Work Plan for Isolation of Lactoccocus Lactis

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Work Plan for Isolation, Purification, Identification and Starter Culture Activity of Lactoccocus lactis

Submitted by:
M.Usman Akram
B.S. (Hons.) Dairy Technology
mh.usman@hotmail.com
Mobile :+923217773736

University of Veterinary and Animal Sciences,
Ravi Campus Pattoki

Lactoccocus lactis
Classification:
Scientific classification|
Kingdom:| Bacteria|
Division:| Firmicutes|
Class:| Bacilli|
Order:| Lactobacillales|
Family:| Streptococcaceae|
Genus:| Lactococcus|
Species:| L. lactis|

Cell Morphology:
* L. lactis cells are cocci that group in pairs and form short chains. * Length of L. lactis ranges from 0.5-1.5 micrometer (µm). * L. lactis does not produce spores (nonsporulating).
* No capsule formation.
* L. lactis grow in microaerophilic conditions
* L. lactis are non motile.
* L. lactis are Gram (+ive).
* L. lactis are catalase (-ive).
* L. lactis have low-GC content.
* L. lactis are acid-tolerant
* Optimum growth temperature is 30oC at pH 6.8.
* L. lactis are not produce CO2 in MRS Broth.
Colony Morphology:
* No pigment/Color.
* Texture is smooth.
* No odor.
* Pinpoint to small size.
* Round form.
* Raised elevation.
* Smooth margins.
Work Plan for Isolation, Purification, Identification and Starter Culture Activity of Lactoccocus lactis

MATERIAL & METHODS
MATERIAL:
Apparatus:
1. Capped test tubes
2. Test tubes holder stand
3. Beakers
4. Microscope
5. Incubator
6. Platinum loop
7. Microtitre plate
8. Durham tubes
9. Conical Flask
10. Measuring cylinder
11. China dish
12. Funnel
13. Spatula
14. Weighing balance
15. Autoclave
16. Aluminium foil
17. Petri dishes with lids
18. pH meter
19. Burner
20. Pipettes
21. Measuring flask with stopper
22. Colony counter
23. Water bath
24. Anerogen kit
Chemicals:
1. Crystal violet
2. Ethanol
3. Saline solution
4. Iodine solution
5. Safranin
6. Hydrogen peroxide (H2O2)
7. MRS media (solid)
8. Distilled water
9. 0.1N HCl
10. 0.1N NaOH
11. Motility Test Medium

Preparation of Cheese Samples:
Take Cheese sample and dilute it into deionized water.
METHODS:
Media and Growth Conditions:
For Lactococcus lactis, MRS Agar (pH 6.8+/-0.1) at 30°C.
Composition of MRS Agar:
MRS agar typically contains (w/v);
1. 1.0 % peptone
2. 0.8 % meat extract
3. 0.4 % yeast extract
4. 2.0 % glucose
5. 0.5 % sodium acetate trihydrate
6. 0.1 % polysorbate 80 (also known as Tween 80)
7. 0.2 % dipotassium hydrogen phosphate
8. 0.2 % triammonium citrate
9. 0.02 % magnesium sulfate heptahydrate
10. 0.005 % manganese sulfate tetrahydrate
11. 1.0 % agar
Isolation of Bacteria:
1. Bacteria were isolated from Cheese samples using the pour plate technique. 2. Briefly, 1ml of each sample was taken and homogenized in 9 ml of 0.9% NaCl. 3. Serial 10 fold dilutions were prepared and 1 ml aliquots from 10-5, 10-6, 10-7 dilutions were used for isolation. 4. Prepare MRS Agar media by dissolving exact quantity in deionized water. 5. Each sample was plated twice.

6. All plates were incubated for 3 days in microaerophilic conditions using Anerogen kit, due to which oxygen level in the jar reduces below 1% with a resulting CO2 level between 9% and 13%. 7. After the incubation, plates with colony forming units (cfu) between 30 and 300 were counted and results were recorded as viability of Cheese bacteria.

Genus Identification Tests:
| Growth at 10oC| Growth at 45oC| 6.5% NaCl| 9.6 pH|
Lactococcus| (+)| (-)| (-)| (-)|

Biochemical Identification Tests:
No.| Biochemical Tests| Lactococcus lactis|
1.| Gram staining| (+)|
2.| Catalase test| (-)|
3.| Gas...
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