9. SIMPULAN
Berdasarkan penelitian yang telah dilakukan, dapat ditarik beberapa kesimpulan berikut ini:
E.coli K99 mampu melekat secara spesifik ke permukaan zona pelusida embrio mencit.
Pencemaran bakteri E.coli K99 pada embrio, baik yang memiliki zona
pelusida utuh mau pun tanpa zona pelusida dapat menghambatnya perkembangan serta mengakibatkan kematian degenerasi embrio. Zona
pelusida pada embrio mampu memberi perlindungan terhadap infeksi bakteri E.
coli K99. Pembasuhan terhadap embrio yang dicemari bakteri
E.coli K99 menggunakan mPBS, tripsin, atau pronase dapat mengeliminasi cemaran bakteri
E.coli K99 dan tidak mengakibatkan kematian embrio. Pembasuhan dengan pronase 0,25 dalam mPBS merupakan larutan paling efektif untuk
menghilangkan bakteri dari permukaan embrio dibandingkan dengan tripsin atau mPBS.
Metode vitrifikasi kriolup dapat digunakan secara efektif untuk kriopreservasi blastosis mencit dan cemaran bakteri
E.coli K99 pada blastosis yang divitrifikasi, tidak mempengaruhi tingkat perkembangan embrio ke tahap
lebih lanjut Vitrifikasi dengan metode kriolup dapat dipakai untuk kriopreservasi
embrio mencit. Vitrifikasi mengakibatkan viabilitas embrio mencit sedikit menurun. Transfer embrio mencit yang telah divitrifikasi ke resipien, berhasil
berkembang menjadi fetus dan lahir menjadi mencit normal Zona pelusida dapat melindungi sel-sel embrio dari infeksi
E.coli K99, walau pun
E.coli K99 terbukti dapat secara spesifik melekat ke permukaan zona pelusida. Bakteri
E.coli K99 yang melekat ke permukaan zona pelusida embrio dapat disingkirkan dengan pencucian menggunakan enzim pronase dan embrio
tersebut dapat berkembang ke tahap lebih lanjut. Embrio yang memiliki zona pelusida utuh dan divitrifikasi dengan metode kriolup, baik yang tercemar
E.coli K99 mau pun tidak, dapat ditransfer dan berkembang sampai lahir.
SARAN
Perlekatan E.coli K99 bersifat spesifik ke permukaan zona pelusida.
Pencemaran bakteri E.coli K99 harus selalu diwaspadai keberadaanya, di
samping agen pencemar lainnya. Untuk itu upaya mencegah pencemaran atau mengeliminasi agen pencemar harus selalu diupayakan sebelum embrio tersebut
ditransfer ke induk resipien. Pencucian embrio dengan enzim pronase sebaiknya dilakukan terhadap
embrio yang akan ditransfer ke induk resipien Pembekuan embrio sebaiknya dilakukan dengan metode vitrifikasi
memanfaatkan larutan vitrifikasi EG 15 dan DMSO 15. Krioprotektan tersebut mampu mematikan agen infeksius yang kemungkinan mencemari permukaan
zona pelusida. Embrio yang divitrifikasi dengan metode kriolup dapat berkembang
secara in vitro maupun in vivo. Namun, adanya viabilitas yang menurun pada
embrio hasil vitrifikasi perlu mendapatkan perhatian agar viabilitasnya dapat ditingkatkan.
Pemanfaatan uji ELISA guna melacak cemaran agen infeksius selain E.coli K99 perlu dikembangkan lebih jauh, guna mencegah tularan penyakit yang
terbawa oleh embrio.
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LAMPIRAN
Lampiran 1 Medium kultur embrio in vitro Kalium simplex optimation medium KSOM
Stok A 2x solution
Bahan mM dlm
KSOMaa gL g50mL
g30mL g20mL g10mL
NaCl BM=58,44
95 11,1000 0,5550 0,3330 0,2220 0,1110
KCl BM=74,56
2,5 0,3720 0,0186 0,0112 0,0074 0,0037
KH2PO4 BM=136,09 0,35 0,0940 0,0047 0,0028 0,0019 0,0009
MgSO4.7H2O BM=246,68
0,2 0,0990 0,0050
0,0030 0,0020
Sodium lactate BM=109,1
10 2,1820 0,1091 0,0655 0,0436 0,0218
Sodium pyruvate BM=110,05
0,2 0,0440 0,0022 0,0013 0,0009 0,0004
D-Glucose BM=180,…
5,5 2,0000 0,1000 0,0600 0,0400 0,0200
Glycyl-Glutamin Hydrate BM=146,1
2 0,5838 0,0292 0,0175 0,0117 0,0058
NaHCO3 BM=84,01
25 4,2000 0,2100 0,1260 0,0840 0,0420
Phenol Red 0,5 2.000
100 60
40 20
Catatan: NaHCO3 dicampur pada saat membuat KSOMaa, dengan mencampurkan setengah dosis saja
Stok B, C, E 100x solution Bahan
mM dlm KSOMaa g5ml
CaCl2.2H2O BM=147,0 = Stok B 1,71
0,1256 HEPES-Sodium Salt BM=238,3 =
Stok C 20 2,3830
EDTA BM=372,2 = Stok E 0,01
0,00186 Catatan: - EDTA dilarutkan dalam 200 µl NaOH 1 N kemudian ditambahkan 4,8
ml DI - Stok B,C, dan E dibuat secara terpisah
KSOMaa Bahan
100mL 30mL 20mL 10mL 5mL
Stok A 50
15 10
5 2,5
Diionise water DI 44,5
13,35 8,9
4,45 2
Stok B
1 0,3 0,2 0,1 0,05 EAA
1 0,3 0,2 0,1 0,05 NEAA
0,5 0,15 0,1 0,05 5mL BSA 0,4
0,4 0,12
0,08 0,04
2,5 Gentamycin 50µgml
µL 100 30 20 10 2
Stok E mL 1
0,3 0,2
0,1 0,05
Catatan: osmolaritas 260; filter medium menggunakan 0,22 µm filter Ekuilibrasi medium sebelum digunakan
Medium dapat digunakan selama 1-2 minggu, jika disimpan pada suhu 4
o
C
KSOMaa + HEPES Bahan 100mL
30mL 20mL
10mL 5mL
Stok A 50
15 10
5 2,5
Diionise water DI 44,5
13,35 8,9
4,45 2
Stok B 1
0,3 0,2
0,1 0,05
Stok C 1
0,3 0,2
0,1 0,05
EAA 1 0,3
0,2 0,1
0,05 NEAA 0,5
0,15 0,1
0,05 0,025
BSA 0,4 g 0,4
0,12 0,08
0,04 0,02
Gentamycin 50µgml µL 100
30 20
10 5
Stok E mL 0,1
Lampiran 2 Medium untuk mengembangkan bakteri E.coli K99
Penyiapan Minca + Iso Vitalex Minca + Is Larutan A, menurut Ginee et al. 1977.
KH
2
PO
4
2.72 g Na
2
HPO
4
20.2 g Air suling
1000 ml Seluruh bahan tersebut diencerkan dalam airsuling dan pastikan pH-nya
7.5, kucurkan 500 ml kedalam labu Erlenmeyer dan selanjutnya di autoklaf pada suhu 121
C selama 15 menit.
Larutan garam-garam trace Minca + Is medium larutan B
MgSO
4
.7H
2
O 10 g
MnCl
2
.4H
2
O 1 g
FeCl
3
.6H
2
O 0.135 g
CaCl
2
2H
2
O 0.4 g
Air suling 1000 ml
Seluruh bahan tersebut dilarutkan dalam airsuling kemudian dimasukan kedalam labu Erlenmeyer dan disucihamakan pada suhu 110
C selama 10 menit.
Minca + Is larutan C
Lima gram casein hydrolysate Oxoid L41 dilarutkan dalam 1000 ml
airsuling, pastikan pH-nya menjadi 7.5 dan disucihamakan pada suhu 121
C selama 15 menit.
Minca + Is larutan D
Dua puluh empatr gram agae Oxoid No 3 Oxoid 113 dilarutkan dalam 920 ml airsuling dengan penguapan 100
C. Pastikan pH-nya menjadi 7.5 dan kemudian kucurkan sebanyak 460 ml kedalam labu Erlenmeyer 1 liter dan
diautoklaf pada suhu 121 C selama 15 menit.
Lima ratus mili liter larutan-A dipanaskan hingga 56 C dan 460 ml larutan-D
dicairkan pada suhu 100 C. Larutan-A dan B dicampur, dan campuran
tersebut didinginkan hingga 56 C. Vial vitox SR90A diencerkan isinya
dengan kandungan vial vitox SR90B dan ditambahkan secara bersamaan dengan 1 ml larutan-B dan 20 ml larutan-C. Medium tersebut dicampur
secara perlahan dan dituang sebanyak 5 ml kedalam botol universal steril, 2 ml kedalam botol bijou untuk membuat agar miring, 20 ml kedalam cawan
petri gelas yang steril atau cawan petri dispossable, dan 150 ml dimasukan
kedalam labu Roux. Setelah media memadat, seluruh media yang disiapkan diinkubasi pada suhu 37
C selama 18-24 jam untuk menguji sterilitas medium tersebut.
Lampiran 3 Penyiapan reagen untuk uji ELISA
1. Normal saline steril sodium khlorida 0.85
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