocuum from positive presumptive coiform tubes/ on agar surface, take care not to pierce or stab the agar medium. Whie preparing pates for different pate count methods, identify each pate with sampe number, diution and other desired information. Microbioogica Anaysis of Mik and Mik Products QUESTIONS i) What is the formua for cacuating microscopic factor in DMC method? ii) Why seria diutions of sampes are required to be prepared in SPC method? iii) What is the reation between Direct Microscopic count and Standard pate count? iv) What is the significance of thermoduric and thermophiic bacteria in pasteurized mik? v) What is the time-temperature combination used for incubation of pates during psychrotrophic bacteria count method? vi) Why is Presumptive coiform test so christened? vii) What are the incubation conditions for yeast and moud count in dairy products?