1 Scope
This standard specifies the method for identification and enumeration of bifidobacterium.
This standard is applicable to identification and enumeration of pure strains of bifidobacterium, strain identification of single bifidobacterium in foods and enumeration of bifidobacterium in foods; namely, one or more different strains of bifidobacterium may be contained in foods.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Constant temperature incubator: 36℃±1℃.
2.2 Refrigerator: 2℃~5℃.
2.3 Balance: with sensibility of 0.01g.
2.4 Aseptic test tube: 18mm×l80mm, 15mm×100mm.
2.5 Aseptic pipette: 1mL (with scale division of 0.01mL), 10mL (with scale division of 0.1mL) or micropipettor and supporting pipette tips.
2.6 Aseptic culture dish: 90mm in diameter.
3 Culture Media and Reagents
3.1 Bifidobacterium medium: see A.1.
3.2 PYG medium: see A.2.
3.3 MRS medium: see A.3.
3.4 Methanol: analytical reagent.
3.5 Trichloromethane: analytical reagent.
3.6 Sulfuric acid: analytical reagent.
3.7 Glacial acetic acid: analytical reagent.
3.8 Lactic acid: analytical reagent.
4 Examination Procedures
See Figure 1 for the examination procedures of bifidobacterium.
Figure 1 Examination Procedures of Bifidobacterium
5 Operation Steps
5.1 Aseptic requirements
All operations shall comply with the aseptic technique procedures.
5.2 Identification of bifidobacterium
5.2.1 Pure strain
5.2.1.1 Sample treatment: for semi-solid or liquid strains, directly inoculate into the bifidobacterium or MRS agar plate; for solid or vacuum freeze-drying strains, add right amount of sterilized normal saline or other appropriate diluent to dissolve the strain powder.
5.2.1.2 Inoculation: inoculate in bifidobacterium or MRS agar plate, conduct anaerobic cultivation at 36℃±1℃ for 48h±2h and extend to 72h±2h when necessary.
5.2.2 Food sample
5.2.2.1 Sample treatment: take 25.0g (mL) into a aseptic conical flask or homogenizing bag containing 225.0mL of normal saline, homogenize at 8 000r/min~10 000r/min for 1min~2min, or flap with a slap type homogenizer for 1min~2min, to prepare the 1:10 homogeneous sample solution. The frozen samples may be firstly unfrozen at 2℃~5℃ for no more than 18h or at a temperature not exceeding 45℃ for no more than 15min.
5.2.2.2 Inoculation or coating: inoculate the said homogeneous sample solution in bifidobacterium or MRS agar plate, or take 0.1mL of homogeneous sample solution of appropriate degree of dilution and uniformly distribute on the bifidobacterium or MRS agar plate. Conduct anaerobic cultivation at 36℃±1℃ for 48h±2h and extend to 72h±2h when necessary.
5.2.2.3 Pure cultivation: select three or more single colonies and inoculate in the bifidobacterium or MRS agar plate. Conduct anaerobic cultivation at 36℃±1℃ for 48h±2h and extend to 72h±2h when necessary.
5.2.3 Strain identification
5.2.3.1 Smear microscopic examination: select single colony of bifidobacterium on bifidobacterium or MRS agar plate and stain. The bifidobacterium is gram positive in the form of short bar, thin bar or sphere and may form various branches or divarication, without acid resistance, spore and power.
5.2.3.2 Biochemical identification: select single colony of bifidobacterium on bifidobacterium or MRS agar plate for biochemical reaction test. The catalase test is negative. See Table 1 for main biochemical reactions of bifidobacterium. Biochemical identification kit or full-automatic microbe biochemical identification system is available.
Table 1 Main Biochemical Reactions of Bifidobacterium Strain
No. Item B.bifidum B.infantis B.longum B.adolescentis B.animalis B.breve
1 L-arabinose - - + + + -
2 D-ribose - + + + + +
3 D-xylose - + + d + +
4 L-xylose - - - - - -
5 Adonitol - - - - - -
6 D-galactose d + + + d +
7 D-glucose + + + + + +
8 D-fructose d + + d d +
9 D-mannose - + + - - -
10 L-sorbose - - - - - -
11 L-rhamnose - - - - - -
12 Dulcitol - - - - - -
13 Inositol - - - - - +
14 Mannitol - - - -a - -a
15 Sorbitol - - - -a - -a
16 α-methyl-D-glucoside - - + - - -
17 N-acetyl-glucosamine - - - - - +
18 Amygdalin - - - + + -
19 Esculin - - + + + -
20 Salicin - + - + + -
21 D-cellobiose - + - d - -
22 D-maltose - + + + + +
23 D-lactose + + + + + +
24 D-melibiose - + + + + +
25 D-sucrose - + + + + +
26 D-trehalose - - - - - -
27 Synanthrin - -a - -a - -a
28 D-melezitose - - + + - -
29 D-raffinose - + + + + +
30 Starch - - - + - -
31 Glycogen - - - - - -
32 Gentiobiose - + - + + +
33 Sodium gluconate - - - + - -
Note: + represents that more than 90% of strains are positive; - represents that more than 90% of strains are negative; d represents that 11%~89% of strains are positive.
a represents that some strains are positive.
5.2.3.3 Determination of organic acid: see Appendix B for the determination of organic acid metabolites (optional) of bifidobacterium.
5.3 Enumeration of bifidobacterium
5.3.1 Pure strain
5.3.1.1 Preparation of solid and semi-solid samples: weight 2.0g of sample through aseptic technique, put in an aseptic homogenizing cup containing 198.0mL of diluent, homogenize at 8 000r/min~10 000r/min for 1min~2min, or put in an aseptic homogenizing bag containing 198.0mL of diluent, slap with a slap type homogenizer for 1min~2min, to prepare the 1:10 homogeneous sample solution.
5.3.1.2 Preparation of liquid sample: take 1.0mL of sample through aseptic technique, add into 9.0mL of diluent and mix well to prepare the 1:10 homogeneous sample solution.
5.3.2 Food sample
5.3.2.1 Sample treatment: take 25.0g(mL) into a aseptic conical flask or homogenizing bag containing 225.0mL of normal saline, homogenize at 8 000r/min~10 000r/min for 1min~2min, or flap with a slap type homogenizer for 1min~2min, to prepare the 1:10 homogeneous sample solution. The frozen samples may be firstly unfrozen at 2℃~5℃ for no more than 18h or at a temperature not exceeding 45℃ for no more than 15min.
5.3.3 Serial dilutions and cultivation
Use 1mL aseptic pipette or micropipettor to prepare decimal serial dilutions of homogeneous sample solution, homogenize at 8 000r/min~10 000r/min for 1min~2min or flap with a slap type homogenizer for 1min~2min. Change a new 1mL aseptic pipette or pipette tip for each serial dilution. According to the estimation of sample concentration, select the homogeneous sample solution of 2~3 appropriate degrees of dilution, take 1.0mL of homogeneous sample solution into aseptic petri dish during decimal serial dilutions and prepare two petri dishes for each dilution. Meanwhile, respectively take 1.0mL of blank diluent and add into two aseptic petri dishes for blank control. Timely cool 15mL~20mL into bifidobacterium or MRS agar medium at 46℃ (put in the thermostatic water bath at 46℃±1℃ for insulation when necessary) and pour to the petri dish and rotate it to mix well. The operation starting from sample dilution to plate pouring shall be completed within 15min. Reverse the plate after the agar is solidified, conduct anaerobic cultivation at 36℃±1℃ for 48h±2h and extend to 72h±2h when necessary. Enumerate all the colonies on the plate after cultivation.
5.3.4 Colony count
5.3.4.1 Observe visually or with magnifier or colony counter when necessary and record the dilution ratio and corresponding colony count. The colony count is expressed by colony-forming units (CFU).
5.3.4.2 Select the plate with colony count between 30CFU~300CFU and free from spreading colony and enumerate the total colony count. For plates with colony count less than 30CFU, record the concrete colony count; for those with colony count more than 300CFU, it may be recorded as countless. The colony count of each dilution shall be the average of two plates.
5.3.4.3 Any plate thereof on which relatively large flake colonies grow should not be used, while those without flake colony shall be taken as the colony count of this dilution degree; if the flake colonies are less than half of the plate and the rest are distributed very uniformly, the plate colony count shall be that of half plate multiplied by 2.
5.3.4.4 When the colonies on the plate show chain growth without obvious boundary, each single chain shall be taken as one colony count.
5.3.5 Expression of results
5.3.5.1 If the colony count on plates for only one dilution degree is within the appropriate counting range, the average value of colony count on two plates shall be calculated and multiplied by corresponding dilution ratio to get the total colony count per g(mL).
Foreword i
1 Scope
2 Apparatus and Materials
3 Culture Media and Reagents
4 Examination Procedures
5 Operation Steps
Appendix A Culture Media and Reagents
Appendix B Detection Method for Organic Acid Metabolites of Bifidobacterium
