Team:NEFU China/Experiments


Enumeration of coliforms (GB 4789.3—2010)

1 Diluting the samples


1.1 Solid and semi-solid samples:Weigh and take 25 g sample, put it in an aseptic homogenizing cup which contains 225 ml phosphate buffer solution or physiological saline, and homogenize it 8000 r/min to 10000 r/min for 1 to 2 min; or put it in an aseptic homogenizing bag which contains 225 ml phosphate buffer solution or physiological saline and homogenize it by flapping with a smack type homogenizer for 1 to 2 min to get 1:10 homogenous sample liquor.


1.2 Liquid samples: Suck 25 ml sample with an aseptic suction tube, put it in an aseptic conical flask (with a certain number of aseptic glass beads placed inside beforehand)

 which contains 225 ml phosphate buffer solution or physiological saline, and blend the solution properly to get 1:10 homogenous sample liquor.


1.3 pH value of the homogenous sample liquor should be between 6.5 and 7.5. Regulate its pH value with 1mol/L sodium hydroxide (NaOH) or 1 mol/L hydrochloric acid (HCL) respectively, when necessary.


1.4 Suck 1 ml 1:10 homogenous sample liquor with a 1ml aseptic suction tube or micro pipettor, empty it in an aseptic test tube (attention: the pointed end of test tube or sucker 

should not touch the diluting liquid) which contains 9 ml phosphate buffer solution or physiological saline slowly along the tube wall, jolt the test tube or beat upon it with a 1 ml 

aseptic suction tube so that it will be homogenized properly to get 1:100 homogenous sample liquor.


1.5 According to estimation of sample pollution, make homogenous sample liquor series diluted by 10 times and above as per the above-stated operating steps. For every increased diluting degree, replace one 1 ml aseptic suction tube or sucker. From preparation of homogenous sample liquor to completion of inoculation, the whole process should be within 15 minutes.


2 Primary fermentation test

For every sample, select homogenous sample liquors with three suitable consecutive dilution degrees (stock solution may be chosen in case of liquid sample), and for every 

dilution degree, inoculate 3 tubes of lauryl sulfate tryptone (LST) broth, 1ml each tube (if more than 1ml is inoculated, double LST broth should be adopted). Make them cultured

 in 36±1°C for 24h ± 2h and observe whether bubbles are generated in the tubes; if there is no any bubble, make them cultured for 48±2h in total. Tubes without bubbles are

 coliform negative and tubes with bubbles go through secondary fermentation test.

3 Secondary fermentation


Take 1 circle of cultures from each of all LST broth tubes which ferment and generate gas within 48h±2h respectively with an inoculation ring, transfer-inoculate them to brilliant green lactose bile (BGLB) broth, culture them in 36°C±1° for 48±2h, observe bubblegeneration.Tubes which generate bubbles are recorded as coliform positive.


4 Reporting most probable number (MPN) of coliforms

According to the number of tubes which are coliform positive verified through 3, search the MPN Table (see Annex B) to report coliform MPN counts in every gram (or ml) of sample.

Media Component (g/L) , 25℃ 

1. Modified Chalmers Agar (MC):

2. Lauryl Sulfate Tryptose Broth (LST) :

3. Brilliant Green Lactose Bile Broth (BGLB):  



The microbiological test of Lactobacillus in yogurt (GB/T16347-1996)

1. Put 25 ml full-shaked sample into sterilized wide mouth bottle containing 225 ml sterile saline aseptically to make uniform dilution of 1:10. Samples are selected for the same brand of yogurt which date 4, 10, 20 days, and expired one day.

2. Suck up 1 ml 1:10 dilution with 1 ml sterile pipette and inject it slowly into a test tube containing 9 ml sterile saline along the tube wall (note not to touch the tip of the pipette tube dilution).

3. Increase by 10-fold dilution increments every time, that is replaced with a 1 ml sterile pipette according to the above steps. So it is total diluted 10-15.

4. Choose dilutions from 10-6 to 10-15 and suck up 1 ml dilution into sterile plates respectively while doing the 10-fold dilution increments. Make two plates each dilution.

5. Inject 15 ml Lactobacillus count medium (modified MC) which was cooled to 50℃ into the plate as soon as the dilutions was shifted into the plate. Rotate it to mix them. Meanwhile, to make a blank comparison, pour the count medium of Lactobacillus into a sterile plate containing sterile saline which is used to test 1 ml dilution. The whole process including adding the culture to the plate to finishing pouring should be done within 20 min.

6. Invert the plate and put it into a 36 ± 1℃ incubator for 72±3 h after the agar has set. Observe the lactobacillus in the plate, select colonies between 30 to 300 and count them. After the calculation, the colonies are randomly taken the Gram stain: (1) fix the smear.(2) stain for 1 min with ammonium oxalate crystal violet. (3) wash with running water.(4) add iodine to cover approximately 1 min. (5) wash with water and absorb the water with absorbent paper. (6) add a few drops of 95% alcohol and gently shake to decolorize it. Wash with water after 20 seconds and absorb the water. (7) stain with fan red for 1 minute,wash it with running water, dry it and then take microscopic examination. Gram-positive bacteria are blue-purple and gram-negative bacteria are red.

7. Do the catalase test: pick up a colony from the solid media into a clean tube, drop 2 ml 3% hydrogen peroxide solution and observe. Those who has bubbles in 30 s are positive, the others are negative.

8. Results identification: The Lactobacillius can be identified according to the following fades: gram-positive, catalase-negative, non-spore sphaerita or bacillus. Calculate the number of Lactobacillus in one plate and multiply the dilution and then we get the number of Lactobacillus of per milliliter of the sample.


Table1  The colony morphology of lactobacillus in the modified MC medium



Genome Extraction

For bacterial gDNA extraction we used the easy DNA Kit according to the manufacturer's instructions.


Plasmids extraction

For the plasmid extraction we used TIANprep Midi Plasmid Kit according to manufacturer's instructions. 

Gel Extraction 

For the Gel Extraction we used TIANgel Midi Purification Kit according to manufacturer's instructions.

AI-2 Quantification

1. E.coli and Bacillus were inoculated into LB media (2%, v/v), respectively, and shaken overnight at 37°C.

2. The bacteria were centrifuged at 6000 g for 3 min. The supernatant was collected and filtered through a 0.22μm membrane

3. Preparing the working solution: A working solution of 10 mM 1, 10-phenanthroline/3.32 mM Fe (III) was prepared by dissolving 0.198 g of 1,10-phenanthroline in 50 ml of deionized distilled water. The solution was adjusted to pH 2 using 1M HCl. Ferric ammonium sulphate (0.16g) was added and the solution was brought to 100 ml using deionized distilled water.

4. For the Fe (III) ion reduction test, 1ml of the cell free supernatant was mixed with 1 ml working solution to develop the full color. The solution was then diluted to 5ml and filtered through a 0.22 μm membrane,followed by scanning for the absorption spectrum against a blank solution within 3 min using a Lambda 25 UV/VIS spectrometer. 


Transformation by electroporation


1. Inoculate 100 μl bacterial culture into 50 ml of MRS and incubate the bacteria at 37°C overnight. 

2. Harvest the cells by centrifugation.

3. Washed the bacteria three times with cold electroporation buffer (PB).

4. Resuspend the cells in PB to reach the  OD600 at about 0.5.

5. Mix 100 μl electrocompetent cells with 10 μl plasmid DNA.

6.Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette. 

7.Electroporate using the following conditions for BTX ECM 630 and Bio-Rad GenePulser electroporators: 2.4kV, 200Ω, 25μF electric pulse in a 0.2 cm cuvette.

8. Immediately add 950 µl of 37°C SMRS to the cuvette, gently mix up and down twice, then transfer to the 1.5 ml tube.

9.Incubate at 37°C for 2 h.

10. Dilute the cells as appropriate then spread 100-200 μl cells onto a pre-warmed selective plate.

11.Incubate the plates at 37°C for 2 to 3 days under anaerobic conditions.

12. Use isolated colonies to check the correct insertion.


Functional identification of the engineered bacteria

1.Inoculate E. Coli CD-2 and E. Coli DH5alpha into 100 ml liquid LB medium, cultured at 37°C 180 rpm for 8 h.

2.Centrifuge at 12,000 rpm for 2 min, then harvest the culture supernatant.

3.The liquid culture supernatant was filtrated through the 0.22 μm filters.

4. Then the supernantant was added into the culture medium of Lactobcillus.

5. Nisin was added at a final concentration of 50 ng/ml.

6.Incubate the engineered Lactobcillus overnight.

7.Centrifuge at 6000 rpm for 5min, discard the supernatant then take photo. 


Functional identification of the engineered bacteria (2)
1.Incubate E. Coli CD-2 and E. Coli DH5alpha on LB agar plate overnight at 37°C.

2. Wash the colonies with fresh MRS.

3. The liquid culture supernatant was filtrated through the 0.22 μm filters.

4. Then the supernantant was added into the culture medium of Lactobcillus.

5. Nisin was added at a final concentration of 50 ng/ml.

6.Incubate the engineered Lactobcillus overnight.

7.Centrifuge at 6000 rpm for 5min, discard the supernatant then take photo.

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