Identofocation of bacteria
- 1. IDENTIFICATION OF BACTERIA Dr amit kumar amitkumarshahi90@gmail.com
- 2. IDENTIFICATION OF BACTERIA • Methods : • A) Culture and identification • B) Automated culture techniques • C) Molecular techniques
- 4. • A) CONVENTIAL CULTURE METHODS: • 1)SITE OF INFECTION ( urine) GRAM STAIN 1)BA & MA :PUS , WOUND SWAB, • 2)PROPER COLLECTION (mid stream urine) ACID FAST STAIN STERILE BODY FLUID , • 3)STANDARD PRECAUTION ALBERT STAIN URINE , RESPIRATORY SAMPLES • 4)COLLECT PRIOR TO ANTIBIOTICS 2) CA: RESPIRATORY AND STERILE BODY FLUID • 5)AVOID CONTAMINTION 3)DCA , TCBS , XLD: STOOL SAMPLE • 6)MUST LABELLED 4) BLOOD CULTURE BOTTLE: BLOOD • 7PROCESSED SOON ( WIHOUT DIRECT MICROSCOPY) IF DELAY TRANSPORT MEDIUN 5)CLED: URINE • SIZE : MILIMETER, PIN POINT: STREPTO , PIN HEAD: STAPHYLO gram&BIOCHEMICALS SHAPE : CIRCULAR OR IRREGULAR SURFACE : SMOOTH , ROUGH GRANULAR • ELEVATION : FLAT RAISED , LOW CONVEX , CONVEX , OR UMBONATE EDGE: CRENATED, ENTIRE , LOBATE , UNDULATE , CILATE OPACITY : TRANSLUCENT, TRANSPARENT OR OpAQUE PIGMENT HEMOLYSIS CONSISTANCY: MUCOID , FRIABLE , firm , butyrous SPECIMEN DIRECT MICROSCOPY CULTURE 37O FOR 48 HRS
- 5. Morphology of bacterial colony
- 6. OVERVIEW STEPS OF CONVENTIAL CULTURE METHODS: • 1) sample collection • 2) direct stain of sample • 3) culture of sample on media for (24-48hrs) • 4) stain of colony • 5) biochemical of colony
- 7. BIOCHEMICAL REACTIONS • Based on: colony morphology (gnb or gpc) • 1) catalase : all organisms • 2) GNB: • A) Sugar fermentation • b) indole test • c)Methyl red test • d) Voges proskauer test • e)Oxidative fermantative test • f)Phenyl pyruvic acid test • g) citrate utilization test • h) Urease hydrolisis test • i) triple sugar iron test
- 8. • 3) Gpc : • A) catalase: staphylococcus aureus B)Dnase test: staphylococcus aureus • C) CAMP( Christie-Atkins munch petersen): group B Streptococcos • D) Bile eschuline test : Enterococcus • E) Sugar fermentation test inuline fermentation: pneumococcus F) PYR test : Streptococcus pyogens and enterococcus G) Bile solubility test : pneumococcus H) Optochain test : pneumococcus I) Bacitracin test : G-A (S) and G-B resistsnce
- 9. • Catalase : CATALASE H2O2 H2O+O2 • catalase + : staphylococcus • Catalase - : streptococcus
- 10. • Oxidase : • oxidase • + organism: pseudomona , vibrio, neisseria ,, bacillus etc -ve organisms: enterobactericeae, acitenobacter etc
- 11. • Indole test :tryptaphanase enzyme breaks aminoacid tryptophane • organism + : E.coli , vibrio clolerae - Organism : klebsiella pneumoniae ,pseudomonas , salmonella 37oc for 48 hrs Kovac’s (para dimethylamino benzaldehyde)
- 12. • Citrate : • Utilized citrate as sole source of carbon • Butt and slant • Media : • Simmon’s citrate agar : colour change to blue due to bromothymol blue • Koser’s medium : turbit • + organisms : klebsiella pneumoniae , citrobacter , enterobacter • - organisms: E.coli , shigella
- 13. • Urea : • Urease urea ammonia alkaline • Media : Christensen’s urea media (phenyl red) • + organisms:proteus , helocobacter pylori • - organisms: E.coli , shigella
- 14. • Triple sugar iron test : gram negative bacteria • Comosition • 3 sugar : glucose , sucrose and lactose • Indicator : phenyl red • Ferric chloride : H2S (Hydrogen sulfide)
- 15. • Interpretation : • K = red (alkaline) • A= yellow (acidic) • K/A= glucose only • A/A=glucose , lactose/sucrose • K/K= non formontive • k/k A/A K/A K/A H2S K/A H2S
- 16. • Methyl red test : sufficient acid during fermentation of glucose at ph4.5 + organism : E.coli , listeria monocytogenes -organism :klebsiella , enterobacter 37oc for 48 hrs Methyl red Glucose phosphate broth
- 17. • Glucose phosphate broth 37oc for 48 hrs 1ml 40% KOH & 3ml 5% alpha nepthonal acetylmethyl carbinol with glucose acetylmethyl carbinol converted to diacetyl after adding of ∝- naphthol, strong alkali (40% KOH) The diacetyl containing compounds found in the peptones of the broth then condense to form a pinkish red polymer Voges–Proskauer (VP) Test
- 18. • Nitrate reduction test: • Nitrate reductase • Nitrate Nitrite • 37OC FOR 24 HRS 6-8 drops of nitrite reagent A 6-8 drops of nitrite reagent B NITRATE POSITIVE : ALL ENTEROBACTERIACEAE NITRATE NEGATIVE: HAEMOPHILUS DUCREYI NITRATE BROTH
- 19. • Phenyl pyruvic acid test (PPA): • phenylalanine deaminase • PHENYLALANINE PYRUVIC ACID (10% ferric chloride)
- 20. Automated culture techniques • Purpose • A) culture • B) identification • C) AST
- 21. 1) AUTOMATED BLOOD CULURE TECHNIQUES Incubate Microbial growth indicated By machine More sensitive Rapid less labour
- 22. • tryptic soy broth • brain hear infusion broth polymetric beads : nutrilized antibiotics in blood sensor microorganism co2 Hydrogen INCREASES (H) DECREASE PH react
- 23. AUTOMATED STSTEM FOR BACTERIAL IDNTIFICATION • MALDI-TOF : (MARIX ASSISTED LASER DESORPTION/IONIZATION TIME OF FLIGNT) • VITEK -2: automated identification and AST system