• 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

1^st half slide à 3+6+8+14+21 = 52

2^nd 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.