GA Bio system A study on the development of bio system for the testing of antimicrobials in milk

Transcription

1 GA Bio system A study on the development of bio system for the testing of antimicrobials in milk

2 Bio system : : ( ) : ( ) : ( ) : ( ) : ( ) : ( ) - 1 -

3 I. Biosystem II. 3 TTC.. disc assay Delvo test, Charm test, BR test. TTC

4 III system. system 2.. 1) 2) 3) 4). system 1) 2) 3) 4). system 1) 2) 3) 4) 5) bio system - 3 -

5 IV ) 807 Gentamicin(1ppm), Sulfamethazine(5000ppm), Oxytetracycline(1ppm), Penicillin G(0.024ppm), Erythromycine(1ppm) Sulfamethazine (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 -

6 37 7, 35 1, 32, system 1) ) Brilliant blue 14 Resazurin, Methyl red, Phenol red, Bromocresol purple, Methylene blue ml 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)

7 . 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

8 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

9 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

10 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 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 % 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 -

11 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

12 CONT ENT S Chapter 1 Introduction Part 1. Objectives and scope of study Objectives of the study Scope of the Study Part 2. Trends of Raw milk quality and antimicrobial test Raw milk quality Antimicrobial testing Chapter 2 Selection of strain and establishment of incubation condition Part 1. Introduction Part 2. Materials and Methods Isolation of lactic bacteria Preparation of antimicrobial solution antimicrobial sensitivity test selection of lactic bacteria for the TTC/TTC-II test Preparation of TTC/TTC-II solution TTC/TTC-II test with the selected lactic bacteria Identification of selected lactic bacteria Optimum incubation condition

13 Part 3. Results and discussion Screening of bacteria for the antimicrobial test Selection and Characterization of selected bacteria Optimum incubation condition for antimicrobial test Chapter 3 Development of indicator for the antimicrobial test Part 1. Introduction Part 2. Materials and Methods Sensitivity test of 1st selected lactic bacteria Screening of lactic bacteria fron raw milk Sensitivity test of 2nd selected lactic bacteria Selection and characterization of indicator Optimization of lactic bacteria and indicator Part 3. Results and discussion Selection and characterization of lactic bacteria Selection and characterization of indicator Optimization of lactic bacteria and indicator Chapter 4 Establishment of bio system with selected strin, medium and indicator Part 1. Introduction Part 2. Materials and Methods Identification of final selected lactic bacteria Coparision of bactria grown in MRS and skim milk

14 3. Sensitivity of selected bacteria at various medium Establishment of optimum medium condition Analysis of residual Sulfadrugs in milk by HPLC Optimum mixture of bacteria, medium and indicator Part 3. Results and discussion Preparation of solution of selected lactic bacteria Preparation of optimum medium Preparation of mixture for the antimicrobial test Bio system of bacteria, medium and indicator References Appendix 1.Trend of antimicrobial testing system in USA Trend of antimicrobial testing system in Australia

15 TTC II test TTC / TTC II test TTC / TTC II test TTC / TTC II test

16 system system MRS Skim milk

17 5. HPLC / / bio system

18 TTC.. disc assay Delvo test, Charm test, BR test. TTC. 2., system system

19 1,,,. 2 system,,,. 3 system,,, bio system

20 % 1, 4 5% 4,

21 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

22 2. /ml /ml , , , , , A 1B A 1B >11 10 > > > A 1B >11 10 > > > A 1B *

23 3. (ml ) ) - : (TBC : Total Bacterial Counts) - : (CFU : Colony Forming Units)

24 4. (ml ) ore/kg 0-3ore/kg -6ore/kg ppl -1.0ppl -2.0ppl $20 $80 +$10 0 -$ /hl -2/hl -3/hl Yen -2-5Yen % 0-1.0% -2/0% Varies Varies ) : IDF Bulletin No. 305/

25 5. ( : count/ml,%) 1 <10 2 <25 3 <50 4 <100 5 > <25 <50 >50 A<3 B<

26 6. ( : count/ml,%) 1 (<25 ) 2 (<50 ) 3 (<75 ) 4 (>75 ) (<20 ) 2 (<50 ) 3 (<60 ) 4 (>60 ) (<20 ) 2 (<50 ) 3 (<50 )

27 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),

28 (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

29 , (Bishop, 1990). 10% 9ml (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,

30 (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)

31 7. Disc Assay TTC TTC II D e l v o 100 / / 64 / / 100 / 10 5, 3-3.5/ 37 / / 100 / 30, 5, 3-3.5/ 37 / / 2,000 / P SP 100 / 2,000 / / 2.5,,, 64 / 3, BR 100 / ,,,, 64 /, 2.75,,

32 Charm AIM,, 96 / 1,500 /,65 / , Charm II 1 / 8 6,000 / 6, 85 / 37, 8, 7,, 18, 3 6,,,,,,,, Penzyme 100 / 2,000 / /

33 Lactek 1 / 7,, 1,500 /, / 3-4, Cite prove Signal ,,, -,,,,,, - Ez-Screen 1 / 5 -, -, Agriscreen : (1994)

34 8. BR Test Charm II Charm Charm CITE Delvo Delvo Disk EZ- La Cowside Farm test P test SP Assay Screen B )

35 . 9, Codex,. 9. (MRLs) / Codex MRLs EU MRLs ( : ppb) / 5/0 10/10 10/10 10/10 50/1,000 20/20 30/0 100 ( 30/0 ) 80/0 10/ /10 ( 10/0 ) 10/0 10/ / / / / / / / /0 ) Codex

36 TTC.. Charm AIM Delvotest 105 Lactek test 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) ) : + =, - = Lactam 0.25%, Tetracycline 4.25%, Chloramphenicol 8.25%, Sulfamethazine 12% -Lactam Penicillin-G Sulfa Chloramphenicol

37 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

38 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

39 2 1,,,., TTC II Disc assay HPLC 12 TTC II 12. TTC II TTC II -lactam ppm 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

40 2 1. sodium azide(juncei chemical Co.Ltd) 0.025% % peptone (Difco) sodium azide 0.025% MRS agar plate( 13) 0.2ml MRS agar plate MRS agar plate TTC / TTC II test. 13. MRS agar Component gram/liter Bacto Proteose Peptone # Bacto Beef Extract 10.0 Bacto Yeast Extract 5.0 Bacto Dextrose 20.0 Tween 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

41 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 H2O + NaOH oxytetracycline H2O + NaOH sulfamethazine 5, H2O + NaOH sulfathiazol 5, H2O + NaOH sulfadimethoxine 5, H2O + NaOH erythromycine H2O + Alcohol chloramphenicol H2O + Alcohol

42 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) % peptone(difco) cfu/ml. MIC(minimum inhibitory concentration) two-fold macrodilution broth (Koneman, 1988) 0.2% yeast extract(difco) tryptic soy broth(difco), (, 1995). oxytetracycline 1ppm, penicillin G 0.024ppm, sulfamethazine 5000ppm, sulfadimethoxine 5000ppm, sulfathiazole 5000ppm, erythromycine 1ppm, chloramphenicol 5ppm, Sigma Chemical Co. (USA)

43 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) TTC / TTC II test. TTC TTC(2,3,5-triphenyltetrazolium chloride, Sigma) 1:25 (4% ) TMP 50.0mg TMP(trimethoprim, Sigma) 100ml methanol 10ml 500 g/ml 1 4 1:9 (10 ) 50 g/ml

44 membrane filter(0.45 m. millipore Co. U.S.A.). 6. TTC / TTC II test TTC test MRS (Difco) 2 10% (Difco) 5% % 1:1(v/v).. TTC test 10% (DIFCO) 8.2ml 0.8ml vortex mixer(fisher scientific) negative control.. 10% (DIFCO) 7.2ml TMP 1ml 0.8ml vortex mixer(fisher scientific) 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)

45 2. Hammes (1992). Gram, (Olympus, Japan) API kit (API biomerieux, France) ATB identification system(biomerieux, France) genus species % (Difco) (30, 32, 35, 37 ), Glucose (2%) ph meter(orion. SA520) ph, 2% ,

46 16. ( ) ( ) Tryptic soy broth(difco) 0.2% yeast extract(difco) 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)

47 No. Penicillin G Oxytetra Sulfame Sulfadi Sulfa Eryt -cycline -thazine -metoxin -thiazole -myc ) + :, :, - :

48 No. Penicillin G Oxytetra -cycline Sulfame -thazine Sulfadi -metoxin Sulfa -thiazole Eryth -myci

49 No. Penicillin G Oxytetra Sulfame Sulfadi Sulfa -cycline -thazine -metoxin -thiazole Ery -m

50 No. Penicillin G Oxytetra Sulfame Sulfadi Sulfa -cycline -thazine -metoxin -thiazole Ery -my

51 No. Penicillin G Oxytetra Sulfame Sulfadi Sulfathiazole myc Eryth -cycline -thazine -metoxin

52 No. Penicillin G Oxytetra Sulfame Sulfadi Sulfa -cycline -thazine -metoxin -thiazole Er -m

53 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 O O O O O O O O O O O O ) + :, - : 15 MRS broth 10% TTC test. 10%

54 ph 4.6 (isoelectric point) ph 4.6 (, 1979).,.. TTC / TTC II test Tryptic soy broth, 10% skim milk 12 TTC / TTC II test

55 19. TTC Antimicrobial agents Lactic bacteria blank Penici -lling Erythr Oxytetracyclinphenicol Chloram- Sulfamethazine -omycin Sulfadimethoxine Sulfathiazol S.thermophilus ) + :, :, - :

56 20. TTC II Antimicrobial agents Lactic bacteria blank TMP blank -ing Penicill Erythro- Oxytetra- mycin Sulfa - methoxine cycline Chloramphenicol Sulfamethazine Sulfathiazol S.thermophilus ) + :, :, - :

57 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)

58 21. API Tests Lactic bacteria 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

59 Lactic bacteria Tests 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 Ketogluconate Ketogluconate ) + :, - :

60 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 Lactobacillus acidophilus 507 Lactobacillus plantarum (1996) S. thermophilus lactic acid bacteria, 7Lactobacillus 1Leuconostoc. Lactobacillus brevis Leuconostoc acetic acic CO2 heterofermenter homofermenter. Lactobacillus plantarum

61 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 ph glucose 37 4, 35 4, 32 1, 30, glucose 37 7, 35 1, 32,

62 23. skim milk(glucose ) ph Lactic bacteria time temp 0h 3h 6h 9h 12h

63 Lactic bacteria S. thermo -philus time temp 0h 3h 6h 9h 12h

64 24. skim milk(glucose ) ph. Lactic bacteria time temp 0h 3h 6h 9h 12h

65 Lactic bacteria S. thermo -philus time temp 0h 3h 6h 9h 12h

66 61% glucose 37 ph. 25. glucose 37 ph Lactic bacteria Glucose ph at 0h ph at 24h Lactic bacteria Glucose ph at 0h ph at 24h G G G G G G G G G G G G G S.thermo G G philus G G G ) + : glucose, - : glucose 9 glucose ph 4, glucose 5 glucose

67 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

68 ,,,,,, 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

69 (Reinbold Reddy, 1974; Sozzi Smiley, 1980), L. acidophilus L.casei. 3 TTC. (Charm Chi, 1982; Bishop White, 1984)., Delvo Penzyme -lactam sulfa.,,,,,, kit MRS broth(difco) 37, 30 incubator 0.02% yeast extract(difco) tryptic soy broth(difco) Sigma(U.S.A.) penicillin G(0.024ppm),

70 oxytetracycline(0.1ppm), sulfamethazine(0.01ppm), sulfathiazole(0.01ppm), sulfadimethoxine(0.01ppm), 10 tryptic soy broth. MRS broth skim milk incubator 10 skim milk(difco). skim milk. 2. sodium azide(junsei chemical Co. Ltd) 0.025% % peptone(difco) sodium azide 0.025% MRS agar plate 0.2ml MRS agar plate MRS agar plate TTC / TTC II test 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)

71 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)

72 26. (%) ph Brilliant blue 0.1 Methyl orange Methyl violet Azolitmin(Litmus) Resazurin Bromophenol blue 0.1 Alcohol Methyl red 0.2 Alcohol Phenol red 0.1 Alcohol Bromocresol purple 0.1 Alcohol Methylene blue 0.1 Alcohol Bromocresol green 0.1 Alcohol Phenolphthalein 0.1 Alcohol Crystal violet 10 Safranine O /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

73 , 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:

74 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 gResazurin +0.05gPhenolred gResazurin g Methyleneblue gResazurin gMethyl red gResazurin g 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%),

75 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 gResazurin g Methylred gResazurin gMethyl red gResazurin g Methylred gResazurin gBromocresolpurple gResazurin g Bromocresol purple gResazurin gBromocresolpurple

76 MRS broth 37, 30 incubator 0.02% yeast extract tryptic soy broth , 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 ,

77 Antibiotic sensitivity Antibiotic Skim MRS test sensitivity test milk (Tryptic soy broth ) (skim milk ) blank regular regular regular regular x o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o + + ) o : (MRS), (Skim milk). + :. - :

78 ( ) ( ) MRS broth 37 incubator skim milk 5%, 24 skim milk 5% regular regular

79 Antibiotic sensitivity Antibiotic sensitivity test (skim milk ) test (skim milk ) regular regular regular regular ) + :. - :

80 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

81 33. (%) 0hr 1hr 4hr 8hr Brilliantblue Phenolphthalein ,0.5ml Methylred 0.2 1ml Resazurin Bromocresol purple 0.1 Phenolred 0.1 ( ) 0.3,0.5ml 1ml Methyleneblue Methylorange Methylviolet Bromocresol green Bromophenol blue Crystal violet SafranineO Litmus

82 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%

83 34. 5 ( ) Blank Methylred 0.2% 0.02% TTC 1 0.5ml 1ml ( ) % + + 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% ( ) %

84 ( ) 425 Blank Methylred 0.2% 0.02% 0.5ml 1 1ml ( ) % + + 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% ( ) %

85 ( ) 3-1 Blank Methylred 0.2% 0.02% 0.5ml 1 1ml ( ) % + + 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% ( ) %

86 ( ) 12-1 Blank Methylred 0.2% 0.02% 1 0.5ml 1ml ( ) % + + 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% ( ) %

87 ( ) Blank Methylred 0.2% ( ml 0.02% ) % + 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% ) %

88 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%)

89 35. Penolred Methylred Resazurin Bromocresolpurple 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr

90 Penolred Methylred Resazurin Bromocresolpurple 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr (1)

91 Penolred Methylred Resazurin Bromocresolpurple 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr

92 Penolred Methylred Resazurin Bromocresolpurple 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr

93 . 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)

94 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%)

95 12-1(1), 15-1, 7-1, 17-1 Resazurin Resazurin Resazurin Resazurin (100) Phenol red ( ) Methyl red ( ) Bromocresol purp ( ) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC ( ) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%)

96 7-2 Resazurin Resazurin Resazurin Resazurin (100) Phenol red ( ) Methyl red ( ) Bromocresol purp ( ) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC ( ) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%)

97 14-1 Resazurin Resazurin Resazurin Resazurin (100) Phenol red ( ) Methyl red ( ) Bromocresol purp ( ) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC ( ) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%)

98 15-2 Resazurin Resazurin Resazurin Resazurin (100) Phenol red ( ) Methyl red ( ) Bromocresol purp ( ) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC ( ) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%)

99 19-2 Resazurin Resazurin Resazurin Resazurin (100) Phenol red Methyl red Bromocresol purp ( ) ( ) ( ) 0hr 3hr 0hr 3hr 0hr 3hr 0hr 3hr SKIM MILK ( 100 ) SKIM+SPC ( ) SPC ( 100 ) )Resazurin(0.005%),Phenolred(0.1%),Methylred(0.01%),Bromocresolpurple(0.005%),Methyleneblue(0.005%)

100 4 system 1.. disc assay, charm II test,, TTC TTC TTC.,

101 TTC / 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

102 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

103 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 g, 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

104 ( / l ) MRS+M17 (50+50) Bacto proteose peptone No.3 5g, Bacto beef extract 5g, Bacto yeast extract 2.5g, Bacto dextrose 10g, Tween g, 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 g, 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

105 96multiwell plate 40 l 96multiwellplate 96 multiwellplate 140 l 120l 96multiwellplate TMPsolution20l 96multiwellplate 60 l 96multiwell plate 20l 37 waterbath 3hr

106 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 ( ) Skim milk + SPC ( ) Skim milk + SPC ( ) 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

107 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 ) 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

108 . 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 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

109 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 cellobiosus CHL API Tests Lactic bacteria Glycerol - - Erythritol - - D Arabinose - - L Arabinose + - Ribose + + D Xylose + - L Xylose - - Adonitol - - -Methyl-D Xyloside - - Galactose + + Glucose + + Fructose

110 Lactic bacteria Tests 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 Ketogluconate Ketogluconate - - ) + :, - :

111 Strep API Tests Lactic bacteria VP HIP ESC PYRA GAL GUR GAL PAL LAP ADH RIB ARA MAN SOR LAC TRE INU RAF AMD GLYG ) + :, - :

112 Tests Lactic bacteria (1) VP HIP ESC PYRA GAL GUR GAL PAL LAP ADH RIB ARA MAN SOR LAC TRE INU RAF AMD GLYG ) + :, - :

113 42. Gram +/ 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

114 . 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

115 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%)

116 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% )

117 , 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% )

118 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% )

119 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%)