Identifikasi Mikroorganisme

secara Morfologi dan Biokimia

Dr. Ari Susilowati, S.Si, M.Si
Dosen Jurusan Biologi FMIPA UNS
• Identifikasi awal dari sebagian besar bakteri yang
dapat dibudidayakan didasarkan pada karakter
morfologis. Karakterisasi fenotipik bakteri
dengan, tes fisiologis dengan tes biokimia
membedakan genus bakteri terkait serta
mengkonfirmasi identitas mereka.
 Pendekatan umum untuk identifikasi bakteri

4 langkah:
• Langkah 1: Pengambilan sampel
• Langkah 2: Membudidayakan pada media isolasi
• Langkah 3: Melakukan teknik identifikasi
• Langkah 4: Hasil = nama bakteri
 Pendekatan umum untuk identifikasi bakteri

Teknik identifikasi yang berbeda:

• Metode fisik
Berdasarkan karakterisasi morfologi bakteri
• Metode genetik
Berdasarkan karakterisasi gen spesifik bakteri
• Metode biokimia
Berdasarkan karakterisasi jalur metabolisme bakteri
Untuk mengidentifikasi bakteri yang tidak diketahui,
hasilnya dibandingkan dengan database
 Bagaimana cara mengidentifikasi bakteri yang tidak
dikenal dengan morfologi

• Bakteri tumbuh sangat cepat ketika disuplai dengan nutrisi

yang berlimpah. Berbagai jenis bakteri akan menghasilkan
koloni yang tampak berbeda, beberapa koloni mungkin
berwarna, beberapa koloni berbentuk lingkaran, dan yang
lainnya tidak teratur.
• Karakteristik suatu koloni (bentuk, ukuran, pigmentasi, dll.)
Disebut morfologi koloni. Morfologi koloni adalah cara para
ilmuwan dapat mengidentifikasi bakteri.
Ada beberapa elemen dasar yang dapat Anda identifikasi untuk
morfologi koloni:
• Bentuk - Apa bentuk dasar dari koloni? Misalnya, melingkar,
berserabut, dll.
• Ketinggian - Apa bentuk penampang koloni?
• Margin - Apa bentuk tepi koloni yang diperbesar?
• Permukaan - Bagaimana permukaan koloni muncul? Misalnya
halus, berkilau, kasar, kusam (kebalikan dari berkilau), rugosa
(berkerut), dll.
• Opacity - Misalnya, transparan (jernih), buram, tembus
pandang (hampir jernih, tetapi pandangan terdistorsi, seperti
melihat melalui kaca buram), warnawarni (berubah warna
dalam cahaya yang dipantulkan), dll.
• Chromogenesis (pigmentasi) - Misalnya, putih, buff, merah,
ungu, dll.
Lihat diagram di bawah untuk contoh ilustrasi bentuk, ketinggian,
dan margin:

3 elemen tambahan morfologi harus diperiksa hanya dalam pengaturan laboratorium

yang diawasi: konsistensi, emulsifikasi, dan bau.
• Colonies of bacteria
and fungus from public
hall air on a petri dish
agar plate isolated on
white background.
Different color, size,
border and type of
colonies. Different
microbial species.
Nutrient agar media
meat-peptone agar
used.
Ukuran, Bentuk, dan Susunan Bakteri

Ada tiga bentuk dasar bakteri: coccus,

bacillus, dan spiral.
• Kokus adalah bakteri bulat atau oval yang memiliki
salah satu dari beberapa pengaturan berbeda
Batang atau bacillus
Bacilli adalah bakteri
berbentuk batang.

• a. bacillus: bacilli tunggal

• b. streptobacillus: basil
disusun berantai

• c. coccobacillus: oval dan

mirip dengan coccus

• Bacillus rata-rata adalah

lebar 0,5-1,0 μm dengan
panjang 1,0-4,0 μm.
Spiral
Spiral datang dalam salah satu
dari tiga bentuk, vibrio,
spirillum, atau spirochete.

• a. vibrio: batang
melengkung atau
berbentuk koma

• b. spirillum: spiral tebal dan

kaku

• c. spirochete: spiral tipis

dan fleksibel
Pengecualian untuk bentuk di atas
• Mereka termasuk bakteri berselubung,
menguntit, berfilamen, berbentuk persegi,
berbentuk bintang, berbentuk gelendong,
berlubang, membentuk trikoma, dan
pleomorfik.
Pengecatan Gram

Prosedur Pengecatan Gram

• Cell wall of
Gram
Positive &
Gram
Negative
Lipoteichoic acids (LTA); Lipopolysaccharides (LPS)
Ultra Structure of Cell wall of GPC & GNB
 Gram negative rod
Gram positive rod

Gram positive cocci Gram negative cocci

 Aplikasi dalam identifikasi
• Bagaimana cara mengidentifikasi bakteri yang tidak
dikenal dengan metode biokimia ??
• 4 langkah:
• 1-Inkubasi bakteri untuk diuji di media dengan nutrisi
yang berbeda (biasanya 5 hingga 20); satu media untuk
satu nutrisi
• 2-Setelah inkubasi, untuk setiap media, tentukan
hasilnya positif atau negatif untuk setiap nutrisi
• 3-Tulis profil biokimia lengkap
• 4-Bandingkan profil dengan database untuk
mengidentifikasi bakteri Anda
 Hasil untuk pewarnaan Gram
Sistem API
• Dengan bakteri yang tidak dikenal untuk diidentifikasi, sistem API mana yang digunakan?
• API Listeria untuk Listeria
• API NH untuk Neisseria
• Cocci Gram-
• Batang Gram +
• API 50CH untuk Bacillus
• Hasil untuk pewarnaan Gram
• Cocci Gram +
• Catalase +
• API Staph untuk Staphylococcus
• Oksidase +
• Batang Gram-
• API 20NE tanpa Enterobacteria
• Oksidase-
• Catalase-
• API 20E untuk Enterobacteria
• API Strep untuk Streptococcus
 API®
• The well-established method for manual
microorganism identification to the species level,
bioMérieux’s API identification products are test kits
for identification of Gram positive and Gram negative
bacteria and yeast. The system offers a large and
robust database now accessible through the
Internet-based APIWEB™ service.
 IMVIC

The IMViC tests are tests used in microbiology

lab testing to identify an organism in the
coliform group. A coliform is a gram negative,
aerobic, or facultative anaerobic rod, which
produces gas from lactose within 48 hours. The
presence of some coliforms indicate fecal
contamination.
 Identifikasi bakteri coliform berdasarkan IMVic tests

Biochemical Test and Identification of E. coli

https://microbiologyinfo.com/biochemical-test-and-identification-of-e-coli/
• Biochemical tests in Microbiology
• https://microbeonline.com/category/bacteriol
ogy/biochemical-tests-in-microbiology/

• Biochemical tests (yang lebih lengkap)

Identifikasi Fungi secara Morfologi
• Fungi are identified by their morphology in
culture. Fungi have mycelium and spores
which are used in the identification. Therefore
you have to search for mycelium (hyphae), the
spores, origin of the spores, asexual or sexual;
and their structure and morphology.
A. Visualization of fungi in tissue preparations
1. Treatment with 10% potassium hydroxide
2. Positive stain with

Lactophenol cotton blue

Grocott silver stain
Hematoxylin
Eosin

Negative stain with India ink

Culture of fungi on
1. Sabouraud's agar (favors fungal growth because of low pH)
2. Mycosel agar (selective for pathogenic fungi because of
chloramphenicol and cycloheximide in medium)
• LPCB Lactophenol Cotton Blue is a stain used for making semi-permanent
microscopic preparation of fungi. The LPCB stain has following three components :
• Phenol : Kills any live organism.
• Lactic acid : Preserves fungal structures.
• Cotton blue : Stains the chitin and cellulose of the fungal cell wall intensely blue.
 Procedure
• Place a drop of 70% ethanol on a clean microscopic glass
slide
• Immerse the specimen in the drop of alcohol
• Add one or at most two drops of the LPCB before the
alcohol dries out
• Holding the coverslip between the index finger and thumb,
touch one edge of the drop of mountant with a coverslip
edge and lower gently avoiding air bubbles
• This preparation is now ready for examination
• Make the initial examination using low power objective.
Switch to higher power (40X) objective for more detailed
examination of spores and other structures
D. Visualization of cultured fungi (25oC and 37oC)
1. Colonial morphology
2. Cellular morphology

a. Hyphal morphology
(1) Aseptate or coenocytic
(2) Septate
(a) Regular connection
(b) Clamp connection
b. Spore morphology
(Spora aseksual)
(1) Conidiospore
(2) Sporangiospore
(3) Arthrospore
(4) Chlamydospore
Kelas utama Fungi:
• Zygomycota (rhizopus)
• Ascomycota
• Basidiomycota (mushrooms)
• Mycorrizhae
• A. Aspergillus; B. Penicillium; C. Geotrichum; D. Trichophyton; E.
Microsporum; F. Epidermophyton and G. Rhizopus.
• Slide culture technique to induce the formation of
spores. (A) Sterilized filter paper, sticks, and slide. (B)
Add sterilized water on the filter paper to maintain
humidity. (C) A small amount of mycelia was
inoculated on 0.5 mm × 0.5 mm PDA agar. (D) Cover a
sterilized coverslip on the agar block, seal the petri
dish, and incubate at the fungal growth temperature
for 7-14 days. The coverslip was taken out and
observed microscopically.
• Slide culture system. An agar-
coated microscope slide is
placed on a bent glass rod
(diameter 4 mm) resting on
moist Whatman No. 1 filter
paper in a Petri dish sealed
with a strip of ParafilmÔ.
Axes for measurement of
colony growth are indicated
by arrowed lines. Bar [10mm.
Preparation of agar block smears. (a) Spore formations and other characteristic
structures were checked by agar block examination of agar plates under a light
microscope. (b) An agar block (15 by 15 mm) was cut using a sterile dissecting knife. (c) A
coverslip (18 by 18 mm) was put onto the agar block after lactophenol cotton blue
staining was performed. (d) The agar block with the coverslip was examined under a light
microscope. (e) The block was dried in air until the thickness of the agar block reached
0.5 mm. (f) The agar block under the coverslip was filled with mounting medium.
Buku Identifikasi fungi
Diseases of a few economically important crops and ornamental plants. a) Leaf spot of
Oryza sativa (rice) -Curvularia sp. ; b) Rust on Plumeria (Frangipani) -Coleosporium
plumeriae ; c) Purple leaf spot of Fragaria (strawberry )-Ramularia grevilleana ; d) Seed
infections of rice -Periconia sp.; e) Rust on Canna-Pucciniathaliae ; f) Leaf blight of Cucurma
(Turmeric) -Diaporthe sp.
Diagrammatic
illustration of the key
steps of molecular
identification of the
plant pathogenic fungi.
 MOLECULAR BACTERIAL AND FUNGAL
IDENTIFICATION

Common methods used for identifying bacterial

and fungal strains are 16S rRNA gene sequencing
and Internal Transcribed Spacer (ITS)
sequencing respectively. These highly conserved
regions are standard tools used to construct
bacterial and fungal phylogenies and taxonomies.
 16S rRNA gene sequence based
identification for bacteria
• As huge varieties of bacteria are present in the environment, the range
and complexity of the techniques to be utilized for their identification
purpose is highly bewildering (Spratt, 2004). However, the use of
nucleotide sequence data from 16S rRNA gene has been regarded to be
the most suitable practice not only to identify but also to draw the
phylogenetic relationship for all microorganisms on earth.

• The reasons behind the use of 16S rRNA gene to be utilized for
identification purpose include i) occurrence of the gene in all organisms
performing the same function, ii) the gene sequence is conserved
sufficiently containing conserved, variable and hyper-variable regions, and
iii) around1500 bp of sequence size which is relatively easy to sequence
and large enough to contain sufficient information for identification Q10
and analysis of phylogeny (Clarridge, 2004). This technique became
popular only after sufficient deposition of 16S rRNA gene sequences in the
database as well as the availability of suitable primers for gene
amplification
Genetic approaches of studying fungal phylogeny mostly include nuclear rDNA
markers. In the case of fungi, rDNA consists of the SSU 18S, ITS, ITS1 + 5.8S + ITS2 and
the LSU 25-28S regions (Hibbett et al., 601 2007). However, ITS has been considered to
be the best fungal barcode Q13 for identification purposes (Porter and Golding, 2012).
It has been reported that the sequence comparison of internal transcribed spacer 604
and D1/D2 26S rDNA spacer sequences (Fig. 4). Analysis of D1/D2 26S rDNA sequences
not only have the advantage of species identification but also permit phylogenetic
analysis.