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. 1. Lactometer test
2. Organoleptic test
3. Temperature
4. Determination of pH
5. Clot on Boiling
6. Alcohol Test
7. Acidity of milk
8. Sediment Test:
9. Ten-Minute Resazurin Test
10.Alizarin-Alcohol Test
11.Direct Microscopic Count
12.Methylene Blue Reduction Test
13.Alkaline Phosphatase test
14.One-Hour Resazurin Reduction Test
15.Standard Plate Count of Milk
16.Coliform Count
17.Enumeration of other types of bacteria
18.Enumeration of yeast and mould
Quality control tests for milk

4. 12. Methylene Blue Reduction Test (MBRT)
Evaluate 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 - 37oC
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, 37oC
Phosphatase present in milk - destroyed during pasteurization- efficacy of pasteurization
10 ml buffer solution containing substrate at 37oC + 2ml milk - 37 oC for 30 minutes
Control set with boiled milk
Colour of the test is compared with the boiled milk control
Excess 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 37oC 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 37oC 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
• Incubated at 37oC 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 37oC for 24-48 hours
Coliforms - pink colonies with or without dark center / green metallic sheen
• Inoculated - in BGLB broth tubes - incubated at 37oC 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 37oC 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 44.5oC 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
• Give MPN of bacteria in milk
• From 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 7oC
• 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 6.5oC for 10 days or 37oC for 3 days
Spore Count- Generally Bacillus spp.,
• Samples kept at 80oC - cooled to room temperature slowly
• Sample plated and incubated at 37oC for 48 hours or 55oC for 48 hour

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

15. 18. Enumeration of yeast and mold
www.labm.com
• Plating milk sample in PDA or Malt extract agar - incubation at 30-32oC for 3 -5 days.
• Microscopic method - Mold mycelia count: Direct 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 mm2
• 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. 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