Isolation and Characterization of Leuconostoc Mesenteroides from Cheese – Part 2

Materials & Methods

1. Sample collection - Cheddar cheese & Mozzarella cheese
2. Serial dilution and spread plate method
3. Isolation of Lactic acid bacteria in MRS medium
4. Selective Isolation of Leuconostoc from other LAB
5. Addition of vancomycin to MRS medium
6. Leuconostoc was isolated and maintained at 4oC
7. Species identification

Biochemical characterization

Sample collection: Cheddar cheese and Mozzarella cheese used in their study were obtained from retail super market (Nilgiris), Chennai. A well vacuum packed cheese was obtained.

Bacterial strains and culture:

Isolation of Leuconostoc:

Culture Media: Leuconostoc are fastidious chemo-organotrophic bacteria. Therefore, culture media must be rich in nutrients such as carbohydrates as energy providers, amino acids as nitrogen compounds, salts and vitamins. The most widely used medium was MRS medium (Man Rogosa Sharpe). The supplements studied in MRS medium are glucose, peptone, yeast extract, beef extract, dipottasium hydrogen phosphate, ammonium citrate, magnesium sulphate, manganese sulphate sodium acetate, and tween 80. Tween 80 usually increases the growth of Leuconostoc by providing oleic acid incorporated into the cell membrane.

Composition of MRS medium

Media gm/lit

Glucose 1.0gm
Peptone 1.0gm
Beef extract 0.8gm
Yeast extracts 0.5gm
Di-potassium hydrogen phosphate
(K2HPo4) 0.2gm
Magnesium sulphate (MgSo4) 0.2gm
Manganese sulphate (MnSo4) 0.005gm
Ammonium citrate 0.2gm
Sodium acetate 0.5gm
Tween 80 0.1gm
Agar 2.0gm
pH 6.8gm

The medium was sterilized at 121oC for 15mins. Leuconostoc was enumerated by spread plate method. Serial dilutions were made from the master dilutions containing 1gm of cheddar cheese in 99ml of distilled water which gives a dilution of 10-1 (serial dilutions were made upto 10-1). 0.1ml of each dilution was spreaded on MRS medium, and incubated at 37oC for 24hrs.

From food sample, all LAB are counted since the complex nutrient requirements and optimal conditions for the growth are roughly comparable for lactobacillus, Pediococcus and Leuconostoc. So, for selective isolation of Leuconostoc from other lactic acid bacteria (vancomycin sensitive), 30mg/ml of vancomycin solution was added to the MRS medium. The Leuconostoc strain isolated was maintained by sub-culturing in MRS medium.

Indicator bacterial strains:

Salmonella typhi, ATCC-21563
Escherichia coli, ATCC-1023
Shigella flexinerrae, ATCC-11461

were received from NCIM and maintained by sub-culturing in nutrient agar slants for further biochemical tests. The incubation period for the test organisms was 37oC for 24hrs. All the cultures were stored at 4oC.

Media used for cultural characterization of Leuconostoc:

MacConkey agar medium
Blood agar medium

Biochemical characterization of Leuconostoc for species identification:

Gram’s staining
Motility test – hanging drop method
Capsule staining – negative staining
Spore staining – Scaffer-fulton method

Biochemical tests:

Indole
Methyl red
Voges Proskauer
Citrate
Nitrate
Urease
Arginine hydrolysis

Carbohydrate fermentation test:

Glucose
Arabinose
Fructose
Sucrose
Maltose
Galactose
Lactose
Mannital

All the above biochemical tests were performed and the results were noted to differentiate Leuconostoc species. The differentiating test among Leuconostoc species are catalase test, arginine test and carbohydrate fermentation tests.

Separate biochemical test were performed for the indicator organisms to confirm the strains and results were noted.