Methylene Blue Reduction Test and Direct Microscopic Count Method

FE 421 FOOD MICROBIOLOGY LABORATORY

Name of student : M. Hakan MAVİŞ

Group : B – 2

Name of experiment : Methylene Blue Reduction Test and Direct Microscopic Count Method

PURPOSE:

The purpose of this experiment was to investigate the coliform in milk and learn methylene blue reduction test and direct microscopic count method for milk.

THEORY:

The Standard Plate Count (SPC) procedure is used to determine the number of bacteria in a sample. In most cases the initial day SPC represents those bacteria that survive pasteurization (thermoduric), although gross contamination after pasteurization can cause high counts. The regulatory standard of < 20,000/ml is generally easily achieved. Most initial day bacteria counts are <500/ml while counts higher than 1000/ml suggest a potential contamination problem, either in the raw milk supply or within the processing equipment.

The ideal milk shows no increase in bacteria counts during refrigerated storage. When milk is held under refrigeration, only bacteria capable of growth under these conditions will grow. While most bacteria prefer warmer temperatures for growth, some bacteria, referred to as psychrotrophs (“cold-loving”), are capable of growth at 45oF or less. The most common types of psychrotrophic bacteria that rapidly spoil milk do not survive pasteurization; thus their presence in milk is the result of post-pasteurization contaminants due to less than adequate sanitation practices. The initial day SPC of fresh pasteurized milk is not a good indicator of the numbers of psychrotrophs present since most bacteria that survive pasteurization are not psychrotrophic (a few types of thermoduric bacteria will grow slowly under refrigeration conditions). A significant increase in the SPC after 7-10 days of refrigeration storage is evidence of psychrotrophic growth and suggests that post-pasteurization contamination has occurred and that shelf-life will be shortened. Generally, when the SPC exceeds 1 – 100 million, the product will become unacceptable due to flavor defects related to bacterial growth. The key to preventing spoilage and extending the shelf-life of a product is to prevent post-pasteurization contamination through a well-designed quality assurance program. It only takes one psychrotrophic bacteria per container of milk to cause spoilage.

The coliform bacteria (coli) count is used as an index of sanitation during the handling and processing of milk products. Coliforms are killed by pasteurization, thus when present in milk, they are regarded as post-pasteurization contaminants resulting from poor sanitation.  Though the standard is “not to exceed 10/ml,” detection of any coliform bacteria suggests that there is some point in processing that has been neglected in regard to effective cleaning and sanitation procedures. As a rule, the detection of coliforms in milk will indicate the potential for a shortened shelf-life due to concurrent contamination with psychrotrophic bacteria. Milks with coliform counts exceeding 10/ml are not tasted on subsequent days in this program.

MATERIALS:

  • Tcyptone glucose yeast agar

  • Milk

  • Methylene blue solution

  • Tubes

  • Pipette

  • Spreader

  • Bunsen burner

  • Test tube rack

  • Etuv

  • Ethyle alcohol

PROCEDURE 1:

1 – Total Count:

Firstly; milk was diluted from non dilution milk to 10-5 dilution in the test tube. In order to make 1 ml non-dilution milk was taken and it was added to 9 ml distilled water and thus 10-1 milk dilution was occurred and this process was continued to 10-5 dilution. After that; 0, 2 ml dilution was taken with pipette from each dilution (non-dilution, 10-1, 10-2, 10-3, 10-4, 10-5) these were inoculated to tcyptone glucose yeast agar with spread plate method. Then; these were incubated at 37 oC for 24 hours. After incubation, between 300 – 30 being microorganisms were counted and in ml numbers of microorganisms were calculated.

2 – Methylene Blue Reduction Test:

Secondly; raw milk and pasteurized milk were studied separately. 10 ml milk was put in the test tube and 3 – 4 drops methylene blue solution was added to milk. Then; milk and methylene blue solution were mixed vigorously, then; these tubes were incubated at 37 oC for 30 minutes. Milk was examined and observed whether decolorization was occurred or not.

RESULTS 1:

Total Count:

non

10-1

10-2

10-3

10-4

10-5

A – raw milk

TNTC

359

284

65

38

9

B – paste. Milk

TNTC

47

2

0

0

0

C – raw milk

TNTC

TNTC

TNTC

198

33

7

Group A)        For 10-2 dilution: (284*102) / 0, 2 ml = 142000

For 10-3 dilution: (65*103) / 0, 2 ml   = 325000

For 10-4 dilution: (38*104) / 0, 2 ml   = 1900000

(142000+325000+1900000) / 3 = 789000

Group B)        For 10-1 dilution: (47*101) / 0, 2 ml   = 2350

Group C)        For 10-3 dilution: (198*103) / 0, 2 ml = 990000

                        For 10-4 dilution: (33*104) / 0, 2 ml   =1650000

(990000+1650000) / 2 = 1320000

Methylene Blue Reduction à this method depends on the ability of microorganisms to change oxidation-reduction potential of medium. Bacteria consume dissolved oxygen in medium and produce some enzyme. These enzymes oxidize substrate and hydrogen removed from substrate and hydrogen was held with methylene blue solution and light blue of milk converted to white or colorless.

PROCEDURE 2:

3 – Coliform Test:

Firstly; milk was diluted from non-dilution to 10-5 dilution, then 0, 2 ml diluted and milks were spread out on violet red bile agar with spread plate method then plates were incubated at 37 oC for 48 hours. After 48 hours number of colonies was counted and in ml number of microorganisms was calculated.

4 – Direct Microscopic Count Method:

In here; firstly; slide was taken and on slide 1 cm2 area was determined and one loopful raw milk was put on this area and raw milk was spread out with distilled water in this area. Then; on slide milk was waited to get dry in air. After that; one loopful xylol was added on each square and waited for 1 min then slide was washed with water. Afterwards; 1 – 2 drops methylene blue was added onto each square and waited for 2 min and again slide was washed with water. Next; slide was got dry in air. Finally; on each square, one drop iol immersion was added and milk was examined under 100X objective.

RESULT 2:

Coliform Test:

non

10-1

10-2

10-3

10-4

10-5

A – raw milk

TNTC

TNTC

TNTC

165

16

2

B – raw Milk

TNTC

TNTC

TNTC

TNTC

10

3

C – raw milk

TNTC

TNTC

TNTC

50

15

2

Group A)        For 10-3 dilution: (165*103) / 0, 2 ml = 825000

Group C)        For 10-3 dilution: (50*103) / 0, 2 ml = 250000

Direct Microscopic Count Method:

1st region

2nd region

3rd region

4th region

5th region

1st half slide

3

6

8

14

21

2nd half slide

2

4

5

10

15

1st half slide à 3+6+8+14+21 = 52

2nd half slide à 2+4+5+10+15 = 36

Average number of microorganisms = (52+36) / 10 = 8, 8

Average number of m/o’s = 8, 8 * 15700 * 100 = 14130000

In here 100 is 0, 01 ml milk.

DISCUSSION:

In this experiment; we examined microbiological properties of milk. In milk number of microorganisms was calculated with total count method and coliform bacteria were investigated with coliform test. Also; methylene blue test was applied the milk. In this test one drop methylene blue solution was dropped into milk and colour of milk was slightly blue. After 30 minute slight blue colour of milk converted to white, this transformation was related with number of microorganisms in milk. Microorganisms were used the dissolved oxygen and microorganisms produced certain enzymes. These enzymes oxidize the substrate, thus hydrogen was removed from substrate. Hydrogen was held with methylene blue solution and color of milk was colourless this process was called decolourization. In addition; while raw milk decolorized for 30 minutes pasteurized milk decolorized for 4 – 5 hours. This showed that; in raw milk, number of microorganisms was more than in pasteurized milk.

Secondly; number of microorganisms was determined with total count method. In each milk dilution, number of microorganisms was calculated with accordance to between 30 – 300 microorganisms and as a result; we observed that raw milk was to contain more number of microorganisms than pasteurized milk.

Next; coliform test was applied the milk and in here number of microorganisms was calculated. Finally; in milk microorganisms was examined with microcopy and dark blue microorganisms were observed and at five regions average number of microorganisms were calculated, and these were determined at the result and calculation.

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