positive
(coccus and rods)
Identifikasi dan deteksi bakteri
Mikrorganisme Spesies
Uji Uji
Fenotip Genotip
CARA KERJA
Koleksi Isolat IMNV
BBPBAP dari empat
Isolasi
pulau Virus
di Indonesia
Amplifikasi
Fragmen Gen MCP
purifikasi
Lane M adalah 100 bp DNA ladder. K- : kontrol negative; K+ : kontrol positif; 1 dan 2
adalah sampel. A. isolat asal Kendal, B. isolat asal Lampung, C. isolat asal Pontianak, D.
isolat asal Banyuwangi, lane 1 adalah produk dari reaksi step pertama 329 bp. Lane 2
adalah produk dari reaksi kedua, step nested 139 bp (BBPBAP Jepara)
Elektroferogram produk RT-PCR
amplifikasi parsial gen MCP
Lane 1,3,5 dan 7 adalah region 9 dan lane 2,4,6,8 adalah region
10. Lane M : 100 bp DNA ladder, lane 1-2: isolat kendal, lane 3-
4: Isolat Lampung, lane 5-6: Isolat Pontianak, lane 7-8: Isolat
Banyuwangi ROZI API2 2021
Salah satu hasil sekuensing
sampel IMNV_Pontianak
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s
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m
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Perbandingan sekuen asam amino parsial gen MCP
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m
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Variasi asam amino parsial gen MCP strain
penelitian dengan strain pembanding
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Strain Penelitian terhadap IMNV Indonesia Strain Penelitian terhadap IMNV Brazil
Lampung :3 Lampung :4
Banyuwangi :5 Banyuwangi :6
Pontianak :1 Pontianak :2
Matriks, presentase Persamaan dan perbedaan Asam Amino
parsial gen MCP Strain Penelitian dengan Strain Pembanding
Analisis Filogenetik
37 IMNV Banyuwangi
44 IMNV Indonesia
41 IMNV Lampung
100 IMNV Pontianak
IMNV Brazil
KHV
WSSV
0.1
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Organizing the Staining Bottles
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Proceed in organized Fashion
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Step 1
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Step 2
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Step 3
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Step 4
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Step 5
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Step 6
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Step 7
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CELL WALL
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Characteristic
Gram positive Gram-negative
Gram reaction
Retain crystal violet dye and stain dark violet or purple can be decolorized to
accept
Peptidoglycan layer
Thick (multilayered) Thin (single-layered)
Teichoic acids
Present in many Absent
Periplasmic space
Absent Present
Outer membrane
Absent Present
Lipopolysaccharide (LPS) content
Virtually non High
Lipid and lipoprotein content
Low High
Toxins produced
Primarily exotoxins Primarily endotoxins
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Gram Positive Gram Negative
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Bacteria
O/F test
– All members of Enterobacteriaceae are O+/F+
– Pseudomonas is O+/F-
Nitrate reductase
– All members of Enterobacteriaceae are nitrate reductase positive
Enterobacteriaceae
Lactose fermenters
Non-lactose fermenter
E. coli, Citrobacter,
Salmonell, Shigella
Klebsiella, Enterobacter
Proteus, Yersinia
MacConkey Agar
Contains
pH indicator
Inhibit growth of G+ve bacteria Cause of differential Acidic: Pink
Cause of selectivity
Lactose feremnters Lactose non feremnters
Pink colonies colorless colonies
Classification of Enterobacteriaceae according to
lactose fermentation (growth on MacConkey Agar)
Enterobacteriaceae
No acid
Acid
Neutral red
Colorless colonies
Pink colonies
Escherichia coli
Salmonella spp
Klebsiella spp
Schigella spp
Enterobacter spp
Proteus spp
Citrobacter spp
Yersinina spp
Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth on MacConkey agar
Method:
– MacConkey agar is inoculated with tested organism
using streak plate technique
– Incubate the plate in incubator at 37 C/24 hrs
Results:
– LF organism appears as pink colonies (e.g. E. coli)
– NLF organism appears as colorless colonies (e.g.
Shigella)
Flame & Cool
1 2
3
4 Flame & Cool
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Method:
A .Klebsiella pneumoniae
Both are lactose fermenters
B .Escherichia coli
C :Salmonella sp. Both Salmonella sp. & Proteus product H2S
D :Proteus mirabilis
E :Ps. aeruginosa Pseudomonas colonies are nearly colorless
.
Growth of Enterobacteriaceae on
EMB agar
Method:
– Inoculate TSI medium with an organism by
inoculating needle by stabbing the butt and
streaking the slant
– Incubate at 37°C for 24 hours
Result
Reaction on TSI s Result
Example
Butt color Slant color H2S
Negative A/Alk/- LNF
Yellow Red
(Glucose fermented) e.g. Shigella
Positive LNF
A/Alk/+
Yellow Red black in butt e.g. Salmonella &
(Glucose fermented with H2S)
Proteus
LF
A/A/-
Yellow Yellow Negative e.g. E. coli, Klebsiella,
(three sugars are fermented)
Enterobacter
Alk/Alk/- Non fermenter e.g.
Red Red
Negative (No action on sugars) Pseudomonas
classification
The classification of streptococci is based on:
1) Hemolysis:
A. Alpha-hemolytic streptococci (α-hemolytic):
Incomplete lysis (partial hemolysis) of red blood
cells (RBCs) in the agar resulting in the formation
of a greenish zone around their colonies.
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classification
2) Cell wall carbohydrates (group-specific carbohydrates):
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Lancefield Group A
Beta-hemolytic Streptococci
(Streptococcus pyogenes)
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properties
Characteristics:
Streptococcus pyogenes (which means pus producing).
Beta-hemolytic.
Gram positive cocci arranged in chains.
Catalase test-negative.
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Virulence factors
Beta-hemolysis group A streptococci have several virulence factors:
1) Streptolysin O:
This enzyme destroys RBCs and WBCs.
2) Streptolysin S:
Responsible for beta-hemolysis.
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Virulence factors
3) M-protein:
Anti-phagocytic.
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Virulence factors
6) Pyogenic exotoxin A:
This toxin is responsible for streptococcal toxic shock syndrome which is similar to
toxic shock syndrome toxin (TSST) caused by S. aureus.
7) Streptokinase (fibrinolysin):
Breaks up fibrin blood clots.
8) Hyaluronidase:
facilitates the spread of the bacteria through tissues by breaking down hyaluronic acid,
an important component of connective tissue.
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Cell surface structure of Streptococcus pyogenes and
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Enterobacteria
ceae
Batang Gram negatif
Sebagian besar
merupakan flora
normal di saluran
cerna manusia dan
hewan
Lainnya merupakan
patogen enterik,
urinarius, dan
respiratorius
Klasifikasi Enterobacteriaceae
(reaksi biokimia dan struktur antigen)
Karakteristik
Motil (kecuali Shigella dan Klebsiella)
Tidak menghasilkan spora
Tumbuh dengan baik pada agar MacConkey
Aerob dan anaerob (anaerob fakultatif)
Memfermentasikan glukosa
Mereduksi nitrat menjadi nitrit
Katalase positif
Oksidase negatif (kecuali Pleisomonas)
Morphology and
Identification
Klebsiella Enterobacter Serratia
Mucoid • motil DNase,
polysaccharide • Citrate (+)
• ornithine
lipase,
capsules decarboxylase gelatinase.
lack of motility (+)
(+)lysine • Gas (+) from
decarboxylase glucose.
• E. aerogenes has
(+)citrate. small capsules.