Minggu, 21 Juni 2009

PRAKTIKUM BAKTERIOLOGI

JURNAL PRAKTIKUM BAKTERIOLOGI KLINIK

PRAKTIKUM : IDENTIFIKASI PNEUMOCOCCUS NAMA :
TANGGAL : TANDA TANGAN :
NO. BAHAN :

HARI I :
1. Pemeriksaan mikroskopik
Bahan pemeriksaan dibuat preparat, kemudian diwarnai dengan pengecetan ……

Hasil pemeriksaan : 1

Bentuk : __________ Bentuk :
Susunan : __________ Warna kapsel :
Sifat : __________ Warna bakteri :
Tersangka : __________



2. Isolasi

Bahan pemeriksaan ditanam pada media :
a.
b.
c.
Dieramkan 370c selama satu malam (18 – 24 jam )

Hari II :
Pengamatan hasil isolasi

Media : ________ Media : ________ Media : _______
Bentuk koloni :
Warna koloni :
Elevasi
Sifat
……………………
……………………



3. Uji kelarutan Empedu


4. Uji Inulin

5. Uji kepekaan (Sensitivity test )

6. Hewan percobaan

Hari III :

Pengamatan Hasil :




Kesimpulan :

Dari bahan nomor : __ didapatkan bakteri :

Diskusi :


Pemeriksa / Penguji



NIP.

PEMERIKSAAN NEISSERIA (Gonococcus, Meningococcus)

Bahan pemeriksaan :

Hapus urogenital (uretra, vagina, serviks), endapan urine, cairn sendi, darah, eksudat mata, hapus tenggorokan.

Identifikasi berdasarkan atas :
1. Pemeriksaan mikroskopik dengan Gram
2. Pembiakan
3. Uji oksidase
4. Uji biokimiawi
5. PPNG tes ( Betalaktamase tes)

Cara kerja :
1. Pemeriksaan mikroskopik

Pemeriksaan mikroskopik dengan pengecetan Gram dari bahan langsung (direct-preparate), hasilnya sebagai berikut :
- Bentuk kokus berpasangan (Diplococcus) seperti buah kopi atau ginjal, Gram negative, biasanya intraseluler dalam lekosit.

2. Pembiakan
Pembiakan yang dipakai :
- Agar coklat (G.C. Agar)
- Agar coklat dari Thayer-Martin
- Cystine trypticase agar (CTA)
- Agar darah

a. Bahan pemeriksaan ditanam pada agar coklat, agar coklat Thayer-Martin dan agar darah sebagai control.
b. Dieramkan pada suhu 350 – 370c selama 2 malam dengan ditambah CO2 (2-10%) didalam eksikator yang dibawahnya diberi kapas basah supaya menjadi lembab. Atau bias juga dengan menyalakan lilin dalam eksikator yang apabila lilin mati berarti O2 telah habis terbakar. Konsentrasi CO2 yang tercapai kira-kira 2% saja.
Agar darah dieramkan seperti biasa (aerob), kalau tumbuh pada perbenihan bakteri berarti Neisseria apathogen.
Pada agar darah coklat dan Thayer-Martin setelah tumbuh tampak koloni bulat, dengan diameter 2 – 3 mm, jernih mengkilat.
c. Dari koloni tersebut selanjutnya ditanam subkultur pada agar coklat dan Thayer-Martin.

3. Uji oksidase
Reagen HCl – tetrametil – p – finildiamin 1% dengan hati-hati diteteskan di atas koloni bakteri. Bila tes positif maka koloni berubah mula-mula merah jambu, kemudian menjadi ungu dan akhirnya hitam sesudah kira-kira 5 menit.
4. Uji biokimiawi
Hasil fermentasi pada CTA :

N. gonorrhoea N. meningitides
Glukosa + +
Maltosa - +
Sakarosa - -

5. PPNG tes ( Betalaktamase tes)
Tes ini gunanya untuk menentukan suku kuman Neisseria gonorrhoea yang resisten terhadap antibiotika penicillin. Suku kuman yang resisten merusak penicillin dengan perantaraan Enzim penicillinase (Betalaktamase) menjadi senyawa yang tidak aktif.
Cara kerja :
1. Dibuat suspensi N. gonorrhoea (umur 2 malam) dalam larutan 0,1 ml penicilin 6000 g / ml
2. Didiamkan dalam suhu kamar selama 30 menit
3. Ditambah 2 tetes larutan kanji 1%
4. Kemudian ditambah lagi 1 tetes larutan lugol (pro Gram) sampai terjadi warna biru.
5. Positif kalau warna biru hilang dalam waktu 10 menit.
Negatif kalau warna biru tetap tidak berubah.

Catatan :
Bahan pemeriksaan untuk N. meningitides adalah :

- liquor
- hapus tenggorok
- darah
JURNAL PRAKTIKUM BAKTERIOLOGI KLINIK

PRAKTIKUM : IDENTIFIKASI NEISSERIA Sp. NAMA :
TANGGAL : TANDA TANGAN :
NO. BAHAN :

HARI I :
1. Pemeriksaan mikroskopik
Bahan pemeriksaan dibuat preparat, kemudian diwarnai dengan pengecetan ……

Hasil pemeriksaan :

Bentuk : __________
Susunan : __________
Sifat : __________
Tersangka : __________

2. Isolasi

Bahan pemeriksaan ditanam pada media :
a.
b.
c.
Dieramkan 370c selama satu malam (18 – 24 jam ) dalam suasana ……….



Hari II :
Pengamatan hasil isolasi

Media : ________ Media : ________ Media : _______
Bentuk koloni :
Warna koloni :
Elevasi
Sifat
……………………
……………………


3. Uji bio kimiawi
Koloni tersangka ditanam pada :

4. Uji oksidase
Koloni tersangka ditetesi ……………….., yang hasilnya adalah ……………….

5. Uji Betalaktamase (PPNG test)

Hari III :

Pengamatan hasil penanaman pada : ………………


Kesimpulan :

Dari bahan nomor : ____ didapatkan bakteri :


Diskusi :




Pemeriksa / Penguji


NIP.

PEMERIKSAAN Corynebacterium diphtheriae

Bahan pemeriksaan :
Hapus tenggorokan, hapus hidung atau dari tempat lain yng mencurigakan .
Identifikasi berdasarkan atas :
1. Pemeriksaan mikroskopik
2. Pembiakan
3. Uji biokimia
4. Uji virulensi
Cara kerja :
1. Pemeriksaan mikroskopik
Dibuat preparat hapus dari bahan pemeriksaan dan diwarnai dengan Albert atau Neisser dan Gram, hasilnya adalah sebagai berikut :

Albert Neisser Gram
Bentuk
Warna batang
Granula
Susunan Batang
Biru kehijauan
Coklat hitam Batang
Kuning coklat
Biru hitam Batang
Ungu
-
Seperti hurup cina atau membentuk hurup V, L, T

2. Pembiakan
Perbenihan yang dipakai :
- Loefler : gunanya untuk menyuburkan bakteri sehingga bila dibuat preparatakan tampak granula yang jelas.
- Agar telurit : gunanya untuk isolasi koloni-koloni Corynebacterium diphtheriae yang selanjutnya ditanam pada gula-gula untuk difteri.
- Telurit cair : berguna sebagai media pengaya.
- Agar darah : gunanya untuk membiak kuman-kuman lainnya seperti Streptococcus haemolyticus dan Staphylococcus aerus
- Gula-gula untik difteri : glukosa serum dan sakarosa serum untuk membedakan C. diptheri dengan kuman sejenis

Bahan pemeriksaan ditanam pada perbenihan di atas, kemudian dieramkan 370c selama 1 malam kecuali agar telurit selama 2 malam. Hasil biakan pada Loefler terlihat koloni-koloni barwarna putih, selanjutnya dibuat preparat Albert atau Neisser.
Dari telurit cair ditanam pada loefler sebagai tanaman ulangan, dan pada agar darah diperiksa adanya kuman-kuman pathogen lainnya.

3. Tes biokimia
Koloni tersangka yang berwarna abu-abu hitam pada agar telurit ditanam pada glukosa serum dan sakarosa serum (atau bisa pula ditambahkan amylum), kemudian dieram pad suhu 370C selama 1 malam.
Hasil pengamatan adalah sebagai berikut :

Glukosa Sakarosa Amylum
C. diphteriae + - +/-
C. Xerosis + + +
C. hofmanii - - -

4. Tes virulensi
a. in vivo : kuman yang berhasil diisolasi disuntikkan pada hewan percobaan.
b. in vitro : Tes elek-Ouchterlony (gel difusi dari elek) kertas saring yang telah dicelupkan kedalam antitoksin C. diptheri diletakkan pada lempeng agar. Kuman yang akan diperiksa ditanam menyilang terhadap kertas saring tadi.
Dieramkan 1 malam untuk difusi, maka terbentuk garis putih pada tempat pertemuan antara toksin dan antitoksin bila kumannya virulen.

JURNAL PRAKTIKUM BAKTERIOLOGI KLINIK

PRAKTIKUM : IDENTIFIKASI C. diphtheriae NAMA :
TANGGAL : TANDA TANGAN :
NO. BAHAN :


HARI I :
1. Pemeriksaan mikroskopik
Bahan pemeriksaan dibuat preparat, kemudian diwarnai dengan pengecetan ……

Hasil pemeriksaan : 1. pengecetan ………………

Bentuk : __________
Susunan : __________
Sifat : __________
Tersangka : __________



Hasil pemeriksaan : 2. pengecetan ………………

Bentuk : __________
Susunan : __________
Sifat : __________
Tersangka : __________

2. Isolasi

Bahan pemeriksaan ditanam pada media :
a.
b.
c.
Dieramkan 370c selama satu malam (18 – 24 jam ) dalam suasana ……….


Hari II :
Pengamatan hasil isolasi

Media : ________ Media : ________ Media : _______
Bentuk koloni :
Warna koloni :
Elevasi
Sifat
……………………
……………………

3. Uji bio kimiawi
Koloni tersangka ditanam pada :

4. Uji keganasan (Virulensi test)

Hari III :

Pengamatan hasil penanaman pada : ………………


Kesimpulan :

Dari bahan nomor : ____ didapatkan bakteri :


Diskusi :



Pemeriksa / Penguji




NIP.

JURNAL PRAKTIKUM BAKTERIOLOGI KLINIK

PRAKTIKUM : IDENTIFIKASI M. tuberculosis NAMA :
TANGGAL : TANDA TANGAN :
NO. BAHAN :


HARI I :
1. Pemeriksaan mikroskopik
Bahan pemeriksaan dibuat preparat, kemudian diwarnai dengan pengecetan ……

Hasil pemeriksaan : 1. pengecetan ………………

Bentuk : __________
Susunan : __________
Sifat : __________
Tersangka : __________



Hasil pemeriksaan : 2. pengecetan ………………

Bentuk : __________
Susunan : __________
Sifat : __________
Tersangka : __________

2. Isolasi

Bahan pemeriksaan ditanam pada media :
a.
b.
c.
Dieramkan 370c selama ……….


Hari II :
Pengamatan hasil isolasi

Media : ________ Media : ________ Media : _______
Bentuk koloni :
Warna koloni :
Elevasi
Sifat
……………………
……………………

3. Uji bio kimiawi
a. Merah netral



b. Niacin test




4. Hewan percobaan


Kesimpulan :

Dari bahan nomor : ____ didapatkan bakteri :


Diskusi :


Pemeriksa / Penguji


NIP.


PEMERIKSAAN Salmonella DAN Shigella

Bahan pemeriksaan untuk Salmonella :
Tinja, darah, sumsum tulang, liquor, urine
Bahan pemeriksaan untuk Shigella :
Tinja
Identifikasi berdasarkan atas

1. pembiakan
2. uji biokimia
3. uji serologi

cara pemeriksaan Salmonella dan Shigella dari tinja
1. pembiakan :
- secara langsung
- tidak langsung
Perbenihan yang dipakai :
- MC agar
- SS agar
- Bismuth Sulfit agar (BSA)
- Selenit dan MKT sebagai media pengaya
- Deretan gula-gula pendek : semi solid, TSIA, manit dan peptone.
Cara kerja :
a. secara langsung tinja ditanam dalam perbenihan MC, SS, dan BSA.
b. Dieramkan 1 malam (18-24 jam) pada suhu 370C, kecuali BSA harus 2 malam
c. Secara tidak langsung tanam 1-3 ml tinja kedalam selenit dan MKT sebagai media pengaya.
d. Dieramkan selama 6 - 16 jam pada suhu 370C, kemudian dari selenit dan MKT tersebut dipindahtanamkan pada perbenihan MC dan SS, kemudian dieramkan 1 malam pada 370C.
e. Diamati adanya koloni-koloni tersangka yaitu yang tampak putih jernih dengan diameter 2-3 mm (Salmonella) atau diameter 1-2 mm (Shigella) dan koloni hitam dengan dasar abu – abu pada BSA (S. typhi)

2. uji biokimia
Dari koloni-koloni tersangka ditanam pada deretan gula-gula yaitu TSIA, Manit, Pepton, dan Semisolid. Dieramkan selama 1 malam pada suhu 370C, kecuali semisolid pada suhu kamar.

3. uji serologi
Hasil biakan pada deretan gula-gula sesudah ditetesi pereaksi, kemudian dicocokkan dengan daftar atau table. Selanjutnya dilakukan “slide-test” aglutinasi dengan antisera spesifik dari biakan TSIA yang tersangka Salmonella atau Shigella menurut daftar / table.

Catatan :
a. bakteri Salmonella dan Shigella (bakteri usus pathogen) cepat mati diluar tubuh hospes, oleh karena itu pengiriman tinja sebaiknya dalam cairan pengawet sebagai transport medium, misalnya Sachs glycerin, Carry dan Blair, atau cairan V.R. (venkantraman ramakrishnan).
b. Beberapa tipe Shigella ada yang tidak “aglutinable”, segera dipasasi beberapa kali melalui agar serum atau suspensi bakteri dimasak dahulu pada 1000C untuk menghilangkan antigen envelopenya.


Daftar / Tabel
Identifikasi Enterobacteriaceae yang pathogen
Dengan semi solid dan gula-gula pendek

TSIA Manit Indol Gerak Enterobacteriaceae
K/M H2S
+ - + S. typhi
K/M H2S +/- +g - + S. paratyphi A, B, C
K/M H2S ++ +g - + S. paratyphi B, C
K/M H2S - +g - + S. paratyphi A
K/M H2S - + - - Sh. Sonnei. S. typhi
K/M H2S - + +/- - Sh. Flexner, Sh. Boydii
K/M H2S - - - - S. shigae
K/M H2S - - + - Sh. Schmitzii
K/M H2S - + + + V. cholerae

Keterangan :
K : kuning (asam)
M : Merah (basa)
+/- : positif atau negative
++ : banyak terbenyuk (kuat)
g : gas
- : negative
: sedikit



PEMERIKSAAN Escherichia coli

Escherichia coli dibedakan dalam 3 macam yaitu :
1. EPEC : Enteropathogenic Escherichia coli, yaitu bakteri coli yang dapat menimbulkan penyakit.
2. ETEC : Enterotoxigenic Escherichia coli, yaitu bakteri coli yang dapat membuat toksin, yang dapat menimbulkan sakit seperti Vibrio cholera eltor.
Dikenal 2 macam toksin, yaitu stabile toxin (ST) dan labile toxin (LT)
ST diperiksa dengan percobaan biologis dengan tikus bayi umur 4 hari, sedangkan LT diperiksa dengan ELISA test.
3. EIEC : Enteroinvasive Escherichia coli, yaitu bakteri coli yang dapat menimbulkan penyakit seperti bakteri disentri.
Tiga macam bakteri ini mempunyai sifat cultural (biakan) dan biokimiawi sama, sehingga untuk mengisolasinya dilakukan cara yang sama. Hanya untuk identifikasi akhir membutuhkan cara yang berbeda tergantung jenisnya..

Bahan pemeriksaan : fasces atau rectal swab.

Cara pemeriksaan :
Hari I
- specimen ditanam pada : Endo, MC dan EMB
- kemudian dieramkan 370C satu malam.

Hari II
- koloni tersangka E. coli diambil, kemudian ditanam pada agar miring dan gula-gula.
- Dieramkan 370C selama satu malam.

Koloni tersangka di :
Endo : besar-besar, cembung, merah tua metalik, smooth.
MC : sedang-besar, keeping-cembung, merah, keruh, smooth.
EMB : sedang, keeping, hijau ketalik tengah berwarna ungu tua, smooth.
Hari III
Pertumbuhan pada media gula-gula diamati, dicatat, kemudian dicocokkan dengan daftar (tabel). Yang cocok dengan daftar, dilanjutkan dengan menentukan apakh bakteri tersebut termasuk EPEC, ETEC, atau EIEC.

Cara untuk menentukan adalah :
EPEC : dilakukan slide aglutinasi dengan antisera terhadap E. coli (baik polivalen maupun monovalen)
ETEC : - Labile toxin dengan ELISA atau RPHA
- Stabile toxin dengan infant mice inoculation.
EIEC : dengan sereny test menggunakan mata marmot

Ciri-ciri biokimiawi E. coli :
Gerak : (+) positif (aktif)
Glukosa : (+) positif
Laktosa : (+) positif
Manit : (+) positif
Maltosa : (+) positif
Sakarosa : (+) positif
Indol (+) positif
TSIA: lereng : kuning
Dasar : kuning
Gas : positif
H2S : negative
Urase : (-) negative
MR : (+) positif
VP : (-) negative
Simmon’s citrate : (-) negative

Catatan L
Sebaiknya pada hari I, disertakan juga penanaman dengan menggunakan media pengaya, setelah dieramkan 370C selama satu malam kemudian dilanjutkan penanaman pada media plate, dan seterusnya.

Ada 11 macam tipe spesifik Coli pathogen (EPEC) yaitu :
Type I : Stoke W 0111 : K58 (B)
II : 972 NCTC 055 : K59 (B)
III : F 41 NCTC 026 : K60 (B)
IV : E990 NCTC 086 : K61 (L)
V : 8623 NCTC 0125 : K70 (B) H19
VI : 8623 NCTC 0126 : K71 (B) H2
VII : 9707 NCTC 0127 : K63 (B)
VIII : 9708 NCTC 0128 : K67 (B) H2
IX : 9114 NCTC 0114 : K (B) H32
X : 9705 NCTC 0119 : K69 (B) H18
XI : C771 NCTC 0142 : K86 (B) H6

JURNAL PRAKTIKUM BAKTERIOLOGI KLINIK

PRAKTIKUM : IDENTIFIKASI NEISSERIA Sp. NAMA :
TANGGAL : TANDA TANGAN :
NO. BAHAN :

HARI I :

1. Isolasi

Bahan pemeriksaan ditanam pada media :
a.
b.
c.
Dieramkan 370c selama satu malam (18 – 24 jam )

Hari II :
Pengamatan hasil isolasi

Media : ________ Media : ________ Media : _______
Bentuk koloni :
Warna koloni :
Elevasi
Sifat
……………………
……………………


2. Uji bio kimiawi
Koloni tersangka ditanam pada :

Hari III

Pengamatan hasil penanaman pada :


3. Uji serologi


Kesimpulan :

Dari bahan nomor : ____ didapatkan bakteri :


Diskusi :

Pemeriksa / Penguji

NIP.

PEMERIKSAAN Vibrio cholera

Bahan pemeriksaan : tinja, muntahan, air, dll
Identifikasi berdasarkan :
1. Pembiakan
2. Uji biokimia
3. Uji serologi
Cara kerja :
1. Pembiakan : - secara tidak langsung
- tidak langsung
perbenihan yang dipakai :
- Aronson agar
- Monsur agar (cholera lauryl sulfat)
- TCBS (thiusulfhate citrate bile sucrose) agar
- Alkali pepton sebagai media pengaya
- Deretan gula-gula (bonterey) pendek dan panjang
a. secara langsung tinja ditanam pada perbenihan Aronson, Monsur dan TCBS agar, dieramkan 370C – satu malam, untuk monsur agar lebih baik dieramkan selama 2 malam.
b. Secara tidak langsung tinja ditanam sebanyak 1 ml kedalam Alkali pepton, kemudian dieramkan 6 – 16 jam pada suhu 370C.
Dari alkali pepton dipindahkan pada perbenihan Aronson dan Monsur, dieramkan 370C selama satu malam
c. Diperiksa adanya pertumbuhan koloni tersangka :
Aronson : merah jernih
Monsur : hitam jerih
CBS : kuning jernih
2. Uji biokimia
Dari koloni tersangka tadi, ditanam pada KIA (TSIA), gula manitol, air pepton dieramkan 370C selama satu malam. Amati, catat hasilnya, jika tersangka V. cholera bila hasilnya sebagai berikut :

KIA : kuning, dan merah; TSIA : kuning
Manit : (+) positif tanpa gas
Pepton : indol (+) positif.
3. Uji serologi
Dari hasil uji bio kimia dilakukan tes aglutinasi dengan anti serum :
V. cholera – O
V. cholera inaba – O
V. cholera Ogawa – O

Jika hasil positif, dilanjutkan dengan uji biokimia menggunakan gula-gula (bonterey) panjang.
Biakan pada perbenihan Aronson, Monsur, TCBS serta hasil biokimia cocok, hanya uji serologi (-) negative, maka kumantersebut Vibrio air (N. AG)

Uji lanjutan V. cholera untuk menentukan biotipe Eltor atau Asiatica.
a. penggolongan Heiberg (secara biokimiawi)
b. Uji voges proskauer pada deretan gula panjang
c. Uji haemaglutinasi dengan eritrosit ayam 2,5%
d. Uji kepekaan (Sensitivity test) dengan polymixin B 50 unit
e. Cholera phage typing dengan grup IV
f. Uji hemolisis dengan erirosit domba 1 % dalam HIB dan HIBG
g. Analisa antigen dengan antiserum V. cholera Inaba dan V. cholera Ogawa.


Aspek pengujian
V. cholera Eltor V. cholera Asiatica
a. Heiberg grup
b. Uji voges proskauer
c. haemaglutinasi dengan eritrosit ayam 2,5%
d. Uji kepekaan polymixin B 50 unit
e. phage typing grup IV
f. Uji hemolisis erirosit domba 1 %
g. Analisa antigen I atau II

Mycobacterium tuberculosis

Part III: Culture
media
The definitive diagnosis of tuberculosis demands that M. tuberculosis be recovered on culture media and identified using differential in vitro tests. Many different media have been devised for cultivating tubercle bacilli and three main groups can be identified, viz egg-based media, agar-based media and liquid media.
The ideal medium for isolation of tubercle bacilli should (a) be economical and simple to prepare from readily available ingredients, (b) inhibit the growth of contaminants, (c) support luxuriant growth of small numbers of bacilli and (d) permit preliminary differentiation of isolates on the basis of colony morphology. For the culture of sputum specimens, egg-based media should be the first choice, since they meet all these requirements. These is increasing evidence that liquid media may give better results with other specimens. While cost prevents their routine use with sputum specimens, it is recommended that both egg-based and liquid medium be used for non-repeatable specimens, eg. cerebrospinal fluid and biopsy material.
It is recommended that all sputum specimens submitted for culture also undergo microscopic examination as outlined in the Technical Series on Microscopy.
Advantages and disadvantages of egg-based media
Advantages
• it is easy to prepare
• it is the least expensive of all media available and supports good growth of tubercle bacilli
• it may be stored in the refrigerator for several weeks provided it was made from fresh eggs and culture bottle caps are tightly closed to minimise drying by evaporation
• contamination during preparation is limited because it is inspissated after being placed in bottles. In addition, the malachite green added to the media suppresses the growth of nonmycobacterial organisms
Disadvantages
• it may take as long as eight weeks before cultures become positive, especially if specimens contain few bacilli or if decontamination procedures have been overly harsh
• when contamination does occur, it often involves the total surface of the medium and the culture is usually lost
Precautions during media preparation
For media of the best quality, chemicals of certified purity, clean glassware and freshly distilled and sterilised water should be used. Directions for preparing media must be followed precisely and without modification. A few general points to obtain good quality media and avoid contamination of reagents and media are as follows:
• Keep the environment as clean as possible. Swab the work surface with a suitable disinfectant (eg. 5% methylated spirits) before dispensing sterile reagents and media. Clean the floor with a wet mop to limit dust
• Use sterile glassware and equipment
• Use reagent grade chemicals and reagents unless otherwise specified
• Check the temperature of inspissators and hot air ovens
• Follow strict aseptic techniques when preparing media, eg. flaming flasks and tubes
• When preparing egg-based media, carefully clean egg shells before breaking
• Do not overheat medium during inspissation
• Do not leave prepared media exposed to light (including ultra-violet light), but store in the refrigerator in the dark when not in use
• Do not skimp on the volume of medium. Place 6-8ml of egg medium in each bottle or 20ml into each test tube
Preparation of egg-based media
LÖWENSTEIN-JENSEN MEDIUM
Löwenstein-Jensen (LJ) medium is most widely used for tuberculosis culture. The modification of the International Union Against Tuberculosis and Lung Disease (IUATLD) is recommended and will be described in detail. LJ medium containing glycerol favours the growth of M. tuberculosis while LJ medium without glycerol but containing pyruvate encourages the growth of M. bovis. Both should be used in countries or regions where patients may be infected with either organism.
Ingredients
Mineral salt solution
Potassium dihydrogen phosphate anhydrous (KH2PO4) 2.4g
Magnesium sulphate (MgSO4. 7H2O) 0.24g
Magnesium citrate 0.6g
Asparagine 3.6g
Glycerol (reagent grade) 12ml
Distilled water 600ml
Dissolve the ingredients in order in the distilled water by heating. Autoclave at 121EC for 30 minutes to sterilise. Cool to room temperature. This solution keeps indefinitely and may be stored in suitable amounts in the refrigerator.
Malachite green solution, 2%
Malachite green dye 2.0g
Sterile distilled water 100ml
Using aseptic techniques dissolve the dye in sterile distilled water by placing the solution in the incubator for 1-2 hours. This solution will not store indefinitely and may precipitate or change to a less-deeply coloured solution. In either case discard and prepare a fresh solution.
Homogenised whole eggs
Fresh hens' eggs, not more than seven days old, are cleaned by scrubbing thoroughly with a hand brush in warm water and a plain alkaline soap. Let the eggs soak for 30 minutes in the soap solution. Rinse eggs thoroughly in running water and soak them in 70% ethanol for 15 minutes. Before handling the clean dry eggs scrub the hands and wash them. Crack the eggs with a sterile knife into a sterile flask and beat them with a sterile egg whisk or in a sterile blender.
Preparation of complete medium
The following ingredients are aseptically pooled in a large, sterile flask and mixed well:
Mineral salt solution 600ml
Malachite green solution 20ml
Homogenised eggs (20-25 eggs, depending on size) 1000ml
The complete egg medium is distributed in 6-8ml volumes in sterile 14ml or 28ml McCartney bottles or in 20ml volumes in 20 x 150mm screw-capped test tubes, and the tops are securely fastened.
Inspissate the medium within 15 minutes of distribution to prevent sedimentation of the heavier ingredients.
Coagulation of medium
Before loading, heat the inspissator to 80EC to quicken the build-up of the temperature. Place the bottles in a slanted position in the inspissator and coagulate the medium for 45 minutes at 80E-85EC (since the medium has been prepared with sterile precautions this heating is to solidify the medium, not to sterilise it). Heating for a second or third time has a detrimental effect on the quality of the medium.
The quality of egg media deteriorates when coagulation is done at too high a temperature or for too long. Discolouration of the coagulated medium may be due to excessive temperature. The appearance of little holes or bubbles on the surface of the medium also indicates faulty coagulation procedures.
Poor quality media should be discarded.
Sterility check
After inspissation, the whole media batch or a representative sample of culture bottles should be incubated at 35E-37EC for 24 hours as a check of sterility.
Storage
The LJ medium should be dated and stored in the refrigerator and can keep for several weeks if the caps are tightly closed to prevent drying out of the medium. For optimal isolation from specimens, LJ medium should not be older than 4 weeks.
For the cultivation of M. bovis, LJ medium is enriched with 0,5% sodium pyruvate. Glycerol is omitted and 8.0g sodium pyruvate is added to the mineral solution.
OGAWA MEDIUM
This medium is cheaper than Löwenstein-Jensen because it is made without asparagine.
Ingredients
Mineral salt solution
Potassium dihydrogen phosphate anhydrous (KH2PO4) 3.0g
Sodium glutamate 3.0g
Distilled water 300ml
Dissolve the ingredients in distilled water by heating. Autoclave at 121EC for 30 minutes to sterilise. Cool to room temperature. This solution keeps indefinitely and may be stored in suitable amounts in the refrigerator.
Malachite green solution, 2%
Malachite green dye 2.0g
Sterile distilled water 100ml
Using aseptic techniques dissolve the dye in sterile distilled water by placing the solution in the incubator for 1-2 hours. This solution will not store indefinitely and may precipitate or change to a less-deeply coloured solution. In either case discard and prepare a fresh solution.
Homogenised whole eggs
Fresh hens' eggs, not more than seven days old, are cleaned by scrubbing thoroughly with a hand brush in warm water and a plain alkaline soap. Let the eggs soak for 30 minutes in the soap solution. Rinse eggs thoroughly in running water and soak them in 70% ethanol for 15 minutes. Before handling the clean dry eggs scrub the hands and wash them. Crack the eggs with a sterile knife into a sterile flask and beat them with a sterile egg whisk or in a sterile blender.
Preparation of complete medium
The following ingredients are aseptically pooled in a large, sterile flask and mixed well:
Mineral salt solution 300ml
Malachite green solution 18ml
Whole hens? eggs (12-16 eggs, depending on size) 600ml
Glycerol 18ml
The resulting pH of the medium is 6.8. The medium is mixed well and distributed in 6-8ml volumes in sterile 14ml or 28ml McCartney bottles or in 20ml volumes in 20x150mm screw-capped test tubes.
Coagulation of medium
Before loading, heat the inspissator to 80EC to quicken the build-up of the temperature. Place the bottles in a slanted position in the inspissator and coagulate the medium for 45 minutes at 80E-85EC (since the medium has been prepared with sterile precautions this heating is to solidify the medium, not to sterilise it). Heating for a second or third time has a detrimental effect on the quality of the medium.
The quality of egg media deteriorates when coagulation is done at too high a temperature or for too long. Discolouration of the coagulated medium may be due to excessive temperature. The appearance of little holes or bubbles on the surface of the medium also indicates faulty coagulation procedures.
Poor quality media should be discarded.
Sterility check
After inspissation, the whole media batch or a representative sample of culture bottles should be incubated at 37EC for 24 hours as a check of sterility.
Storage
The medium should be dated and stored in the refrigerator and can keep for several weeks if the caps are tightly closed to prevent drying out.
In laboratories where centrifuges are not available, a simple culture technique could be employed as follows: Sputum specimens are decontaminated with equal volumes of 4% NaOH and inoculated directly onto modified or acid-buffered Ogawa medium. This technique shows a fairly comparable oase yield when compared with concentrated culture techniques.
ACID-BUFFERED OGAWA MEDIUM
Ingredients
Modified Ogawa Acid-buffered Ogawa
Potassium dihydrogen phosphate 2g 3g
Magnesium citrate (KH2PO4) 0.1g -
Sodiumglutamate 0.5g 1.0g
Glycerol 4ml 6ml
Distilled water 100ml 100ml
Homogenised whole eggs 200ml 200ml
2% Malachite green solution 4ml 6ml
Final pH 6.4 6.2
Preparation
Dissolve the ingredients in the distilled water and boil for 30 minutes. Cool to room temperature and add the homogenised eggs and malachite green solution. Transfer 6-8ml volumes to suitable bottles and inspissate at 85EC for 45-60 minutes.
A variety of more expensive and labour intensive culture methods1are available to countries with the required financial and human resources. Some of these will be discussed briefly:
OPTIONS
HERMAN KIRCHNER LIQUID MEDIUM
This medium is most useful and least expensive of the liquid media for culture and tubercle bacilli. It has the additional advantage that it can support a large inoculum.
DUBOS OLEIC ACID-ALBUMIN LIQUID MEDIUM
This medium is recommended for the cultivation of tubercle bacilli from cerebrospinal, pleural and peritoneal fluid. It may be prepared from basic ingredients or may be obtained commercially as a ready-to-use base to which sterile albumin or serum is added.
MIDDLEBROOK 7H-10 AND 7H-11 AGAR MEDIUM
Middlebrook 7H-10 may be made from basic ingredients or may be prepared from commercially available 7H-10 agar-powdered base and Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment. 7H-11 is a 7H-10 agar enriched by the addition of enzymatic digest of casein. It is best to prepare 7H-10 and 7H-11 medium in small quantities of 200 to 400ml to minimise the amount of heat needed to melt the agar. Boiling the basal medium before autoclaving (either to solubilise the agar or to provide stocks of prepared base that may be stored and boiled for later use) should be avoided because the repeat heating produces medium of inferior quality.
When Middlebrook 7H-10 or 7H-11 medium is used for isolation cultures must be incubated in an atmosphere of 10% CO2. Exposure of Middlebrook 7H-10 or 7H-11 agar to either daylight of heat results in the release of formaldehyde in sufficient concentration to inhibit the growth of mycobacteria.


OPTION
SELECTIVE MEDIUM
Specimens which are excessively contaminated may be inoculated onto selective antibiotic-containing media. Use may be made of antibiotics to which mycobacteria are not sensitive but which are capable of destroying the contaminants, eg. penicillin (50-100 units/ml), nalidixic acid (35Fg/ml) or polymyxin (20-25Fg/ml).
The antibiotics may be:
• Added to egg medium before inspissation
• Added to the surface of the medium slant or
• Mixed with the inoculum
Mycobactosel medium contains several antibiotics, eg. cycloheximide (0.4mg/ml), lincomycin (0.002mg/ml) and nalidixic acid (0.035mg/ml), while mycobactosel agar is commercially available. Antibiotic enriched medium should be stored in the refrigerator in the dark for a maximum of four weeks.
1 Kent PT, Kubica GP. Public health mycobacteriology: Guide for the Level III Laboratory. US Department of Health and Human Services, Centres for Disease Control, USA, 1985.
OPTION
RADIOMETRIC METHOD FOR TUBERCULOSIS CULTURE
Recent development in the diagnosis of tuberculosis include an automated system for detecting early growth of mycobacteria by a radiometric method (BACTEC: Beckton Dickinson). Sputum or other homogenates are decontaminated as necessary and added to vials containing Middlebrook 7H12 medium, an antibiotic mixture (to avoid the growth of other organisms) and 14C-labelled palmitic acid. The medium is prepared commercially (BACTEC 12B: Beckton Dickinson) in rubber-sealed bottles and inoculated with a syringe and hypodermic needle. If mycobacterial growth occurs, 14C palmitic acid is utilised and 14CO2 is produced. The air space above the medium in each bottle is sampled automatically by the BACTEC machine at fixed intervals and the amount of radioactive gas is estimated and recorded. Infectious aerosols are contained in the apparatus and captured in HEPA filters before the air is exhausted.
Growth of mycobacteria may be detected within 5-7 days, but positive results require further testing to distinguish between tubercle bacilli and other mycobacteria. In the BACTEC machine, p-nitro-a-acetylamino-ß-priophenone (NAP) is used and tubercle bacilli can be differentiated within five days. NAP inhibits the growth of M. tuberculosis and usually does not affect the growth of MOTT bacilli.
Comparative tests have shown that the method is very successful and reliable and that confirmatory results for M. tuberculosis can be obtained within two weeks. However, the BACTEC machine is very expensive to purchase and to operate. In addition, two hazards must be considered if the machine is to be used for routine tuberculosis bacteriology: the use of hypodermic needles for the inoculation of media carries the risk of needle-stick injury, while the culture media is radioactive and presents a problem in terms of waste disposal.
In summary, the BACTEC method is invaluable for the detection of tubercle bacilli in material such as cerebrospinal fluid where rapid results are crucial in the management of the patient. However, the high cost of both the apparatus and the radio-labelled medium prohibits its routine use in most high tuberculosis prevalence countries.

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