GA0060-0992 Bio system A study on the development of bio system for the testing of antimicrobials in milk
Bio system. 1998. 12. : : ( ) : ( ) : ( ) : ( ) : ( ) : ( ) - 1 -
I. Biosystem II. 3 TTC.. disc assay Delvo test, Charm test, BR test. TTC. - 2 -
III. 1... system. system 2.. 1) 2) 3) 4). system 1) 2) 3) 4). system 1) 2) 3) 4) 5) bio system - 3 -
IV. 1.. 1) 807 Gentamicin(1ppm), Sulfamethazine(5000ppm), Oxytetracycline(1ppm), Penicillin G(0.024ppm), Erythromycine(1ppm) 107. 107 2Sulfamethazine (0.01ppm), Sulfadimethoxine (0.01ppm), Sulfathiazole (0.01ppm), Oxytetracycline (0.1ppm), Penicillin G (0.024ppm), Erythromycine (1ppm), Chloramphenicol (0.1ppm) 15. 2) 15 10% (Difco) 12 TTC test. Tryptic soy broth TTC test/ TTC II test 8 Lactobacillus para. paracasei 2, Lactobacillus brevis 1, Lactobacillus plantarum 3, Leuconostoc mesen. cremoris 1, Lactobacillus acidophilus 1. 3) S. thermophilus 9 (30, 32, 35, 37 ), Glucose (2%) 3 24 ph, glucose 37 4, 35 4, 32 1, 30, glucose - 4 -
37 7, 35 1, 32, 30 1 37.. system 1) 1 807 21 2 725 43 64. 2) Brilliant blue 14 Resazurin, Methyl red, Phenol red, Bromocresol purple, Methylene blue 5. 0.5ml 0.75ml resazurin 0.005%, phenol red 0.01%, Methyl red 0.01%, Bromocresol purple 0.005%, Methylene blue 0.005%. 3) 5 Resazurin(0.005%) 8 Resazurin Resazurin, Resazurin + Methyl red (1:1) Resazurin + Bromocresol purple (1:1). - 5 -
. system 1) Tryptic soy broth skim milk / Enterococcus casseliflavus 2, Enterococcus faecium 4, Lactobacillus plantarum 1, Lactobacillus cellobiosus 1Streptococcus thermophilus 1. MRS broth Skim milk starter MRS 16. 2) Resazurin(0.005%) SPC broth, MRS broth, M17 broth, SPC + MRS(1:1), SPC + M17(1:1), MRS + M17(1:1), Skim milk), SPC + Skim milk(1:1), MRS + Skim milk(1:1), M17 + Skim milk(1:1) MRS broth, M17 broth, MRS + Skim milk(1:1), SPC + Skim milk(1:1), MRS + Skim milk(1:1) SPC Skim milk. 3) / (SPC +Skim milk) (resazurin, methyl red, bromocresol purple) Skim milk SPC, Skim milk SPC Resazurin. - 6 -
4) bio system bio system multiwell plate Enterococcus casseliflavus, Enterococcus faecium, Lactobacillus plantarum, Lactobacillus cellobiosus Streptococcus thermophilus MRS 20ul, SPC + Skim milk 140ul, Resazurin(Resazurin + Methyl red {1:1} Resazurin + Bromocresol purple {1:1}) 60ul 40ul 37 3. - 7 -
S UMMA RY Subject : A study on the development of bio system for the testing of antimicrobials in milk This study was carried out to establish effective bio system for the detection of antimicrobials in milk. Lactic bacteria was isolated from the raw milk and selected based on the sensitivity of antimicrobials in milk. Various kinds of medium and indicators were tested its sensitivity on the antimicrobials and selected. Based on the results of sensitivity, lactic bacteria, medium and indicators were selected and antimicrobials testing bio system were established. The results obtained were summarized as follows; 1. Among 807 strains of lactic bacteria isolated from the raw milk, 15 were selected as a testing organism based on the sensitivity on the antibiotics(sulfamethazine 0.01 ppm, sulfadimetoxin 0.01 ppm, sulfathiazole 0.01 ppm, sulfathiazole 0.01 ppm, erythromycine 1 ppm, chloramphenicol 0.1 ppm). 2. Among 15 strains of lactic bacteria, 8 of them shown coagulability and sensitivity on the skim milk and tryptic soy broth. They were identified as Lactobacillus para paracasei, Lactobacillus brevis, Lactobacillus plantarum, Leuconostoc mesen. cremoris, Lactobacillus acidophilus. - 8 -
3. Through the ph changes during the fermentation of lactic bacteria in skim milk at various temperatures, optimun temperature for the incubation of selected lactic bacteria were revealed as 37 4. Totally 64 strains of lactic bacteria were selected from 1532 strains of bacteria isolated from raw milk during 2 years study. 5. Various kinds of indicators(14) were tested its sensitivity for the antimicrobial testing and optimum indicator for the detection of antimicrobials were resazurin, methyl red and bromocresol purple and its optimum concentration and dosage were 0.1-0.005% and 0.5 ml respectively. 6. Practical indicator for the detection of antimicrobials were Resazurin, Resazurin + Methyl red(1:1) and Resazurin + Bromocresol purple(1:1) compared to the other kinds of indicators. 7. Through the sensitivity test on the tryptic soy broth and skim milk medium, selected strains were identified as Enterococcus casseliflavus, Enterococcus faecium, Lactobacillus plantarum, Lactobacillus cellobiosus, and Streptococcus thermophilus. And MRS broth was optimum medium for the preparation of starter cultures of selected lactic bacteria. 8. Through the various kinds of medium test with the Resazurin indicator, MRS broth, M17 broth, MRS + Skim milk(1:1) and SPC + Skim - 9 -
milk were revealed as good medium and SPC+Skim milk was one of the best medium for the antimicrobial test in milk. 9. SPC+Skim milk and Resazurin were the optimum medium and indicator for the antimicrobial testing of raw milk. 10. The optimum bio system for the testing of antimicrobials in milk is composed of 20ul of lactic starter culture(enterococcuscasseliflavus, Enterococcus faecium, Lactobacillus plantarum, Lactobacillus cellobiosus, Streptococcusthermophilus), 140ul of medium broth(spc + Skim milk), 60ul of indicator solution(resazurin, Methyl red, Bromocresol purple) and 40 ul of sample milk in the multiwell plate. The mixed solution of the multiwell plate is incubated at 37 for 3 hours then the result will be defined according to the changes of indicator in the mixed solution. - 10 -
CONT ENT S Chapter 1 Introduction --------------------------------------------17 Part 1. Objectives and scope of study ---------------------------17 1. Objectives of the study ---------------------------------17 2. Scope of the Study --------------------------------------17 Part 2. Trends of Raw milk quality and antimicrobial test -------18 1. Raw milk quality ----------------------------------------18 2. Antimicrobial testing -----------------------------------26 Chapter 2 Selection of strain and establishment of incubation condition -----------------------------------------------38 Part 1. Introduction --------------------------------------------38 Part 2. Materials and Methods -----------------------------------39 1. Isolation of lactic bacteria ----------------------------39 2. Preparation of antimicrobial solution -------------------40 3. antimicrobial sensitivity test --------------------------41 4. selection of lactic bacteria for the TTC/TTC-II test ----42 5. Preparation of TTC/TTC-II solution ----------------------42 6. TTC/TTC-II test with the selected lactic bacteria -------43 7. Identification of selected lactic bacteria --------------43 8. Optimum incubation condition ----------------------------44-11 -
Part 3. Results and discussion ----------------------------------44 1. Screening of bacteria for the antimicrobial test --------44 2. Selection and Characterization of selected bacteria -----52 3. Optimum incubation condition for antimicrobial test -----60 Chapter 3 Development of indicator for the antimicrobial test -----66 Part 1. Introduction --------------------------------------------66 Part 2. Materials and Methods -----------------------------------68 1. Sensitivity test of 1st selected lactic bacteria --------68 2. Screening of lactic bacteria fron raw milk --------------69 3. Sensitivity test of 2nd selected lactic bacteria --------69 4. Selection and characterization of indicator -------------70 5. Optimization of lactic bacteria and indicator -----------71 Part 3. Results and discussion ----------------------------------75 1. Selection and characterization of lactic bacteria -------75 2. Selection and characterization of indicator -------------79 3. Optimization of lactic bacteria and indicator -----------89 Chapter 4 Establishment of bio system with selected strin, medium and indicator -----------------------------------------------99 Part 1. Introduction --------------------------------------------99 Part 2. Materials and Methods ----------------------------------100 1. Identification of final selected lactic bacteria -------100 2. Coparision of bactria grown in MRS and skim milk -------100-12 -
3. Sensitivity of selected bacteria at various medium -----101 4. Establishment of optimum medium condition --------------105 5. Analysis of residual Sulfadrugs in milk by HPLC --------106 6. Optimum mixture of bacteria, medium and indicator ------107 Part 3. Results and discussion ---------------------------------108 1. Preparation of solution of selected lactic bacteria ----108 2. Preparation of optimum medium --------------------------121 3. Preparation of mixture for the antimicrobial test ------129 4. Bio system of bacteria, medium and indicator -----------135 References -----------------------------------------------------142 Appendix 1.Trend of antimicrobial testing system in USA -----------148 2.Trend of antimicrobial testing system in Australia -----152-13 -
1 1 -----------------------------------------------------17 -----------------------------------17 1. ------------------------------------------------------17 2. ------------------------------------------------------17 2 --------------------------------18 1. -----------------------------------------------------18 2. -------------------------------------------------26 2 1 2 ---38 -----------------------------------------------------38 ---------------------------------------------39 1. -----------------------------------39 2. TTC II test -------------------------------------------------------40 3. ------------------------------------------41 4. TTC / TTC II test ---------------------42 5. TTC / TTC II test ---------------------------------42 6. TTC / TTC II test ----------43 7. ---------------------------------------43 8. ---------------------------------44-14 -
3 ---------------------------------------------44 1. ---------44 2. ------------------------52 3. ----------------------------60 3 1 2 system ---------------------66 -----------------------------------------------------66 ---------------------------------------------68 1. 1 -------------------------68 2. -----------------------------------69 3. 2 ---------------------------69 4. ------------------70 5. ----------------71 3 ---------------------------------------------75 1. -----------------------75 2. ----------------------------------79 3. ----------------87 4 1 2 system ---99 -----------------------------------------------------99 --------------------------------------------100 1. ---------------------------------------100 2. MRS Skim milk ---------------------100 3. -----------------------------101 4. -------------------------------------------105-15 -
5. HPLC ------------------------106 6. / / --------------------------107 3 --------------------------------------------108 1. -----------------------------108 2. -----------------------------121 3. -------------------------------129 4. bio system --135 ------------------------------------------------------142 1. -----------------------------------148 2. -----------------------------------152-16 -
1 1 1. 3 TTC.. disc assay Delvo test, Charm test, BR test. TTC. 2., system system. - 17 -
1,,,. 2 system,,,. 3 system,,, bio system. 2 1. 1995 1. 200. - 18 -
. 1993 6 1997 75% 1, 4 5% 4,5.. - 19 -
1. Petri film Spiral count Bacto scan Breed count Petri foss Petri foss Petri foss Bacto scan SPC Fossomatic Fossomatic Fossomatic Fossomatic Fossomatic Somacount Disc assay TTC (1 ) Dairy Board MMB * : 3-4 / * : Milkoscan, Multispec * : Delvo test, BR test, Charm aim test * : Lactec test, Cite probe MMB Disc assay Charm test Charm test Disc Assay MMB - 20 -
2. /ml /ml 1 10 +39 1 25 0 2 10 25 +16 2 25 50 0 3 25 50 + 6,93. 6. 1 3 50 75 0 4 50 100 0 75-11 100-11,95.10.16,96. 7. 1,97. 3. 1,98. 1 50 11 1A 1B 2 3 4 3 +52 10 +41 10 25 +17 25 50 + 9 50 100 0 75-11 100-31 1 2 3 20 0 20 50 0 50 75 0 83 11 1A 1B 2 3 4 3 >11 10 >24 10 25 > 8 25 50 >40 50 1 2 3 4 20 +30 20 50 0 50 60-10 60-30 83 60 1A 1B 2 3 4 3 >11 10 >24 10 25 > 8 25 50 >40 50 1 2 3 20 +30 20 50 0 50-30 83 60 1A 1B 2 3 4 3 553 10 540 10 25 512 25 50 502 50 419 * 4 3 1-21 -
3. (ml ) 1 2 3 4 5 6 3 10 10 30 3 30 - - 40 2 10 2 10 - - - 20 2 5 2 5 - - - - 30 1 5 7 5 5 7 5 - - - 10 1 2 2 5 5 - - - 52 4 2 5 2 5 5 10 10 20 20 50 50-10 1 30 30 - - - - 4 4 10 10 25 25 50 50-100 1-2 10 25 25 50 10 50 - - - 2 ) - : (TBC : Total Bacterial Counts) - : (CFU : Colony Forming Units) - 22 -
4. (ml ) 1 2 3 4 5 30 30 40 40 75 75 - +3ore/kg 0-3ore/kg -6ore/kg 40 40 40 50 50 100 100 - - 0-0.5ppl -1.0ppl -2.0ppl 10 30 40 40 75 75 - +$20 $80 +$10 0 -$10 100 6 50 50 60 60 70 70 75-75 1 4 0-1/hl -2/hl -3/hl 30 30 50 50 - - 100 2 3 0-1 -2Yen -2-5Yen 30 50 50 70 30 70 - - 2 +0.5% 0-1.0% -2/0% 20 20 50 50 - - - 2 +30 0-30 20 20 30 30 50 50 - Varies 50 60 60 80 80 100 50 100 - Varies ) : IDF Bulletin No. 305/1995 75-10 1-23 -
5. ( : count/ml,%) 1 <10 2 <25 3 <50 4 <100 5 >100 1993. 6 1993 1994 1995 26.7 44.7 60.4 66.8 18.7 20.5 18.7 17.7 15.5 13.1 9.9 7.8 17.6 12.3 7.1 5.3 21.5 9.6 4.1 3.1 1 <25 <50 >50 A<3 B<10 1996 1997 1998 40.6 47.3 60.5 30.2 28.8 20.5 15.5 13.3 9.5 6.9 5.5 5.5 5.0 4.9 4.0-24 -
6. ( : count/ml,%) 1 (<25 ) 2 (<50 ) 3 (<75 ) 4 (>75 ) 1993.6 1993 1994 1995.10 1995 30.03 26.93 26.65 20 23.28 34.17 32.16 32.77 40 33.81 17.87 19.12 22.39 75 25.18 17.93 21.73 18.11 75 17.72 1996 23.4 30.46 31.35 14.75 1996.7 1 (<20 ) 2 (<50 ) 3 (<60 ) 4 (>60 ) 1996 22.7 44.7 9.75 22.7 1997.31 (<20 ) 2 (<50 ) 3 (<50 ) 1998 24.4 46.7 28.9 - - 25 -
2..,,,,,. TTC, TTC ( ) sulfa. TTC, Delvo test Penzyme test., Disc Assay, Delvo test, BR test, Charm test, HPLC (Van Eenennaam, 1993; Macaulay Packard, 1981; Read, 1971). (Delvo test, BR test) (Disc Assay) (Hammonds Adenwala, 1990), (Charm test, HPLC test), Lactec test, Charm test.,., kit. (Disk Assay), (TTC, Delvo, BR, Charm AIM), - 26 -
(Charm ), (Penzyme), (Lactek, Cite Prove, Signal. Ez-Screen, Agri-Screen) (, 1993; Bishop, 1990). 1) BsDA(B. stearothermophilus disk assay) 0.08IU/ml penicillin 2.5 (Gilbertson, 1995). 82 2, penicillinase (, 1993). 2) ) Charm Farm Test (, 1993). ) Delvotest (Bishop, 1990), B. stearothermophilus var. calidolactis (Carlsson Björck, 1992) Delvotest P sulfa Delvotest SP.(Larocque, 1986).,, (Bishop, 1990). ) BR Test (Brilliant Black Reduction Test) TTC (, 1993) agar diffusion. (Bishop, 1990). ) TTC test (Streptococcus thermophilus) TTC(2,3,5-triphenyltetrazolium), TTC Triphenyl formazan., TTC - 27 -
, (Bishop, 1990). 10% 9ml 80 5 37 (10% + ) 1ml 37 2 TTC 4% 0.3ml 37. TTC succinate dehydrogenase triphenyl formazon,. 7 (, 1993). TTC TMP(trimethoprim) Streptococcus thermophilus TTC triphenyl formazon TTC II test p-aminobenzoc acid(paba) penicillinase (Bishop, 1990). 3) Membrane-matrix antibiotic-binding protein -lactam CITE Probe Tetracycline CITE Probe (, 1993). 4) ELISA( ) ELISA (Bishop, 1990; Charm Chi, 1986). ) Lactek system, - 28 -
(Bishop, 1990;, 1991). ) Agri-Screen Test well polyclonal (Bishop, 1990). ) Signal T gentamicin, neomicin, sulfamethazine 3 high-affinity well (Bishop, 1990). ) Cite prove Cite (Bishop, 1990). ) Ez-Screen test 5 gentamicin, sulfadimethoxin, sulfamethazine (Bishop, 1990). 5) Charm II Test (, 1993; Brady Katz, 1989), - lactam, macrolide, sulfonamide, aminoglycoside, tetracycline chloramphenicol (Senyk, 1990; Collins-Thompson, 1988; Carlsson Björck, 1991). (Bishop, 1990). 6) Penzyme method -lactam (Bishop, 1990; Senyk, 1990; Suhren Walte, 1996). - 29 -
7. Disc Assay TTC TTC II D e l v o 100 / 3.5-4 50 / 64 / 2.5 83-87 / 100 / 10 5, 3-3.5/ 37 / 2.5 83-87 / 100 / 30, 5, 3-3.5/ 37 / 2.5 100 / 2,000 / P 3-3.5 SP 100 / 2,000 / 3.5-4 64 / 2.5,,, 64 / 3, BR 100 / 3.5-4 -,,,, 64 /, 2.75,, - 30 -
Charm AIM,, 96 / 1,500 /,65 / 3.5-4 3, Charm II 1 / 8 6,000 / 6, 85 / 37, 8, 7,, 18, 3 6,,,,,,,, Penzyme 100 / 2,000 / 1-1.5 47 / 15-31 -
Lactek 1 / 7,, 1,500 /, / 3-4, Cite prove Signal - - - -,,, -,,,,,, - Ez-Screen 1 / 5 -, -, Agriscreen - - - : (1994) - 32 -
8. BR Test Charm II Charm Charm CITE Delvo Delvo Disk EZ- La Cowside Farm test P test SP Assay Screen B ) - 33 -
. 9, Codex,. 9. (MRLs) / Codex MRLs 4-100 100 EU MRLs 4 4 4 30 30 30 100 ( : ppb) / 5/0 10/10 10/10 10/10 50/1,000 20/20 30/0 100 ( 30/0 ) 80/0 10/0 25 100 10/10 ( 10/0 ) 10/0 10/0 40 50/0 100 200 50 50/50 100 30/0 500 150/150 200 125/0 0 0 0/0 200 200 100/100 50 30/0 ) Codex. - 34 -
TTC.. Charm AIM Delvotest 105 Lactek test 10. 10. Charm AIM Delvotest Lactek test + - - (%) -Lactam 1(0.25) 399(99.75) Tetracycline 17(4.25) 383(95.75) Chloramphenicol 33(8.25) 367(91.75) 400(100) Sulfamethazine 48(12.00) 352(88.00) ) : + =, - = 10 4 -Lactam 0.25%, Tetracycline 4.25%, Chloramphenicol 8.25%, Sulfamethazine 12% -Lactam Penicillin-G Sulfa Chloramphenicol. 11. - 35 -
11. AMINOGLYCOSIDES Amikacin Dihydrostreptomycin Gentamicin Kanamycin Neomycin Streptomycin BACITRACIN BETA-LACTAMS CHLORAMPHENICOL FOLIC ACID ANALOG MACROLIDES LINCOSAMIDES Amoxicillin Ampicillin Cefadroxil Cefotaxime Ceftiofur Cephalexin Cephaloridine Cephalosporin Cephapirin Cephradine Cloxacillin Dicloxicillin Hetacillin Nafcillin Oxacillin Penicillin Piperacillin Ticarcillin Florfenicol Thiamphenicol Chloramphenicol glucuronide Chloramphenicol Methotrexate Pyrimethamine Trimethoprim Clindamycin Erythromycin Lincomycin - 36 -
Oleandomycin Pirlimycin Spiramycin Tylosin NOVOBIOCIN SPECTINOMYCIN SULFA DRUGS TETRACYCLINES Dapsone Sulfabromomethazine Sulfacetamide Sulfachlorpyridazine Sulfadiazine Sulfadimethoxine Sulfadoxine Sulfaethoxypyridazine Sulfamerazine Sulfamethazine Sulfamethizole Sulfamethoxazole Sulfanilamide Sulfapyridine Sulfaquinoxaline Sulfathiazole Sulfisoxazole Chlortetracycline Demeclocycline Doxycycline Methacycline Minocycline Oxytetracycline Rolitetracycline Tetracycline - 37 -
2 1,,,., TTC II Disc assay HPLC 12 TTC II 12. TTC II TTC II -lactam 0.0024ppm 0.004ppm Sulfa 0.05ppm 0.01ppm Oxytetracycline - 0.1ppm -lactam Sulfa Oxytetracycline TTC II Bulk Tank Charm AIM, Charm II, Delvo, Lactec, Penzyme test. TTC II,. kit. - 38 -
2 1. sodium azide(juncei chemical Co.Ltd) 0.025% 35 24 0.1% peptone (Difco) 102-104 sodium azide 0.025% MRS agar plate( 13) 0.2ml 35 48. MRS agar plate 3 35 48. MRS agar plate TTC / TTC II test. 13. MRS agar Component gram/liter Bacto Proteose Peptone #3 10.0 Bacto Beef Extract 10.0 Bacto Yeast Extract 5.0 Bacto Dextrose 20.0 Tween 80 1.0 Ammonium Citrate 2.0 Sodium Acetate 5.0 Magnesium Sulfate 0.1 Manganese Sulfate 0.05 Dipotassium Phosphate 2.0 Sodium Azide 0.25 Bacto Agar 15.0-39 -
2. TTC II test penicillin G, oxytetracycline, sulfamethazine, sulfathiazole, sulfadimethoxine, erythromycine, chloramphenicol Sigma (U.S.A). 14. concentration(ppm) Antimicrobial agents first selection second selection Solvents penicillin G 0.024 0.024 H2O + NaOH oxytetracycline 1 0.1 H2O + NaOH sulfamethazine 5,000 0.01 H2O + NaOH sulfathiazol 5,000 0.01 H2O + NaOH sulfadimethoxine 5,000 0.01 H2O + NaOH erythromycine 1 0.25 H2O + Alcohol chloramphenicol 5 0.1 H2O + Alcohol - 40 -
penicillin G 60ppm, oxytetracycline 100ppm, sulfamethazine 50ppm, sulfathiazole 50ppm, sulfadimethoxine 50ppm, erythromycine 260ppm, chloramphenicol 100ppm stock solution 4 1 membrane filter(0.45 m, millipore Co. U.S.A). 3. MRS broth 2 MRS broth(difco) 35 18 0.1% peptone(difco) 105-106cfu/ml. MIC(minimum inhibitory concentration) two-fold macrodilution broth (Koneman, 1988) 0.2% yeast extract(difco) tryptic soy broth(difco), 35 48 (, 1995). oxytetracycline 1ppm, penicillin G 0.024ppm, sulfamethazine 5000ppm, sulfadimethoxine 5000ppm, sulfathiazole 5000ppm, erythromycine 1ppm, chloramphenicol 5ppm, Sigma Chemical Co. (USA). - 41 -
15. Tryptic soy broth Component gram/liter Bacto tryptone 17 Bacto soytone 3 Bacto dextrose 2.5 Sodium chloride 5 Dipotassium phosphate 2.5 Yeast extract 2 4. TTC / TTC II test MRS broth(difco) 2 10% (Difco) 24. 5. TTC / TTC II test. TTC TTC(2,3,5-triphenyltetrazolium chloride, Sigma) 1:25 (4% ) 7 2.. TMP 50.0mg TMP(trimethoprim, Sigma) 100ml methanol 10ml 500 g/ml 1 4 1:9 (10 ) 50 g/ml. - 42 -
membrane filter(0.45 m. millipore Co. U.S.A.). 6. TTC / TTC II test TTC test MRS (Difco) 2 10% (Difco) 5% 37 12 10% 1:1(v/v).. TTC test 10% (DIFCO) 8.2ml 0.8ml vortex mixer(fisher scientific) 15 85 5. negative control.. 10% (DIFCO) 7.2ml TMP 1ml 0.8ml vortex mixer(fisher scientific) 15 85 5. negative control TMP control sample negative control sample TMP. 1ml 37 incubator 2. 4% TTC 0.3 ml negative control TMP control sample ( 1 ) TMP contol ( ),. 7. MRS broth(difco) - 43 -
2. Hammes (1992). Gram, (Olympus, Japan) API kit (API biomerieux, France) ATB identification system(biomerieux, France) genus species. 8. 10% (Difco) (30, 32, 35, 37 ), Glucose (2%) ph meter(orion. SA520) ph, 2% 3 24. 3 1.. 1 14, 681. 126 807.. - 44 -
16. ( ) ( ) 29 33 53 31 43 20 56 36 46 30 41 29 55 46 133 126. Tryptic soy broth(difco) 0.2% yeast extract(difco) 121 15 Penicillin G(0.024ppm), Oxytetracycline(0.1ppm), Sulfamethazine(0.01ppm), Sulfadimethoxine(0.01ppm), Sulfathiazole(0.01ppm), Erythromycine(0.25ppm), Chloramphenicol(0.1ppm), MRS broth (Difco) 18 105-106 48. 807 107. - 45 -
17. 107 No. Penicillin G Oxytetra Sulfame Sulfadi Sulfa Eryt -cycline -thazine -metoxin -thiazole -myc 1-3 + + + + + - 2-2 + + + + + - 5-1 + + + + + - 5-2 + + + + - 5-4 + + + + + - 9-1 + + + + + - 10-1 + + + + + - 19-1 + + + + + - 4-1 + + + + + - 6-3 + + + + - 4-1 + + + + + - 5-1 + + + + + - 10-1 + + + + + - 10-2 + + + + + - 15-1 + + + + + - 15-2 + + + + + - 15-3 + + + + + - 16-2 + + + + + - 22-3 + + + + + - ) + :, :, - : - 46 -
No. Penicillin G Oxytetra -cycline Sulfame -thazine Sulfadi -metoxin Sulfa -thiazole Eryth -myci 23-1 + + + + + - 24-1 + + + + + - 26-3 + + + + + - 2-2 + + + + + - 4-3 + + + + + - 4-4 + + + + + - 7-1 + + + + + - 10-1 + + + + + - 12-6 - - - - - - 13-2 - - - - - - 13-3 + + + + + - 13-4 + + - + + - 13-6 + + + + + - 13-7 + + + + + - 13-8 + + + + + - 1-1 + + + + + - 2-2 + + + + + - 15-2 + + + + + + - 47 -
No. Penicillin G Oxytetra Sulfame Sulfadi Sulfa -cycline -thazine -metoxin -thiazole 56-2 + + + + + 56-3 + + + + 56-5 - - - - - 56-6 + - - - - 130-3 + + + 215-3 - - + 218-1 + + + + + 218-2 + + + + + 232-1 + + + + + 232-3 - - - - - 233-3 + + + + + 237-1 + + + + + 239-1 + + + + + 243-1 + + 243-3 + + + + + 243-4 + + + + + 268-1 - - - - 284-2 + + + + + Ery -m - 48 -
No. Penicillin G Oxytetra Sulfame Sulfadi Sulfa -cycline -thazine -metoxin -thiazole 284-1 + + + + + 295-1 + + + + + 295-2 + + + + + 295-3 + + + + + 295-4 + + + + + 299-3 + + + + + 299-4 + + + + + 299-5 + + + + + 299-6 + + + + + 309-2 - - - - - 346-2 + + + + + 346-5 + + + + + 372-1 + + + + + 1 2 + + + + + 3 + + + + + 4 + + + + + 7 + + + + + Ery -my - 49 -
No. Penicillin G Oxytetra Sulfame Sulfadi Sulfathiazole myc Eryth -cycline -thazine -metoxin 673 + + + + + - 228 + + + + + - 704 + + + + + - 699 + + + + + - 393 + + + + + + 217 + + + + + - 341 + + + + + - 493 + + + + + + 491 + + + + + - 695 + + + + + - 425 + + + + + + 698 + + + + + + 231 + + + + + + 164 + + + + + + 229 + + + + + + 234 + + + + + - 163 + + + + + + - 50 -
No. Penicillin G Oxytetra Sulfame Sulfadi Sulfa -cycline -thazine -metoxin -thiazole 466 + + + + + 659 + + + + + 230 + + + + + 347 + + + + + 235 + + + + + 30 672 + + + + + 665 + + + + + 128 + + + + + 467 + + + + + 203 + + + + + 661 + + + + + 671 + + + + + 708 + + + + + 507 674 + + + + + 473 + + + + + 476 + + + + + Er -m - 51 -
2.. TTC / TTC II Tryptic soy broth(difco) TTC / TTC II test 10% (Difco). 18. Lactic bacteria coagulation selected bacteria for TTC / TTC II method 6-3 + O 12-6 + O 1-13-2 + O 13-4 + O 56-3 + O 56-5 + O 56-6 + O 215-3 + O 232-3 + O 243-1 - 243-4 - 268-1 + O 309-2 + O 507 + O ) + :, - : 15 MRS broth 10% 12 12 TTC test. 10% - 52 -
ph 4.6 (isoelectric point) ph 4.6 (, 1979).,.. TTC / TTC II test Tryptic soy broth, 10% skim milk 12 TTC / TTC II test. - 53 -
19. TTC Antimicrobial agents Lactic bacteria blank Penici -lling Erythr Oxytetracyclinphenicol Chloram- Sulfamethazine -omycin Sulfadimethoxine Sulfathiazol 6-3 + + + + + 12-6 + + + + + + 13-2 + + + + + + + + 13-4 + + + + + + + + 56-3 + + + + + + + + 56-5 + + 56-6 + 215-3 + + + + + + + 232-3 + + + + + + + 268-1 + + + + + + + 309-2 + + + + + + + + 507 + - - S.thermophilus + + + + ) + :, :, - : - 54 -
20. TTC II Antimicrobial agents Lactic bacteria blank TMP blank -ing Penicill Erythro- Oxytetra- mycin Sulfa - methoxine cycline Chloramphenicol Sulfamethazine Sulfathiazol 6-3 + + - - + + + - - 12-6 + + - - - - - - + 13-2 + + + - - + + - - 13-4 + + - - - - - - + 56-3 + + + + + + 56-5 + + + - + + - 56-6 + + + - + - - + 215-3 + + + + 232-3 + + 268-1 + + 309-2 + + + + + + 507 + + - - - - + + - S.thermophilus + + - - ) + :, :, - : - 55 -
12 TTC TMP(Trimethoprim, Sigma) TTC II TTC II TTC. TTC II TMP TTC sulfa TTC II (, 1989).. Tryptic soy broth(difco) TTC test/ TTC II test 8 Gram Gram rod. API 50 CHL kit(api biomerieux, France) ATB identification system(biomerieux, France) 21. - 56 -
21. API Tests Lactic bacteria 6-3 12-6 13-4 56-3 56-5 56-6 309-2 507 Glycerol - - - - - - - - Erythritol - - - - - - - - D Arabinose - - - - - - - - L Arabinose - - - - + - - - Ribose + + + + + - - + D Xylose - - - - - - - - L Xylose - - - - - - - - Adonitol - - - - - - - - -Methyl-D Xyloside - - - - - - - - Galactose + + + + + - + + Glucose + + + + + + + + Fructose + + + + + - + + Mannose + + + + + - + + Sorbose - - + + - - - - Rhamnose + - - - - - - - Dulcitol - - - - - - - - Inositol - - - - - - - - Mannitol + + + + + - - + Sorbitol + + + + + - - + - 57 -
Lactic bacteria Tests 6-3 12-6 13-4 56-3 56-5 56-6 309-2 507 -Methyl-D Mannoside - - - - - - - + -Methyl-D Glucoside - - - - - - - - N Acetyl Glucosamine + + + + + - + + Amygdalin + + + + + - - + Arbutin + + + + + - - + Esculin + + + + + + + + Salicin + + + + + - - + Cellobiose + + + + + - - + Maltose + + + + + - + + Lactose + + + + + + + + Melibiose - + + - + - - + Sucrose + + + + + - + + Trehalose + + + + + - + + Inulin + + - + - - - - Melezitose + + + + + - - - Raffinose - + + + - - + + Starch - - - - - - - - Glycogen - - - - - - - - Xylitol - - - - - - - - Gentiobiose - + - + - - - - D Turanose + + + + + - - + D Lyxose - - - - - - - - D Tagatose + + + + - - - - D Fucose - - - - - - - - L Fucose - - - - - - - - D Arabitol - - - - - - - - L Arabitol - - - - - - - - Gluconate - - - - - - - - 2 Ketogluconate - - - - - - - - 5 Ketogluconate - - - - - - - - ) + :, - : - 58 -
Gram (Olympus. Japan) API kit. 22. Lactic bacteria 6-3 Lactobacillus para.paracasei 12-6 Lactobacillus brevis 13-4 Lactobacillus plantarum 56-3 Lactobacillus para.paracasei 56-5 Lactobacillus plantarum 56-6 Leuconostoc mesen. cremoris 309-2 Lactobacillus acidophilus 507 Lactobacillus plantarum (1996) S. thermophilus lactic acid bacteria, 7Lactobacillus 1Leuconostoc. Lactobacillus brevis Leuconostoc acetic acic CO2 heterofermenter homofermenter. Lactobacillus plantarum. - 59 -
3. lactose cell -galactosidase -P-galactosidase glucose galactose galactose-6-phosphate galactose (, 1988). O Leary (1976) glucose lactose S. thermophilus lactose glucose L. bulgaricus glucose lactose. (1988) lactose glucose. lactose. S. thermophilus 9 (30, 32, 35, 37 ), Glucose (2%) 3 24 ph 23 24. ph glucose 37 4, 35 4, 32 1, 30, glucose 37 7, 35 1, 32, 30 1 37. - 60 -
23. skim milk(glucose ) ph Lactic bacteria 6-3 12-6 time temp 0h 3h 6h 9h 12h 30 6.55 6.34 6.42 6.37 6.30 6 32 6.55 6.36 6.44 6.38 6.32 6 35 6.55 6.42 6.37 6.26 6.12 5 37 6.55 6.37 6.39 6.27 6.18 5 30 6.55 6.35 6.43 6.43 6.42 6 32 6.55 6.37 6.46 6.46 6.40 6 35 6.55 6.45 6.43 6.39 6.26 6 37 6.55 6.42 6.44 6.36 6.28 6 30 6.55 6.39 6.45 6.44 6.45 6 309-2 507 32 6.55 6.40 6.45 6.45 6.39 6 35 6.55 6.49 6.49 6.46 6.43 6 37 6.55 6.46 6.49 6.37 6.47 6 30 6.55 6.41 6.43 6.41 6.35 6 32 6.55 6.43 6.44 6.38 6.31 6 35 6.55 6.47 6.45 6.33 6.16 6 37 6.55 6.38 6.34 6.19 6.15 6-61 -
Lactic bacteria 13-4 56-3 56-5 56-6 S. thermo -philus time temp 0h 3h 6h 9h 12h 30 6.55 6.35 6.37 6.30 6.19 6 32 6.55 6.40 6.42 6.31 6.20 6 35 6.55 6.47 6.36 6.25 6.04 5 37 6.55 6.41 6.39 6.25 6.15 5 30 6.55 6.47 6.46 6.37 6.26 6 32 6.55 6.47 6.46 6.37 6.20 6 35 6.55 6.50 6.50 6.39 6.25 6 37 6.55 6.48 6.47 6.33 6.28 6 30 6.55 6.41 6.43 6.41 6.35 6 32 6.55 6.43 6.44 6.38 6.31 6 35 6.55 6.47 6.45 6.33 6.16 6 37 6.55 6.41 6.40 6.23 6.13 5 30 6.55 6.42 6.45 6.40 6.34 6 32 6.55 6.44 6.45 6.41 6.32 6 35 6.55 6.47 6.43 6.33 6.16 5 37 6.55 6.41 6.39 6.26 6.13 5 30 6.55 6.33 5.93 5.53 5.28 5 32 6.55 6.21 5.74 5.40 5.16 5 35 6.55 6.13 5.52 5.20 4.96 4 37 6.55 5.98 5.32 5.08 4.83 4-62 -
24. skim milk(glucose ) ph. Lactic bacteria 6-3 12-6 309-2 507 time temp 0h 3h 6h 9h 12h 30 6.53 6.42 6.42 6.35 6.30 32 6.53 6.49 6.49 6.41 6.35 35 6.53 6.46 6.41 6.31 6.20 37 6.53 6.46 6.39 6.29 6.15 30 6.53 6.28 6.39 6.36 6.34 32 6.53 6.40 6.49 6.46 6.40 35 6.53 6.45 6.43 6.34 6.18 37 6.53 6.49 6.46 6.38 6.22 30 6.53 6.41 6.49 6.46 6.46 32 6.53 6.49 6.54 6.53 6.54 35 6.53 6.51 6.49 6.49 6.40 37 6.53 6.48 6.51 6.49 6.43 30 6.53 6.40 6.43 6.35 6.28 32 6.53 6.50 6.51 6.45 6.36 35 6.53 6.53 6.47 6.36 6.24 37 6.53 6.44 6.43 6.31 6.23-63 -
Lactic bacteria 13-4 56-3 56-5 56-6 S. thermo -philus time temp 0h 3h 6h 9h 12h 30 6.53 6.40 6.43 6.35 6.28 32 6.53 6.50 6.51 6.45 6.36 35 6.53 6.53 6.47 6.36 6.24 37 6.53 6.54 6.49 6.36 6.21 30 6.53 6.41 6.40 6.21 6.14 32 6.53 6.43 6.38 6.13 6.15 35 6.53 6.50 6.43 6.27 6.18 37 6.53 6.41 6.34 6.23 6.11 30 6.53 6.41 6.44 6.36 6.28 32 6.53 6.46 6.44 6.32 6.22 35 6.53 6.44 6.39 6.25 6.10 37 6.53 6.51 6.43 6.29 6.12 30 6.53 6.43 6.37 6.40 6.36 32 6.53 6.45 6.50 6.47 6.42 35 6.53 6.46 6.51 6.44 6.33 37 6.53 6.47 6.44 6.35 6.20 30 6.53 6.26 5.81 5.48 5.28 32 6.53 6.24 5.68 5.38 5.08 35 6.53 6.12 5.44 5.12 4.88 37 6.53 5.94 5.35 5.00 4.70-64 -
61% 37 37 37. glucose 37 ph. 25. glucose 37 ph Lactic bacteria 56-6 56-3 6-3 12-6 13-4 Glucose ph at 0h ph at 24h Lactic bacteria Glucose ph at 0h ph at 24h G + 5.70 G + 5.56 6.55 56-5 6.55 G - 5.75 G - 5.52 G + 6.10 G + 5.78 6.55 309-2 6.55 G - 6.00 G - 5.10 G + 5.50 G + 5.60 6.55 507 6.55 G - 5.57 G - 5.58 G + 5.07 S.thermo G + 4.45 6.55 6.55 G - 5.10 -philus G - 4.38 G + 5.30 6.55 G - 5.45 ) + : glucose, - : glucose 9 glucose ph 4, glucose 5 glucose. - 65 -
3 system 1,. (Fox, 1995; Gutierrez, 1990; Watts, 1995).., (, 1993)., (Marth Ellickson, 1959). starter (Kosikowski, 1982; Tamime Robinson, 1985; Valladao Sandine, 1994; Krienke, 1950;, 1984; Schiffmann, 1992). 1,000 penicillin, streptomycin, neomycin, chloramphenicol, tetracycline, sulphonamide, cloxacillin ampicillin. - 66 -
,,,,,, DNA RNA.. penicillin penicillinase penicillinase antibiotic-resistant yoghurt strain (, 1991).,,, calving bolluses,.(brady Katz, 1988)., penicillin (Ryan, 1986). Streptococcus Bacillus (Chagonda Ndikuweraca, 1989; Chen Chang, 1994; Vidal Collins-thompson, 1987), S. thermophilus (Reinbold Reddy. 1974), penicillin. L. delbrueckii subsp. bulgaricus S. salivarius subsp. thermophilus - 67 -
(Reinbold Reddy, 1974; Sozzi Smiley, 1980), L. acidophilus L.casei. 3 TTC. (Charm Chi, 1982; Bishop White, 1984)., Delvo Penzyme -lactam sulfa.,,,,,, kit. 2 1. 1 MRS broth(difco) 37, 30 incubator 0.02% yeast extract(difco) tryptic soy broth(difco) 105-106 48. Sigma(U.S.A.) penicillin G(0.024ppm), - 68 -
oxytetracycline(0.1ppm), sulfamethazine(0.01ppm), sulfathiazole(0.01ppm), sulfadimethoxine(0.01ppm), 10 tryptic soy broth. MRS broth skim milk 37 30 incubator 10 skim milk(difco). skim milk. 2. sodium azide(junsei chemical Co. Ltd) 0.025% 35 24 0.1% peptone(difco) 102-104 sodium azide 0.025% MRS agar plate 0.2ml 35 48. MRS agar plate 3 35 48. MRS agar plate TTC / TTC II test. 3. 2 MRS broth(difco) 37 incubator skim milk(difco) 5% 24 skim milk 5% 48. penicillin G (0.024ppm), oxytetracycline(0.1ppm), sulfamethazine(0.01ppm), sulfathiazole(0.01ppm), sulfadimethoxine(0.01ppm) - 69 -
10 membrane filter(0.45 m. Whatman) skim milk. 4. 1/2 Tryptic soy broth(difco) skim milk(difco) MRS broth(difco) skim milk starter. Brilliant blue(bio-rad), Methyl orange(janssen chemica), Methyl violet(wako chemical), Azolitmin(Litmus. BDH chemical), Resazurin (Sigma), Bromophenol blue(showa chemical), Methyl red(sigma), Phenol red(crown), Bromocresol purple(junsei chemical), Methylene blue (Samchun chemical), Bromocresol green(junsei chemical), Phenolphthalein(Samchun chemical), Crystal violet(junsei chemical), Safranine O(Junsei chemical). - 70 -
26. (%) ph Brilliant blue 0.1 Methyl orange 0.1 3.1 4.4 Methyl violet 0.05 0.1 1.5 3.2 Azolitmin(Litmus) 0.2 5.0 8.0 Resazurin 0.1 3.8 6.5 Bromophenol blue 0.1 Alcohol 3.0 4.6 Methyl red 0.2 Alcohol 4.2 6.3 Phenol red 0.1 Alcohol 6.8 8.0 Bromocresol purple 0.1 Alcohol 5.2 6.8 Methylene blue 0.1 Alcohol Bromocresol green 0.1 Alcohol 3.8 5.4 Phenolphthalein 0.1 Alcohol 8.2 10.0 Crystal violet 10 Safranine O 0.25 5.. 1/2 1. Phenol red(0.1%), Methyl red(0.01%), Resazurin (0.005%), Bromocresol purple(0.005%), Methylene blue(0.005%) 5, 27. Phenol red Bromocresol purple 100% alcohol, Methylene blue 50% alcohol - 71 -
, Methyl red 100% alcohol alcohol, Resazurin. Oxytetracycline(0.1ppm), Penicillin G (0.024ppm), Sulfamethazine(0.01ppm), Sulfathiazole(0.01ppm), Sulfadimethoxine(0.01ppm) Sigma(U. S. A.), stock solution membrane filter(0.45 m. Whatman). 27. Phenolred (0.1%) 100%alcohol Methylred (0.01%) 100%alcohol 100%alcohol Resazurin(0.005%) Bromocresolpurple (0.005%) Methyleneblue (0.005%) 100%alcohol 50% alcohol. Phenol red(0.1%), Methyl red(0.01%), Resazurin (0.005%), Bromocresol purple(0.005%), Methylene blue(0.005%) 5 resazurin 1:1. - 72 -
28. Resazurin Resazurin (100) Resazurin+Phenolred (50+50) Resazurin+Methyleneblue (50+50) Resazurin+Methylred (50+50) Resazurin+Bromocresol purple (50+50) ( /100ml) 0.005g Resazurin 0.0025gResazurin +0.05gPhenolred 0.0025gResazurin +0.0025g Methyleneblue 0.0025gResazurin +0.005gMethyl red 0.0025gResazurin +0.0025g Bromocresol purple Phenol red(0.1%), Methyl red(0.01%), Resazurin (0.005%), Bromocresol purple(0.005%), Methylene blue(0.005%) 3 Methyl red(0.01%), Resazurin(0.005%), Bromocresol purple (0.005%),. - 73 -
29. 3 Methylred (100) Resazurin (100) Bromocresol purple (100) Resazurin+Methylred (25+75) Resazurin+Methylred (50+50) Resazurin+Methylred (75+25) Resazurin+Bromocresol purple (25+75) Resazurin+Bromocresol purple (50+50) Resazurin+Bromocresol purple (75+25) ( /100ml) 0.01gMethyl red 0.005g Resazurin 0.005g Bromocresol purple 0.00125gResazurin +0.0075g Methylred 0.0025gResazurin +0.005gMethyl red 0.00375gResazurin +0.0025g Methylred 0.00125gResazurin +0.00375gBromocresolpurple 0.0025gResazurin +0.0025g Bromocresol purple 0.00375gResazurin +0.00125gBromocresolpurple - 74 -
3 1.. 1 807 1996 MRS broth 37, 30 incubator 0.02% yeast extract tryptic soy broth 105-106 48, MRS broth skim milk 37, 30 incubator skim milk. penicillin G(0.024 ppm) oxytetracycline(0.1ppm), sulfamethazine(0.01ppm), sulfathiazole(0.01ppm), sulfadimethoxine(0.01ppm) 10 tryptic soy broth. 807 21, 30. - 75 -
30. 21 Antibiotic sensitivity Antibiotic Skim MRS test sensitivity test milk (Tryptic soy broth ) (skim milk ) blank regular regular regular regular x10 10 4-1 o + + + o + - 9-5 o + + + o + - 7-2 o + + + o + - 9-1 o + + + o + - 14-3 o + + + o + - 3-1 o + + + o + - 7-2 o + + + o + - 15-2 o + + + o + - 17-1 o + + + o + - 17-2 o + + + o + - 4-1 o + + + o + - 4-1 o + + + o + - 12-1 o + + + o + - 2 o + + - 3 o + + - 56-3 o + + - 368-2 o + + + o + - 376-2 o + + + o + - 425 o + + - o + + 163 o + + + o + - 125 o + - - o + + ) o : (MRS), (Skim milk). + :. - : - 76 -
. 2 16 725.. 31. ( ) ( ) 60 28 32 71 66 106 37 11 42 39 35 22 35 38 80 23. 2 MRS broth 37 incubator skim milk 5%, 24 skim milk 5% 48 725 43 regular regular 10 32. - 77 -
32. 43 Antibiotic sensitivity Antibiotic sensitivity test (skim milk ) test (skim milk ) regular regular regular regular 10 10 1-2 + - 7-2 + - 3-1 + - 9-3 + - 5-3 + - 4-1 + - 5-5 - - 5-2 + - 6-1 + - 8-4 + - 8-3 + - 11-1 - - 10-1 - - 2-2 + - 11-1 + - 6-1 + - 17-1 + - 7-2 + - 18-1 + - 1-2 + - 19-2 + - 1-3 + - 19-3 + - 3-2 - - 30-2 + - 4-1 + - 8-1 + - 6-3 + - 9-1 + - 8-1 + - 9-2 + - 10-1 + - 12-1 + - 16-2 + - 14-1 - 7-1 + - 5-2 + - 36-2 + - 8-2 + - 15-1 + - 11-1 - - 15-2 + - 13-1 - ) + :. - : - 78 -
2. TTC ( ) skim milk 2 (2% ) 37 10%. Brilliant blue(bio-rad), Methyl orange(janssen Chimica), Methyl violet(wako Chemical), Azolitmin(Litmus, BDH Chemical), Resazurin(Sigma), Bromophenol blue(showa Chemical), Methyl red(sigma), Phenol red(crown), Bromocresol purple(junsei Chemical), Methylene blue(samchun Chemical), Bromocresol green(junsei Chemical), Phenolphthalein(Samchun Chemical), Crystal violet(junsei Chemical), Safranine O(Junsei Chemical) 14,. TTC II (10%) (37 water bath) 2 33 skim milk 5ml 0.3ml, 0.5ml, 0.75ml 1ml Resazurin, Methyl red, Phenol red, Bromocresol purple, Methylene blue. 0.3ml, 1ml 0.5ml 0.75ml. - 79 -
33. (%) 0hr 1hr 4hr 8hr Brilliantblue 0.1 - - - Phenolphthalein 0.1 - - - 0.3,0.5ml Methylred 0.2 1ml 0.0004 - - - Resazurin 0.1 0.0005 - - - Bromocresol purple 0.1 Phenolred 0.1 ( ) 0.3,0.5ml 1ml 0.0004 - - - Methyleneblue 0.1 0.0004 - - - Methylorange 0.1 - - - Methylviolet 0.05 - - - Bromocresol green Bromophenol blue 0.1 - - - 0.1 - - - Crystal violet 10 - - - SafranineO 0.25 - - - Litmus 0.2 - - - - 80 -
5 Resazurin, Methyl red, Phenol red, Bromocresol purple, Methylene blue 3 Skim milk, skim milk., skim milk. TTC, 425, 3-1, 12-1, 14-3 skim milk 85 5 (skim milk) 0.5ml, 1ml 37 2 ( 34) 0.5ml 1ml 1ml. resazurin 0.1% 0.01% 0.005%, phenol red 0.01% 0.005% 0.1%, Methyl red 0.01%, Bromocresol purple 0.005% Methylene blue 0.005%. - 81 -
34. 5 ( ) Blank Methylred 0.2% 0.02% TTC 1 0.5ml 1ml ( ) + + 0.01% + + Resazurin 0.1% 0.01% ( ) 0.005% Bromocresol purple 0.1% 0.01% ( 0.005% ) Phenolred 0.1% 0.01% ( ) ( ) ( ) 0.005% ( ) ( ) Methylene blue 0.1% 0.01% ( ) - - - - 0.005% - - - 82 -
( ) 425 Blank Methylred 0.2% 0.02% 0.5ml 1 1ml ( ) + + 0.01% + + Resazurin 0.1% 0.01% ( ) 0.005% Bromocresol purple 0.1% 0.01% 0.005% ( ) Phenolred 0.1% 0.01% ( ) ( ) ( ) 0.005% ( ) ( ) Methylene blue 0.1% 0.01% ( ) - - - - 0.005% - - - 83 -
( ) 3-1 Blank Methylred 0.2% 0.02% 0.5ml 1 1ml ( ) + + 0.01% + + Resazurin 0.1% 0.01% ( ) (3 ) 0.005% Bromocresol purple 0.1% 0.01% 0.005% ( ) Phenolred + 0.1% 0.01% ( ) ( ) ( ) 0.005% ( ) ( ) Methylene blue 0.1% 0.01% ( ) - - - - 0.005% - - - 84 -
( ) 12-1 Blank Methylred 0.2% 0.02% 1 0.5ml 1ml ( ) + + 0.01% + + Resazurin 0.1% 0.01% ( ) (3 ) 0.005% Bromocresol purple 0.1% 0.01% ( ) 0.005% Phenolred + 0.1% 0.01% ( ) ( ) ( ) 0.005% ( ) ( ) Methylene blue 0.1% 0.01% ( ) - - - 0.005% - - - 85 -
( ) Blank Methylred 0.2% ( 14-3 0.5ml 0.02% ) + 0.01% + 1 1ml + (3 ) (3 ) + (3 ) (3 ) Resazurin 0.1% ( (3 ) (3 ) 0.01% ) (3) (3 ) 0.005% (3 ) (3 ) Bromocresol purple 0.1% 0.01% ( ) 0.005% Phenolred 0.1% + ( 0.01% ) ( ) ( ) 0.005% ( ) ( ) Methylene blue 0.1% - - ( 0.01% ) - - 0.005% 3 - - 86 -
3.. 1/2 1, Phenol red(0.1%), Methyl red (0.01%), Resazurin(0.005%), Bromocresol purple(0.005%), Methylene blue(0.005%), Resazurin (0.005%), Methyl red(0.01%) Bromocresol purple(0.005%). Phenol red(0.1%) Methylene blue(0.005%). 5 Resazurin(0.005%), Methyl red(0.01%) Bromocresol purple(0.005%).. - 87 -
35. Penolred Methylred Resazurin Bromocresolpurple 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr 7-2 15-2 17-1 56-5 268-1 239-1 14-1 - 88 -
Penolred Methylred Resazurin Bromocresolpurple 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr 7-2 393 12-1(1) 11-1 8-4 5-2 10-1 - 89 -
Penolred Methylred Resazurin Bromocresolpurple 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr 8-1 9-1 7-1 15-1 15-2 368-2 7-2 - 90 -
Penolred Methylred Resazurin Bromocresolpurple 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr 4-1 30-2 10-1 8-3 8-1 11-1 10-2 - 91 -
. 8 Resazurin Resazurin(100), Resazurin + Methyl red (50+50) Resazurin + Bromocresol purple (50+50). Phenol red Methylene blue Resazurin + Phenol red (50+50) Resazurin + Methylene blue(50+50). - 92 -
36. 5 * STANDARD Resazurin Resazurin Resazurin Resazurin (100) + + + Phenolred (50 +50) Methyl red (50+50) Bromocresol purple (50+50) 0 hr 3hr 0hr 3hr 0hr 3hr 0hr 3 hr SKIM MILK (100) SKIM+SPC (50+50) SPC (100) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%) - 93 -
12-1(1), 15-1, 7-1, 17-1 Resazurin Resazurin Resazurin Resazurin (100) + + + Phenol red (50 + 50) Methyl red (50 + 50) Bromocresol purp (50 + 50) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC (50 + 50) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%) - 94 -
7-2 Resazurin Resazurin Resazurin Resazurin (100) + + + Phenol red (50 + 50) Methyl red (50 + 50) Bromocresol purp (50 + 50) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC (50 + 50) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%) - 95 -
14-1 Resazurin Resazurin Resazurin Resazurin (100) + + + Phenol red (50 + 50) Methyl red (50 + 50) Bromocresol purp (50 + 50) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC (50 + 50) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%) - 96 -
15-2 Resazurin Resazurin Resazurin Resazurin (100) + + + Phenol red (50 + 50) Methyl red (50 + 50) Bromocresol purp (50 + 50) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC (50 + 50) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%) - 97 -
19-2 Resazurin Resazurin Resazurin Resazurin (100) + + + Phenol red Methyl red Bromocresol purp (50 + 50) (50 + 50) (50 + 50) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC (50 + 50) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%) - 98 -
4 system 1.. disc assay, charm II test,, TTC TTC TTC., 1-2 2-4. 1 1-2 1. - 99 -
TTC. 2 1. / MRS broth(difco) 2 Gram, (Olympus, Japan) API kit(api biomerieux, France) ATB identification system (biomerieux, France) genus species. 2.MRS Skim milk /. Skim milk(100%, Difco), Skim milk + SPC(50% + 50%, Difco), SPC(100%, Difco) 3, Methyl red(0.01%, Sigma), Resazurin(0.005%, Sigma), Bromocresol purple(0.005%, Junsei chemical) 3. MRS broth Skim milk MRS Skim milk 37 3. - 100 -
3. 1/2. SPC broth(100%, Difco), MRS broth(100%, Difco), M17 broth(100%, Difco), SPC + MRS(50% + 50%), SPC + M17(50% + 50%), MRS + M17(50% + 50%), SPC + Skim milk(50% + 50%), Skim milk(100%, Difco), MRS + Skim milk(50% + 50%), M17 + Skim milk(50% + 50%). 121, 15,. M17 broth 50 10% lactose solution(difco). 10% lactose solution membrane filter(0.45 m, Whatman). Resazurin(0.005%). 96 multiwell plate(sterile) multichannel pipettor 40 l 140 l, TMP 120 l TMP20 l. 60 l plate 20 l 37 waterbath 3 1. - 101 -
37. ( / l ) SPC broth (100) Bacto tryptone 5g, Bacto yeast extract 2.5g, Bacto dextrose 1g MRS broth (100) Bacto proteose peptone No.3 10g, Bacto beef extract 10g, Bacto yeast extract 5g, Bacto dextrose 20g, Tween 80 1g, Ammonium citrate 2g, Sodium acetate 5g, Magnesium sulfate 0.1g, Manganese sulfate 0.05g, Dipotassium phosphate 2g M17 broth (100) Bacto tryptone 5g, Bacto soytone 5g, Meat digest 5g, Yeast digest 2.5g, Ascorbic acid 0.5g, Magnesium sulfate 0.25g, Disodium- -glycerophosphate 19g, 10% lactose solution 50ml SPC+MRS (50+50) Bacto proteose peptone No.3 5g, Bacto beef extract 5g, Bacto yeast extract 3.75g, Bacto dextrose 10.5g, Tween 80 0.5g, Ammonium citrate 1g, Sodium acetate 2.5g, Magnesium sulfate 0.05g, Manganese sulfate 0.025g, Dipotassium phosphate 1g, Bacto tryptone 5g SPC+M17 (50+50) Bacto tryptone 5g, Bacto soytone 2.5g, Meat digest 2.5g, Yeast digest 1.25g, Ascorbic acid 0.25g, Magnesium sulfate 0.125g, Disodium- -glycerophosphate 9.5g, 10% lactose solution 25ml, Bacto yeast extract 1.25g, Bacto dextrose 0.5g - 102 -
( / l ) MRS+M17 (50+50) Bacto proteose peptone No.3 5g, Bacto beef extract 5g, Bacto yeast extract 2.5g, Bacto dextrose 10g, Tween 80 0.5g, Ammonium citrate 1g, Sodium acetate 2.5g, Magnesium sulfate 0.175g, Manganese sulfate 0.025g, Dipotassium phosphate 1g, Bacto tryptone 2.5g, Bacto soytone 2.5g, Meat digest 2.5g, Yeast digest 1.25g, Ascorbic acid 0.25g, Disodium- -glycerophosphate 9.5g, 10% lactose solution 25ml Skim Milk (100) Skim milk 100g Skim+SPC (50+50) Skim milk 50g, Bacto trytone 2.5g, Bacto yeast extract 1.25g, Bacto dextrose 0.5g Skim+MRS (50+50) Skim milk 50g, Bacto proteose peptone No.3 5g, Bacto beef extrat 5g, Bacto yeast extrat 2.5g, Bacto dextrose 10g, Tween 80 0.5g, Ammonium citrate 1g, Sodium acetate 2.5g, Magnesium sulfate 0.05g, Manganese sulfate 0.025g, Dipotassium phosphate 1g Skim+M17 (50+50) Skimmilk50g,Bactotryptone2.5g,Bactosoytone2.5g,Meatdigest2.5g, Yeastdigest1.25g,Ascorbic acid 0.25g, Magnesium sulfate 0.125g, D isodium- -glycerophosphate 9.5g, 10% lactose solution 25ml - 103 -
96multiwell plate 40 l 96multiwellplate 96 multiwellplate 140 l 120l 96multiwellplate TMPsolution20l 96multiwellplate 60 l 96multiwell plate 20l 37 waterbath 3hr 1. - 104 -
4. SPC broth skim milk. 121 / 15. Oxytetracycline(0.1ppm), Penicillin G (0.024ppm), Sulfamethazine(0.01ppm), Sulfathiazole(0.01ppm), Sulfadimethoxine(0.01ppm) membrane filter(0.45 m, Whatman). 38. ( / l ) Skim milk (100) Skim milk + SPC (75 + 25) Skim milk + SPC (50 + 50) Skim milk + SPC (25 + 75) Skim milk 100g Skim milk 75g, Bacto tryptone 1.25g, Bacto yeast extract 0.625g, Bacto dextrose 0.25g Skim milk 50g, Bacto tryptone 2.5g, Bacto yeast extract 1.25g, Bacto dextrose 0.5g Skim milk 25g, Bacto tryptone 3.75g, Bacto yeast extract 1.875g, Bacto dextrose 0.75g SPC broth (100) Bacto tryptone 5g, Bacto yeast extract 2.5g, Bacto dextrose 1g - 105 -
5. HPLC. Sigma (St. Louis, MO, USA) sulfamerazine, sulfadimethoxine, sulfathiazole 10mg 100ml methanol 100 g/ml stock solution. (mixture of standards) stock solution 1ml methanol 100ml 1 g/ml.. 1) 10ml 125ml. 2) 50ml ( - (2+1, volume/volume) ) 1. 3) vent. 4) 1 vent 1, 5. 5) ( - ). 6) 25ml 2-5 2.. 7) 5 ml 2 8) rotary evaporator 32. 9) 0.1 M potassium dihydrogen phosphate 1ml 1 vortex. 10) hexane 5ml 1 vortex. 11) 2 1 vortex 15-106 -
. 12) 0.45 m filter LC.. HPLC Table 39. Operating conditions for sulfonamides by HPLC Instrument Column JASCO HPLC system(jasco Co. Ltd., Japan) CrestPak C18S(150 mm x 4.6 I.D.)(JASCO, Japan) Eluent 0.017 M H3PO4/acetonitrile (70/30) Flow rate Detector 1.0ml/min UV(270nm) Column Temp. 35 Attenuation Sample size 512 mv F.S 20 l 6. / / / 8 SPC broth(difco) skim milk(difco), Resazurin(0.005%), Bromocresol purple(0.005%) / /. 96 multiwell plate(sterile) multichannel pipettor 40 l 140 l, TMP 120 l TMP20 l. 60 l plate 20 l 37 waterbath 3. - 107 -
3 1.. Tryptic soy broth(difco) / skim milk(difco) / Gram (Olympus, Japan) Gram. 2 rod, 6 Streptococcus. rod API 50 CHL kit(api biomerieux, France), streptococcus API Strepkit(API biomerieux, France). Enterococcus casseliflavus 2, Enterococcus faecium 4, Lactobacillus plantarum 1, Lactobacillus cellobiosus1 40 41. 40. 50 CHL API Tests Lactic bacteria 7-2 14-1 Glycerol - - Erythritol - - D Arabinose - - L Arabinose + - Ribose + + D Xylose + - L Xylose - - Adonitol - - -Methyl-D Xyloside - - Galactose + + Glucose + + Fructose + + - 108 -
Lactic bacteria Tests 7-2 14-1 Mannose - + Sorbose - - Rhamnose - + Dulcitol - - Inositol - - Mannitol - + Sorbitol - + -Methyl-D Mannoside + + -Methyl-D Glucoside - - N Acetyl Glucosamine - + Amygdalin - + Arbutin - + Esculin + + Salicin - + Cellobiose + + Maltose + + Lactose + + Melibiose + + Sucrose + + Trehalose - + Inulin - + Melezitose - + Raffinose + + Starch - - Glycogen - - Xylitol - - Gentiobiose - + D Turanose - + D Lyxose - - D Tagatose - - D Fucose - - L Fucose - - D Arabitol - + L Arabitol - - Gluconate + + 2 Ketogluconate - - 5 Ketogluconate - - ) + :, - : - 109 -
41. 20 Strep API Tests Lactic bacteria 7-1 15-1 15-2 VP + + + HIP - - - ESC + + + PYRA + + + GAL + + + GUR - - - GAL + + + PAL - - - LAP + + + ADH + + + RIB + + + ARA + + + MAN + + + SOR - - - LAC + + + TRE + + + INU - - - RAF - - - AMD - - - GLYG - - - ) + :, - : - 110 -
Tests Lactic bacteria 17-1 19-2 12-1(1) VP + + + HIP - - - ESC + + + PYRA + + + GAL - - + GUR - - - GAL + + + PAL - - - LAP + + + ADH + + + RIB + + + ARA + + + MAN + + + SOR - - - LAC + + + TRE + + + INU - - - RAF + - + AMD - + + GLYG - - - ) + :, - : - 111 -
42. Gram +/- 17-1 Gram+ Streptococcus Enterococcus casseliflavus 19-2 Gram+ Streptococcus Enterococcusfaecium 7-1 Gram+ Streptococcus Enterococcusfaecium 15-1 Gram+ Streptococcus Enterococcusfaecium 15-2 Gram+ Streptococcus Enterococcusfaecium 12-1(1) Gram+ Streptococcus Enterococcus casseliflavus 14-1 Gram+ rod Lactobacillusplantarum 7-2 Gram+ rod Lactobacillus cellobiosus - 112 -
. MRS Skim milk /. Skim milk (100%), Skim milk + SPC (50% + 50%), SPC (100%) 3, Methyl red (0.01%), Resazurin(0.005%), Bromocresol purple(0.005%) 3. MRS, Skim milk. MRS broth Skim milk starter MRS 2 Skim milk 3 MRS starter. - 113 -
43. / MRS Skimmilk MRS SKIMMILK Methylred (0.01%) regular regular X 10 SKIM MILK (100%) Resazurin (0.005%) regular regular X10 Bromocresol purple (0.005%) regular regular X 10 Methylred (0.01%) regular regular X 10 Resazur (0.005 regular r SKIM+SPC (50%+50%) SPC (100%) - 114 -
44. standard 3 MRS SKIMMILK Methyl red (0.01%) Resazurin (0.005%) Bromocresolpurple (0.005%) Methylred (0.01%) Resaz (0.00 regular regular X10 SKIMMILK (100% ) regular regular X 10 regular regular X10 regular regular X10 regular SKIM+SPC ( 50%+50%) SPC (100% ) - 115 -
45. 3 17-1, 14-1 MRS SKIM MIL Methyl red (0.01%) Resazurin (0.005%) Bromocresolpurple (0.005%) Methyl red (0.01%) Resa (0. 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr 0hr SKIMMILK (100% ) SKIM+SPC ( 50%+50%) SPC (100% ) - 116 -
19-2 MRS SKIM MIL Methyl red (0.01%) Resazurin (0.005%) Bromocresolpurple (0.005%) Methyl red (0.01%) Resa (0. 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr 0hr SKIMMILK (100% ) SKIM+SPC ( 50%+50%) SPC (100% ) - 117 -
15-1, 15-2, 12-1(1) MRS SKIMMI Methylred (0.01%) Resazurin (0.005%) Bromocresolpurple (0.005%) Methylred (0.01%) R ( 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr 0hr SKIM MILK (100%) SKIM+SPC (50%+50%) SPC (100%) - 118 -