MODUL PRAKTIKUM MIKROBIOLOGI TERPADU

Nama Lengkap :
NIM/BP :
Kelompok :
Topik : 2 (dua)
Dosen Penanggung jawab :

Asisten :

PROGRAM STUDI SARJANA TEKNOLOGI PANGAN DAN HASIL PERTANIAN

FAKULTAS TEKNOLOGI PERTANIAN
UNIVERSITAS ANDALAS
2024
 TOPIK 2
PENGARUH SUHU TERHADAP TOTAL MIKROBA
A. Tujuan Praktikum
Pada praktikum Pengaruh suhu terhadap total mikroba, terdapat beberapa kompetensi
yang harus dicapai oleh praktikan, di antaranya adalah”
1. Mahasiswa mampu menghitung jumlah media dan membuat media PCA (Plate
Count Agar)
2. Mahasiswa memahami prinsip sterilisasi dan mampu melakukan sterilisasi basah
dan sterilisasi kering menggunakan autoklaf dan oven
3. Mahasiswa mampu membuat media yang sesuai dengan analisis yang dibutuhkan
4. Mahasiswa mampu melakukan pengenceran serial
5. Mahasiswa mampu melakukan analisa kuantitatif dengan metode TPC
6. Mahasiswa mengetahui cara menghitung koloni mikroba yang terbentuk dari
sampel dan melakukan analisis
B. Landasan Teori
Mikroorganisme adalah organisme yang bersifat mikroskopis yang bersifat
menguntungkan dan ada juga yang bersifat merugikan bagi kepentingan manusia.
Pertumbuhan mikroba sangat dipengaruhi oleh berbagai faktor lingkungan, termasuk
suhu. Berikut beberapa hal yang perlu diperhatikan mengenai pengaruh suhu terhadap
pertumbuhan mikroba:
a. Optimum Suhu: Setiap jenis mikroba memiliki suhu optimum di mana
pertumbuhannya paling aktif. Misalnya, bakteri dan ragi tumbuh optimal pada suhu
sekitar 20°C. Pada suhu ini, mereka berkembang biak dengan baik dalam media dan
lingkungan yang stabil.
b. Variasi Respons: Mikroba tidak hanya bervariasi dalam persyaratan nutrisi, tetapi
juga menunjukkan respons yang berbeda terhadap suhu. Beberapa mikroba
mungkin lebih tahan terhadap suhu ekstrem, sementara yang lain lebih sensitif.
c. Pentingnya Kontrol Suhu: Dalam industri pangan, pengendalian suhu sangat
penting untuk mencegah pertumbuhan mikroba patogen. Suhu yang tidak tepat
dapat menyebabkan pertumbuhan berlebihan dan bahkan keracunan makanan.
d. Pengaruh Penyimpanan: Total bakteri pada makanan dapat meningkat secara
signifikan saat disimpan pada suhu ruang dalam waktu yang lama. Oleh karena itu,
penyimpanan makanan pada suhu yang tepat sangat penting untuk mengurangi
risiko kontaminasi mikroba.
Terdapat beberapa metode yang dapat digunakan untuk menghitung jumlah mikroba
dalam bahan makanan, salah satunya adalah metode hitung cawan (Total Plate Count)
dengan mengukur jumlah sel yang ada. Metode hitung cawan sudah lama digunakan
dalam mikrobiologi pangan dengan asumsi bahwa mikroorganisme tersebar secara
homogen di dalam bahan. Metode ini memiliki kelebihan yakni kapasitas untuk
menghitung jumlah bakteri jika terlalu banyak ataupun jika terlalu sedikit dapat
menggunakan faktor pengenceran. Selain itu, metode ini juga hanya menghitung
bakteri hidup. Disisi lain, metode ini juga memiliki kekurangan yaitu perhitungan
kumpulan sel bakteri dapat salah hitung sebagai koloni tunggal sehingga dilaporkan
sebagai CFU/mL daripada sel/ml. Selain itu, metode ini membutuhkan waktu yang
lebih lama sekitar 2-3 hari. Metode hitung cawan termasuk metode yang digunakan
untuk penanaman bakteri dengan menggunakan media padat yakni dengan prinsip
kerjanya berdasarkan pembuatan seri pengenceran (homogenisasi) sampel dengan
kelipatan 10. Hasil perhitungan dengan menggunakan hitung cawan ini dalam bentuk
Colony forming unit (CFU). CFU ini menunjukkan jumlah koloni yang tumbuh tiap
gram atau mililiter sampel yang dihitung dari jumlah cawan, faktor pengenceran, dan
volume yang digunakan.

Dalam mikrobiologi, terdapat dua metode yang umum digunakan untuk mengisolasi
dan menghitung jumlah koloni mikroba di dalam sampel, yakni:
a. Metode tuang (Pour plate) : metode ini diawali dengan menuangkan sampel yang
telah diencerkan ke dalam cawan petri, kemudian menuangkan media agar yang
sesuai ke dalam petri yang berisi sampel. Setelah media mengeras, koloni bakteri
akan tumbuh baik di permukaan maupun di dalam medium.
b. Metode sebar (Spread Method ) : pada metode ini, medium agar terlebih dahulu
dituang pada cawan petri dan dibiarkan membeku. Langkah selanjutnya adalah
sampel yang telah diencerkan ditanamkan pada permukaan media yang telah
membeku. Koloni bakteri akan tumbuh di permukaan agar.

Gambar 1. Metode tuang dan sebar

Dalam melakukan perhitungan jumlah mikroba dalam pangan, salah satu perlakuan
yang dilakukan adalah pengenceran. Larutan pengencer digunakan untuk mengurangi
jumlah mikroba dalam sampel agar memungkinkan perhitungan yang lebih akurat.
Beberapa jenis larutan pengencer yang umum digunakan adalah larutan fisiologis 0,9%
NaCl, Buffer Fosfat, Buffer Laktat Ringer, Buffer Pepton Air, dan Buffer butterfield.
Pemilihan larutan pengencer tergantung pada jenis sampel, tujuan analisis, dan kondisi
lingkungan yang diinginkan untuk pertumbuhan mikroba.

Kelangsungan hidup dan pertumbuhan mikroorganisme dipengaruhi oleh adanya

nutrisi dan faktor lingkungan. Bahan nutrisi yang tersedia dapat berupa bahan alami
 dan dapat berupa bahan sintesis. Berdasarkan komposisi atau susunan bahannya, media
terbagi atas media alami, sintetis, dan semi sintetis. Berdasarkan bentuknya, media
dibagi menjadi media cair, padat, dan semi padat. Sedangkan berdasarkan kegunaanya,
media dibagi menjadi media umum, media selektif, media differensial, dan media
pengaya.

Proses pengerjaan di laboratorium juga tidak terlepas dari proses steril yang mana
semua alat-alat dan bahan yang digunakan harus dalam keadaan steril. Suatu alat dan
bahan dikatakan steril apabila bahan tersebut bebas dari mikroorganisme. Sterilisasi
dapat dilakukan dengan berbagai cara, yaitu cara kimia, mekanik, atau fisik.
C. Prosedur Kerja
Pada praktikum pengamatan pengaruh suhu terhadap total mikroba pada produk pangan
hewani/nabati, tujuan akhir dari pengamatan ini adalah untuk melihat dan menganalisis
bagaimana perbedaan jumlah total mikroba pada produk hewani/nabati pada perlakuan
suhu yang berbeda-beda yakni suhu ruang, suhu 40 C, dan suhu minus 10-17 0 C.
Analisis dilakukan dengan dengan menggunakan metode hitung cawan (Total Plate
Count).
1. Pembuatan Media PCA (Plate Count Agar)
Alat dan Bahan:
- Timbangan
- Gelas ukur 500 ml
- Labu erlenmeyer 500 ml dan 1000 ml
- Tabung reaksi
- Kaca pengaduk
- Autoklaf
- Beaker glass 500 ml dan 1000 ml
- Hot plate
- Aquadest
- Kapas
- Kain kasa
- Alkohol 70%
2. Pembuatan/penyediaan pengenceran bertingkat/berseri (Serial Dilution)
Alat dan Bahan:
- Alu dan mortar
- Tabung reaksi
- Mikropipet dan tip
- Kapas
- Aluminum foil
- Sampel
- Larutan pengencer
- Rak tabung reaksi
- Vortex
 Penentuan besarnya atau banyaknya tingkat pengenceran tergantung pada perkiraan
jumlah mikroba dalam sampel. Perbandingan yang digunakan adalah 1 : 9. Berikut
tahapan pengerjaan yang akan saudara lakukan:
- Menghaluskan sampel dengan menggunakan mortar
- Menimbang sampel sebanyak 1 gram
- Memasukkan sampel ke dalam tabung reaksi yang telah berisi Na-Fis 10 ml
secara aseptis
- Menghomogenkan larutan sampel dengan me-vortex tabung
- Mengambil larutan dalam tabung sebanyak 1 ml dengan menggunakan pipet,
kemudian memasukkan larutan ke dalam tabung reaksi lain yang telah berisi
larutan pengencer, dilakukan secara aseptis
- Melakukan hingga pengenceran ke 7
- Setelah selesai melakukan pengenceran, ambil 1 ml suspensi menggunakan
pipet pada pengenceran 10-6 dan 10-7.
- Memasukkan suspensi ke dalam cawan petri
- Menuangkan media hangat (suhu 45 – 500 C) ke dalam cawan yang telah berisi
suspensi dan tutup
- Homogenkan campuran media dan suspensi dengan cara menggoyangkan atau
memutar cawan petri secara perlahan dengan membentuk angka delapan di atas
meja yang rata dan dalam kondisi asepti, lalu biarkan media memadat.
- Melakukan inkubasi pada cawan yang telah berisi suspensi dan media selama 2
x 24 jam. Cawan petri diletakkan dalam kondisi terbalik
- Menghitung koloni bakteri

Catatan tambahan:
Pemindahan dilanjutkan hingga tabung pengenceran terakhir dengan cara yang sama, hal yang perlu
diingat bahwa pipet ukur yang digunakan harus selalu diganti, artinya setiap tingkat pengenceran
digunakan pipet ukur steril yang berbeda/baru. Prinsipnya bahwa pipet tidak perlu diganti jika
memindahkan cairan dari sumber yang sama.
 Gambar 2. Teknik pengenceran bertingkat dan penanaman pada media
Dalam analisis ini, teknik penanaman suspensi yang digunakan adalah metode cawan tuang
(pour plate method). Adapun tujuan dari teknik ini adalah untuk menyebarkan sel-sel bakteri
tidak hanya pada permukaan medium agar saja, melainkan sel terendam di dalam medium (di
dalam agar) sehingga terdapat sel yang tumbuh di permukaan agar yang kaya akan O2 dan
ada juga yang tumbuh di dalam agar dengan kandungan oksigen sedikit. Teknik ini
membutuhkan agar yang belum padat untuk dituang bersama suspensi bakteri ke dalam
cawan petri lalu kemudian dihomogenkan dan dibiarkan memadat.

Gambar 3. Teknik pour plate

3. Perhitungan jumlah mikroba

Perhitungan jumlah mikroba dapat dilakukan dengan berbagai cara baik secara
langsung dengan hitung mikroskopik menggunakan Haemocytometer, dan secara
tidak langsung dengan metode hitung cawan (plate count). Selain itu, juga terdapat
perhitungan dengan metode MPN dan turbidimetri menggunakan alat
spektrofotometer.
Prinsip metode hitung cawan adalah jika sel mikroba yang masih hidup
ditumbuhkan pada media agar, maka sel mikroba itu akan berbiak membentuk
koloni yang dapat dilihat dan dihitung menggunakan mata telanjang, dan disebut
dengan “colony forming unit” = cfu. Metode hitung cawan dapat dilakukan dengan
metode tuang dan metode permukaan. Perhitungan jumlah mikroba dianggap valid
jika dalam satu cawan tumbuh koloni sebanyak 30-300. Jika pertumbuhan mikroba
terlalu padat, maka harus dilakukan pengenceran terlebih dahulu.

Faktor pengenceran = FP
FP = pengenceran awal x pengenceran selanjutnya x jumlah yang
ditumbuhkan (volume yang dimasukkan ke dalam cawan petri (0,1 ml atau 1
ml)

Koloni per mL (cfu/mL) = Jumlah koloni x (1/FP)

Catatan penting untuk aturan ALT !

a. Cawan yang dipilih dan dihitung adalah yang mengandung jumlah koloni
antara 30-300
b. Beberapa koloni yang bergabung menjadi satu merupakan suatu kumpulan
koloni yang besar dimana jumlah koloni diragukan, dapat dihitung sebagai satu
koloni
c. Suatu deretan (rantai) yang terlihat sebagai suat garis tebal dihitung sebagai
satu koloni
d. Data yang dilaporkan harus mengikuti peraturan yaitu hanya terdiri dari dua
angka, yaitu angka pertama di depan koma dan angka ke dua di belakang koma
e. Jika semua pengenceran menghasilkan angka kurang dari 30 koloni, hanya
jumlah koloni yang terendah yang dihitung. Hal ini mendakan pengenceran
yang dilakukan melebihi kebutuhan. Oleh karena itu, koloni pada pengenceran
terendah yang dihitung. Hasilnya dilaporkan sebagai kurang dari 30 dikalikan
dengan besarnya pengenceran, tetapi jumlah sebenarnya harus dicantumkan
dalam tanda kurung
f. Jika semua pengenceran menghasilkan lebih dari 300 koloni pada cawan petri,
berarti pengenceran yang dilakukan terlalu rendah. Kemudian pilih cawan
dengan pengenceran tertinggi kemudian dibagi menjadi beberapa bagian sektor
(2,4, atau 8) dan dihitung jumlah koloni dari satu sektor. Hasil perhitungan
kemudian dikali dengan jumlah sektor untuk mendapatkan jumlah koloni
dalam cawan petri. Oleh karena itu, jumlah koloni pada pengenceran yang
tertinggi yang dihitung. Hasilnya dilaporkan sebagai lebih dari 300 dikalikan
dengan faktor pengenceran.
g. Jika terdapat lebih dari satu cawan yang mempunyai jumlah koloni yang
memenuhi syarat, maka kalibrasi jumlah koloni dengan tingkat pengenceran.
Bagilah hasil pengenceran yang tinggi dengan yang lebih rendah. Jika hasil
pembagian >1 maka diambil hasil pengenceran tertinggi, jika hasil pembagian
 <1 maka jumlah koloni kedua pengenceran dirata-ratakan untuk mendapatkan
kesimpulan ALT.
Satu koloni yang terpisah dari koloni lainnya dapat diamati tiper pertumbuhan pada masing-
masing media, diantaranya dilakukan terhadap konsistensi, bentuk koloni, warna, dan
permukaan koloni.

D. Hasil dan Pengamatan

Tabung pengenceran (10-x) volume sampel yang faktor pengenceran
dimasukkan (ml)

Perhitungan koloni per pengenceran

1. Pengenceran ......................
a. Jumlah koloni

b. Faktor pengenceran :

c. Perhitungan :

2. Pengenceran ...............
a. Jumlah koloni :
 b. Faktor pengenceran :

c. Perhitungan :

3. Pengenceran ............
a. Jumlah koloni :

b. Faktor pengenceran :

c. Perhitungan :

Tingkat pengenceran Kesimpulan

Jumlah koloni
(CFU/g)
 LEMBAR TUGAS PENDAHULUAN/AKHIR PRAKTIKAN
Nama Lengkap :
Kelompok :
Hari/tanggal :

Jawablah pertanyaan di bawah ini dengan tepat, jelas, dan benar.

1. Saudara hendak melakukan pengamatan jumlah total mikroba pada jajanan-jajanan
anak-anak di sekitar SDN 03 Kota Padang. Setelah mengambil sampel di lapangan,
sampel-sampel tersebut saudara bawa ke laboratorium dengan tujuan untuk
menganalisis total mikroba yang terdapat di masing-masing sampel. Tuliskan
langkah-langkah prosedur kerja yang akan saudara lakukan hingga mendapatkan hasil
total mikroba yang terdapat pada sampel.
Jawab :
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
 ..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
2. Sebagai seorang ahli dalam analisis mikrobiologi dalam bahan pangan, Anda diminta
untuk melakukan pengujian angka lempeng total sampel terasi ikan. Pengenceran
yang Anda lakukan adalah 10-2, 103, dan 10-4. Semua pengenceran yang ditanam pada
media PCA, menghasilkan nilai Terlalu Banyak Untuk Dihitung (TBUD) atau
melebihi 300 koloni. Jelaskan apa penyebab dan solusi ideal yang saudara tawarkan
dari permasalahan ini?
Jawab:
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
 ..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
3. Berapakah jumlah koloni per gram dari sampel dengan data berikut

Pengenceran : 10-4 (metode tuang)

..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
 ..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
4.
 LOGBOOK PRAKTIKAN
Nama Lengkap :
Kelompok :
Hari/tanggal :

1. Kegiatan Pembuatan Media dan Sterilisasi

Pada laman ini, saudara tuliskan media yang digunakan, jumlah media yang dibuat dan
perhitungannya, serta proses singkat pembuatan media. Bagaimana proses sterilisasi
alat-alat yang saudara gunakan serta cara mengemas alat sebelum disterilisasi. Catat hal
penting yang saudara lakukan.
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
2. Kegiatan pengenceran dan penanaman suspensi sampel
Pada laman ini, tuliskan poin-poin penting yang saudara lakukan pada kegiatan
pengenceran mulai dari persiapan hingga proses pengenceran. Apa larutan pengencer
yang digunakan, berapa jumlah tabung reaksi yang saudara gunakan, apa metode yang
saudara gunakan, dll.
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
3. Kegiatan pengamatan jumlah mikroba dengan ALT
Detail pengerjaan yang dilakukan mencakup : jumlah tabung pengenceran yang
ditanam, volume media yang ditambhakan dan kekeruhan media sebelum dan sesudah
inokulasi, pengamatan terhadap bentuk, warna, dan ukuran koloni. Tuliskan poin-poin
penting dan fenomena yang saudara temukan selama melaksanakan pengamatan.
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................
..........................................................................................................................................