Microbiological analysis of milk part II

Microbiological analysis of milk part II

1. Microbiological Analysis of Milk – Part II of II Dr. Dhanya KC Assistant Professor Department of Microbiology St. Mary’s College, Thrissur-680020 Kerala

2. Analyses of milk - to ensure quality Platform tests or milk reception tests At milk reception sites - rapid tests Lactometer test, Organoleptic test, COB Alcohol Test, Ten-Minute Resazurin Test, etc. Microbiological quality Analyses of milk Direct tests (Quantitative) Assessment of the actual number of bacteria DMC, SPC, etc Indirect tests (Qualitative ) - Based on the metabolic activity of the microbes MBRT , resazurin test, etc www.dairyprocessinghandbook.tetrapak.com

3. Lactometer test Organoleptic test Temperature Determination of pH Clot on Boiling Alcohol Test Acidity of milk Sediment Test : Ten-Minute Resazurin Test Alizarin-Alcohol Test Direct Microscopic Count Methylene Blue Reduction Test Alkaline Phosphatase test One-Hour Resazurin Reduction Test Standard Plate Count of Milk Coliform Count Enumeration of other types of bacteria Enumeration of yeast and mould Quality control tests for milk

4. 12. Methylene Blue Reduction Test ( MBRT) E valuate microbial load of milk Methylene blue - blue in its oxidised state, colorless - reduced state Time taken for reduction - number and types of bacteria growing in milk 1 ml. Methylene blue + 10 ml of milk - mixed - 37 o C Observe every 30 minutes for colour Two control tubes - 10 ml milk + 1 ml MB - heat for 3 minutes 10 ml. of milk + 1 ml of tap water MBR Time (Hours) Quality of Milk 5 and above Very good 3 and 4 Good L and 2 Fair 0.5 and below Poor www.bamgalajol.info

5. 13. Alkaline Phosphatase test Phosphatase converts the substrate P- nitrophenyl phosphate to p- nitrophenol p- nitrophenol - Yellow coloured in alkaline solution At pH 9.5, 37 o C Phosphatase present in milk - destroyed during pasteurization- efficacy of pasteurization 10 ml buffer solution containing substrate at 37 o C + 2ml milk - 37 o C for 30 minutes Control set with boiled milk C olour of the test is compared with the boiled milk control E xcess yellow colour - improper pasteurization. www.researchgate.net

6. 14 One-Hour Resazurin Reduction Test Resazurin - oxidation-reduction indicator - reduction in stages – due to bacterial activity Time taken for reduction - bacterial activity in milk Resazurin + Milk - incubated at 37 o C for 1 hour - colour changes recorded Reduction - series of colour change – blue, lilac , mauve, purple, pink, colourless Rapid reduction to pink and colourless - indicates High bacterial content and poor keeping quality Leucocyte cells present during mastitis and late lactation One hour Resazurin Disc No. Quality of Milk 4 or higher Good 3.5 to 1 Fair 0.5 to 0 Poor www.sserc.org.uk

7. 15. Standard Plate Count (SPC) of Milk Total Plate Count (TPC) or Aerobic Plate Count (APC) or Total Viable Count ( TVC) Milk sample - thoroughly mixed– to evenly distribute microbes Dilutions prepared Inoculated into media - Pour plate Incubated at 37 o C for 48h Colonies counted - Colony forming unit/ml of milk calculated Disadvantages Only rough estimate of microbial count Time consuming, laborious and difficult All types of bacteria will not grow Sample may not be representative Advantages Enumeration of viable microbes Colonies – differentiation of microbes Suitable for samples with low bacterial number, such as pasteurized milk

8. SPC/ ml Grade Not exceeding 2,00,000 Very good 2,00,000 - 10,00000 Good 10,00000 - 50,00000 Fair Over 50,00000 Poor Microbiological Standards for Grading of Raw Milk www.quizlet.com

9. 16. Coliform Count Coliforms in milk - unsanitary conditions or practices - production, processing or storage Protocol for determining coliforms Presumptive test Confirmatory test Completed test Test for fecal coliforms Most probable number (MPN) for enumeration of low counts Differentiation of Escherichia coli and Enterobacter aerogenes

10. Presumptive test: Sample of milk - inoculated in   MacConkeys  broth or Bile salt lactose peptone broth I ncubated at 37 o C for 24-48 hour Production of acid and gas - presumptive evidence of coliforms in milk www.ugenelab.com.sg www.slideplayer.com Confirmatory test Positive presumptive test tubes - selected for confirmatory test. Sample from Presumptive tube inoculated -on EMB or Endo agar plates Incubated at 37 o C for 24-48 hours Coliforms  - pink colonies with or without dark center / green metallic sheen Inoculated - in BGLB broth tubes - incubated at 37 o C for 48 hours Formation of gas - confirmatory result for coliforms

11. Completed test Positive confirmatory test tubes or typical colonies from agar plates Inoculated into   MacConkeys  broth tubes and nutrient agar slants Incubated at 37 o C for 24-48 hours Acid and gas production in  broth - positive completed test Gram negative, non-spore forming  cocco -bacillary rods – positive completed test Test for fecal coliforms (Eijkman’s test) Inoculum from positive presumptive tube-in BGLB or   MacConkeys  broth tubes Incubated at 44.5 o C   for 24 hours Gas production - indicate the presence of fecal coliforms

12. Most probable number (MPN ) A count of the number of positive presumptive tubes G ive MPN of bacteria in milk F rom  MPN table www.researchgate.net Differentiation of  E coli and Enterobacter  aerogenes Two types of coliforms in milk Differentiated on the basis of IMViC   test

13. 17. Enumeration of other types of bacteria www.austincc.edu Counting Proteolytic bacteria - Mostly   psychrotrophs   - grow at 7 o C Proteolytic microbial count on Casein  agar plates or Milk agar plates - incubation for 6 days Casein breakdown - white zone of precipitation Counting Lipolytic bacteria – Mainly Psychrotrophs Tributyrin  agar - incubation at 6.5 o C for 10 days or 37 o C for 3 days Spore Count- Generally   Bacillus  spp., Samples kept at 80 o C - cooled to room temperature slowly S ample plated and incubated at 37 o C for 48 hours or 55 o C for 48 hour

14. 17. Enumeration of other types of bacteria Thermophilic Count: Generally Bacillus spp and Clostridium spp Incubation at 45 o C or 55 o C for 48 h . Thermoduric Count: Micrococcus ,  Microbacterium , Bacillus , etc S amples placed at 63 o C for 30 min - cooled down to 5 o C Plated and incubated at 37 o C for 48 h . Psychrotrophic Count: Primarily of family  Pseudomonadaceae , Neisseriaceae   etc. Sample plated and incubated at 7-10 o C for 7-10 days or 15 o C for 3 days

15. 18. Enumeration of yeast and mold www.labm.com Plating milk sample in PDA or Malt extract agar - incubation at 30-32 o C for 3 -5 days . Microscopic method - Mold mycelia count: D irect microscopic count Macroscopic method - Methylene blue borax test 5 ml warm milk mixed with hot methylene blue and borax - shake well Agglutinated mold mass gathered into a circular disc Diameter of disc measured in mm 2 Visual mold test, modified methylene blue borax test Milk mixed with methylene blue and alkaline salts - stirred and heated Filtered – visually measure the mycelium

16. Thank you.. References Chemical & Microbiological Analysis of Milk & Milk Products, Ramakant Sharma, International Book Distributing Co., First Edition 2006 Food Microbiology, Third Edition, Martin R. Adams and Maurice O. Moss University of Surrey, Surrey, Guildford, UK. Manual by Dr.Sunitha Gover , NDRI, Karnal http://ecoursesonline.iasri.res.in/mod/page/view.php?id=65171