Team:NEFU China/
Protocols
The microbiological test of Lactobacillus in yogurt (GB/T16347-1996)
1. Put 25ml full-shaked sample into sterilized wide mouth bottle containing 225ml sterile saline aseptically to make uniform dilution of 1:10.Samples are selected for the same brand of yogurt which date 4days, 10days, 20days,and expired one day.
2. Suck up 1ml 1:10 dilution with 1ml sterile pipette and inject it slowly into a test tube containing 9ml 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 minutes.
6. Invert the plate and put it into a 36 + 1℃ incubator for 72+3 hours 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 minute.(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. We do the catalase test: pick up a colony from the solid media into a clean tube, drop 2ml 3% hydrogen peroxide solution and observe. Those who has bubbles in 30s 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
|
Lactobacillus types |
Modified MC |
|
Bacillus |
The bottom of the plate is pink，colonies are small, round，red，they have star-like edge， their diameter can be 2 +1mm,they may have a faint halo |
|
Sphaerita |
The bottom of the plate is pink，colonies are small， round，red，they have neat edge，they may have a faint halo |
The Most Probable Number(MPN) Method of Coliform bacteria in yogurt(GB/T 4789.3-2008)
1. Sample Preparation:
(1)Put 25 ml full-shaked sample into sterilized wide mouth bottle containing 225ml sterile saline aseptically to make uniform dilution of 1:10.Adjust pH to 6.5~7.5 with 1M NaOH or 1M Hcl. Samples are selected for the same brand of yogurt which expiration date, expiration date 35℃ 0.5h,and expired one day.
(2)Suck up 1ml 1:10 dilution with 1 ml sterile pipette and inject it slowly into a test tube containing 9ml 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 1ml sterile pipette according to the above steps. The whole process including adding the culture to the plate to finishing pouring should be done within 20 minutes.
2. Fermentation Test:
Sample Dilution: 10-1, 10-2, 10-3, respectively. Inoculate each 1 ml Sample to 3 tubes of LST. Put them into a 36 + 1℃ incubator for 24±2 hours. Observe gas producing situation in tubes. If no bubble produced, continue incubate to 48 h±2 h.Record the number of producing bubble tubes in 24h and 48h. (No gas producing tubes are Coliform bacteria Negative. Gas producing tubes carry out next test.)
3. Refermentation Test:
Inoculate all Gas producing tubes in 48h±2h to BGLB tubes. Put them into a 36 + 1℃ incubator for48±2h.Observe gas producing situation in tubes.(Gas producing tubes are Coliform bacteria Positive.)
4. The MPN of Coliform bacteria report:
According to the number of Coliform bacteria Positive tubes, Search MPN Key. Report the MPN of Coliform bacteria in per ml of sample.
Table2 The MPN Key of Coliform bacteria in per ml(g) of sample
|
Number of Positive tubes |
MPN |
95% Confidence Limit |
Number of Positive tubes |
MPN |
95% Confidence Limit |
||||||
|
0.10 |
0.01 |
0.001 |
|
Upper |
Lower |
0.10 |
0.01 |
0.001 |
|
Upper |
Lower |
|
0 |
0 |
0 |
<3.0 |
--- |
9.5 |
2 |
2 |
0 |
21 |
4.5 |
42 |
|
0 |
0 |
1 |
3.0 |
0.15 |
9.6 |
2 |
2 |
1 |
28 |
8.7 |
94 |
|
0 |
1 |
0 |
3.0 |
0.15 |
11 |
2 |
2 |
2 |
35 |
8.7 |
94 |
|
0 |
1 |
1 |
6.1 |
1.2 |
18 |
2 |
3 |
0 |
29 |
8.7 |
94 |
|
0 |
2 |
0 |
6.2 |
1.2 |
18 |
2 |
3 |
1 |
36 |
8.7 |
94 |
|
0 |
3 |
0 |
9.4 |
3.6 |
38 |
3 |
0 |
0 |
23 |
4.6 |
110 |
|
1 |
0 |
0 |
3.6 |
0.17 |
18 |
3 |
0 |
1 |
38 |
8.7 |
180 |
|
1 |
0 |
1 |
7.2 |
1.3 |
18 |
3 |
0 |
2 |
64 |
17 |
180 |
|
1 |
0 |
2 |
11 |
3.6 |
38 |
3 |
1 |
0 |
43 |
9 |
180 |
|
1 |
1 |
0 |
7.4 |
1.3 |
20 |
3 |
1 |
1 |
75 |
17 |
200 |
|
1 |
1 |
1 |
11 |
3.6 |
38 |
3 |
1 |
2 |
120 |
37 |
420 |
|
1 |
2 |
0 |
11 |
3.6 |
42 |
3 |
1 |
3 |
160 |
40 |
420 |
|
1 |
2 |
1 |
15 |
4.5 |
42 |
3 |
2 |
0 |
93 |
18 |
420 |
|
1 |
3 |
0 |
16 |
4.5 |
42 |
3 |
2 |
1 |
150 |
37 |
420 |
|
2 |
0 |
0 |
9.2 |
1.4 |
38 |
3 |
2 |
2 |
210 |
40 |
430 |
|
2 |
0 |
1 |
14 |
3.6 |
42 |
3 |
2 |
3 |
290 |
90 |
1000 |
|
2 |
0 |
2 |
20 |
4.5 |
42 |
3 |
3 |
0 |
240 |
42 |
1000 |
|
2 |
1 |
0 |
15 |
3.7 |
42 |
3 |
3 |
1 |
460 |
90 |
2000 |
|
2 |
1 |
1 |
20 |
4.5 |
94 |
3 |
3 |
2 |
1100 |
180 |
4100 |
|
2 |
1 |
2 |
27 |
8.7 |
42 |
3 |
3 |
3 |
>1100 |
420 |
--- |
Media Component (g/L) , 25℃
1. Modified Chalmers Agar (MC):
Soy Peptone 5.0g
Beef Extract Powder 3.0g
Yeast Extract Powder 3.0g
Glucose 20.0g
Actose 20.0g
CaCO3 10.0g
Agar 15.0g
Neutral Red 0.05g
pH 6.0±0.1
2. Lauryl Sulfate Tryptose Broth (LST) :
Tryptone 20.0g
Nacl 5.0g
Actose 5.0g
K2HPO4 2.75g
KH2PO4 2.75g
Sodium Lauryl Sulfate 0.1g
pH 6.8±0.2
3. Brilliant Green Lactose Bile Broth (BGLB):
Tryptone 10.0g
Actose 10.0g
Ox-gall Powder 20.0g
Brilliant Green 0.0133g
pH 7.2±0.1
Genome Extraction
For bacterial genome extraction we used TIANamp Genomic DNA Kit, Invitrogen according to manufacturer's instructions.
Plasmids extraction
For bacterial plasmid extraction we used EndoFree Maxi Plasmid Kit and TIANprep Midi Plasmid Kit.
Gel Extraction
We used TIANgel Midi Purification Kit according to manufacturer's instructions.
Transformation by electroporation
1. Inoculate 100 μl bacterial culture into 50 ml of MRS and incubate thr 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 an OD600 of about 50.
5. Mix 100μl electrocompetent cells with 10 μl plasmid DNA.
6. Incubate the cuvettes for electroporation on ice and the above mix as well
7. Subjected the sample to a 2.4 kV, 200 Ω, 25 μF electric pulse.
8. Add 950 μl SMRS as quick as possible.
9. Incubate for 2 h at 37°C.
10. Plate on MRS supplemented with the appropriate antibiotic.
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 medium, 180rpm, incubate for 8 hours.
2. Centrifuge at o harvest the culture supernatant.
3. Pass the liquid through a 0.22 μm filtering membrane.
4. Add the supernantant to the culture medium of Lactobcillus.
5. Add nisin to the final concentration of 50 ng/ml.
6. Incubate the engineered Lactobcillus overnight.
7. Centrifuge at 6000 rpm for 5 min.
1. Incubate E. Coli CD-2 and E. Coli DH5alpha on LB agar plate.
2. Wash the colonies with fresh MRS.
3. Pass the colony wash through a 0.22 μm filtering membrane.
4. Add the supernantant to the culture medium of engineered Lactobcillus.
5. Add nisin to the final concentration of 50 ng/ml.
6. Incubate the engineered Lactobcillus overnight.
7. Centrifuge at 6000 rpm for 5 min.