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it contains how the sample is processed and how it is subjected to staiing, biochemical reactions, what are the culture medias used, and thier methods. it also includes the antimicrobial susceptibility testing

it contains how the sample is processed and how it is subjected to staiing, biochemical reactions, what are the culture medias used, and thier methods. it also includes the antimicrobial susceptibility testing


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  2. 2. APPROACH TO LABORATORY DIAGNOSIS • The laboratory diagnosis of infectious diseases involves two main approaches: • Bacteriological approach:- Identified by microscopic observation, macroscopic identification, biochemical reactions and molecular diagnosis approaches. • Immunological approach:- Identified by detection of antibodies against organism in the patient’s serum.
  3. 3. Lab diagnosis is useful for : • Identification: causative agent responsible for disease. • Treatment : accurate antimicrobial therapy. • Surveillance purpose : to assess the disease burden in the community by estimating the prevalence and incidence of the infections. • For outbreak investigation : diphtheria outbreaks in the community, MRSA outbreak in the hospitals. • To start PEP(post-exposure prophylaxis): for infectious diseases such as anthrax, and plaque.
  4. 4. TEST CYCLE Preanalytical (sampling, labelling, transport, storage, specimen clinical information) Analytical (macroscopic, microscopic, culture, identification, interpretation, antibiogram,…) Postanalytical (final report to physician)
  5. 5. • Laboratory diagnosis comprises of different steps : • Specimen collection • Direct detection (microscopy ) • Culture • Identification (biochemical tests, automated identification) • Antimicrobial susceptibility test • Serology • Molecular methods
  6. 6. A) SPECIMEN COLLECTION • Depends upon the type of underlying infections. • Proper collection of sample is of paramount importance for the isolation of the bacteria in culture.
  7. 7. GENERAL PRINCIPLES • Standard precautions should be followed for collecting and handling all specimens. • Should collect before antibiotic starts • Contamination with indigenous flora should be avoided. • Swabs are though convenient but considered inferior to tissue, aspirate and body fluids. • Container : sterile, tightly sealed, leakproof, wide mouth, screw capped containers should be used.
  8. 8. • Labelling : with name, age, gender, treating physician, diagnosis, antibiotic history, type of specimen and desired investigation name. • Rejection : if contaminated or compromised or improperly labelled. • If anaerobic culture is requested, proper anaerobic collection containers with media should be used. • Specimen should not be sent in container containing formalin for microbiological analysis.
  9. 9. TYPES OF INFECTIONS SPECIMEN COLLECTED Bloodstream infection, sepsis, endocarditis Paired blood culture specimen(8-10 ml) Infectious disease requiring serology Blood (2ml/investigation) Diarrheal diseases Stool, rectal swab meningitis CSF Infections of other sterile body area Eg., pleural fluid, synovial fluid, peritoneal fluid Skin & soft tissue infections Pus or exudate, wound swabs, aspirates from abscess and tissue bits. Anaerobic infections Aspirates, tissues, blood & sterile body fluid, bone marrow. URT infections Throat swab, nasopharyngeal swab, per-nasal swab. LRT infections Sputum, endotracheal aspirate, BAL, protected specimen brush UTI Midstream urine, suprapubic aspirate, urine from catheter tube Genital infections Urethral swabs, cervical swabs, exudate from genital ulcers. Eye infections Conjunctival swabs, corneal scrapings, aqueous or vitreous fluid. Ear infections Swabs from outer ear, aspirate from inner ear.
  10. 10. Specimen transport:- • Most of specimen, should not exceed 2 hours; exception:- Immediate transport (<15 min) include CSF, and body fluids, ocular specimens, tissue specimens, suprapubic aspirate, bone specimen. Urine added with preservative (boric acid) - up to 24 hours. Stool culture: transported with in 1 hour, but with transport medium - up to 24hrs. Rectal swabs – up to 24hrs. For anaerobic culture: specimen put into RCMB or any other anaerobic transport medium and transported immediately.
  11. 11. SPECIMEN STORAGE:- • Can be stored at room temperature immediately after receipt, Up to 24 hrs. exceptions are : Blood cultures – incubated at 37°C . Sterile body fluids, bone, vitreous fluid , suprabic aspirate – immediately plated or incubated 37°C. Corneal scraping – immediately plated at bed-side on to BA or CA. Stool culture – stored up to 72hrs at 4°C. Urine, LRT specimen, gastric biopsy – stored up to 24 hrs at 4°C.
  12. 12. DIRECT METHOD • Microscopic detection:-  bacterial motility :– * Hanging drop method
  13. 13.  Morphology and staining reaction:- SIMPLE STAIN: methylene blue stain
  14. 14. • NEGATIVE STAINING :- nigrosine/ India ink Demonstration of bacterial/yeast capsules which do not take up simple stain. E.g., S.pneumoniae, Cryptococcus
  15. 15. • GRAM STAIN:- differentiate between G+ve and G-ve bacteria.
  16. 16. • Uses of gram stain:- o To differentiate G+ve and G-ve bacteria. o To start empirical treatment o For identification of fastidious org., which takes time to grow on culture. o For anaerobic organisms which do not grow on ordinary medium. o Analysing quality of specimen( sputum specimen) Gram positive organisms:- Staphylococcus Streptococcus Enterococcus Bacillus Clostridium Candida Gram negative organisms:- Neisseria Shigella Salmonella Escherichia coli Klebsiella Vibrio
  17. 17. ACID-FAST STAIN:- Diffrentiate acid fast org. from non acid-fast org. Acid-fast organism
  18. 18. Modifications : o cold method (kinyoun’s method) – intermittent heating is not needed. o acid-alcohol alternatively used as decolourizer. o malachite green used as counter stain. o Different con. Of sulphuric acid. Acid fast organisms :- Mycobacterium tuberculosis Mycobacterium leprae Nocardia Bacterial spores
  19. 19. • ALBERT STAIN : demonstrate the metachromatic granules of Corynebacterium diphtheriae. Green bacilli with bluish black metachromatic Granules of C. diphtheriae
  20. 20. CULTURE • Specimens are inoculated on to various culture media and incubated. • Colonies grown are subjected to identification and AST. • Different culture media:- • SIMPLE/BASAL MEDIA:- support growth of non-fastidious bacteria. *Peptone water *Nutrient broth *Nutrient agar
  21. 21. • ENRICHMENT MEDIA :-liquid media added with inhibitory agents which selectively allow org. to grow. tetrathionate broth – salmonella typhi selenite-F broth – isolation of Shigella alkaline peptone water – isolation of Vibrio cholerae
  22. 22. MEDIA ISOLATION Lowenstein-Jensen medium Mycobacterium tuberculosis Thiosulphate citrate bilesalt sucrose agar Vibrio cholerae Deoxycholate citrate agar Salmonella & shigella Xylose lysine deoxycholate agar Salmonella & Shigella Potassium tellurite agar Corynebacterium diphtheriae
  23. 23. • TRANSPORT MEDIA :- transport of clinical specimen suspected to contain delicate organism or when delay is expected. Organism Transport medium Neisseria Amies medium and Stuart's medium Vibrio cholerae V R medium Autoclaved sea water Cary Blair medium Shigella, salmonella Buffered glycerol saline Cary Blair medium
  24. 24. • DIFFERENTIAL MEDIA :- differentiate between 2 groups of bacteria using an indicator. MacConkey agar – Differential and low selective medium. Isolation of enteric gram negative bacteria. Differentiate lactose fermenters (pink colonies) and Non lactose fermenters (colorless colonies). Indicator – neutral red CLED(cysteine lactose electrolyte deficient agar) – Differentiate between LF and NLF colonies. For urine specimen.
  25. 25. • ANAEROBIC CULTURE MEDIA:- contain reducing substances which take up oxygen & create lower redox potential and allows growth of obligate anaerobes. o Robertson cooked meat broth o Brain heart infusion broth o Egg yolk agar o Thioglycollate broth
  26. 26. BLOOD CULTURE • Recovery of bacteria from blood is difficult as they are in lesser quantity in blood and many are fastidious; so enriched media are used. • Two types 1) Conventional blood culture 2) Automated blood culture
  27. 27. Conventional blood culture media • Monophasic medium – contain BHI broth • Biphasic medium – liquid phase with BHI broth solid agar slope with BHI agar • Disadvantage : - subcultures are made onto BA and MA periodically for one week. Higher risk of contamination due to opening of cap everytime when subcultures are made
  28. 28. Automated blood culture technique Continuous automated monitoring. Incubated bottles are periodically tilted automatically. Once positive for microbial growth, instrument gives signal. Composition – tryptic soy broth / BHI broth, polymeric resin beads Specimens – blood, CSF, synovial fluid, pleural fluid Gives higher yield of positive culture It is Rapid Less labor intensive – fully automated.
  29. 29. Automated system 1) BacT/ALERT 3D 2) BacT/ALERT VIRTUO 3) BACTEC Disadvantages:- • High cost of instrument and culture bottles. • Inability to observe the colony morphology.
  30. 30. Culture method • It involves inoculating specimen onto appropriate culture media, incubating culture plates in appropriate conditions. 1.Selection of media:- depends upon type of specimen to be processed. commonly used are BA & MA. 2. Inoculation of specimen:- with the help of bacteriological loop (platinum or nichrome wire) loop is heated till red hot and made cool.
  31. 31. Inoculation method 1.STREAK CULTURE • Used for obtaining individual isolated colonies from a mixed culture. • Intermittent heating is done between the streak.
  32. 32. 2. LIQUID CULTURE Bacterial growth is detected by observing turbidity in the medium. Advantages: • Specimen containing small quantity of bacteria • Specimen containing antibiotics • When large yield of bacteria are required Disadvantages: • Do not provide pure culture • No visible colonies
  33. 33. LAWN CULTURE For antimicrobial susceptibility testing by disc diffusion method by using swab POUR PLATE TECHNIQUE For quantifying bacterial load Serial dilutions are added onto molten agar STROKE CULTURE Carried out on agar slope by streaking in zig zag fashion STAB CULTURE Stabbing semi solid agar butt
  34. 34. ANAEROBIC CULTURE METHOD • McIntosh and Filde’s anaerobic jar • Anoxomat • GasPak system • GENbag • Anaerobic glove box and anaerobic workstation • Reducing agents – RCM broth. • Pre-reduced Anaerobically Sterilized media
  35. 35. Incubatory condition • Aerobes and facultative anaerobes – 37°C aerobically overnight in an incubator. • For capnophilic bacteria – candle jar is used.(3-5% CO2) • For microaerophilic bacteria – requires 5% O2. • For obligate anaerobes – anaerobic culture methods used.
  36. 36. COLONY MORPHOLOGY After overnight incubation examined under bright illumination for : • Size (mm) • Shape - circular/irregular • consistency - dry , moist or mucoid • Density - opaque, translucent or transparent • Haemolysis on BA • Colour of the colony • Pigment production
  37. 37. Culture smear & motility testing Colonies grown on culture media are subjected to gram staining and motility testing using hanging drop method.
  38. 38. Culture Identification Two types: Biochemical identification Automated identification system Identification of bacteria from culture is made either by conventional biochemical tests or by automated identification system.
  39. 39. Biochemical identification 2. OXIDASE TEST Detect presence of cytochrome oxidise enzyme. It turns deep purple with in 10 sec. * Positive - Vibrio, Pseudomonas etc. * Negative - Enterobacteriaceae family etc. 1. CATALASE TEST Catalase producing bacteria mixed with a drop of 3% hydrogen peroxide & look for effervescence * Positive – Staphylococcus, Enterobacteriaceae family etc. * Negative – Streptococcus
  40. 40. 4. CITRATE UTILISATION TEST • Ability of bacteria to utilise citrate as sole source of carbon • Medium – simmon’s citrate medium • Indicator – bromothymol blue • Produce colour change from green to blue • Positive – klebsiella, Citrobacter • Negative – E.coli, shigella
  41. 41. 5. UREA HYDROLYSIS TEST • Ability of bacteria to split urea to produce ammonia and makes medium alkaline • Medium – Christensen urea medium • Indicator – phenol red • Colour changes to pink. • Positive – Klebsiella, Proteus • Negative – E.coli , Shigella
  42. 42. 6. TRIPLE SUGAR IRON AGAR TEST Contains 3 sugars – glucose, sucrose, lactose (1:10:10) Ability of organism to ferment sugars and to produce hydrogen sulphide. Medium – TSI medium Indicator – phenol red Interpretation :- A/A – (acid/acid) both slant & butt change to yellow. K/A – alkaline slant (red)/acid butt (yellow) K/NC – Alkaline slant (red) /Alkaline butt (red) Gas production – cracks on medium H2S production – medium changes to Black.
  43. 43. AUTOMATED SYSTEM 1. MALDI-TOF 2. VITEK 2 3. Phoenix 4. Microscanwalkaway system
  44. 44. ANTIMICROBIAL SUSCEPTIBILITY TEST • Done for pathogenic bacteria. • Types: 1. Disc diffusion method (kirby-Bauer’s method) 2. Dilution test (broth & agar dilution) 3. E-test 4. Automated AST (VITEK)
  45. 45. DISK DIFFUSION METHOD • Most widely used method • Suitable for rapidly growing pathogenic bacteria • Mostly performed from colony • Medium – Mueller-Hinton agar(MHA) • Inoculum – suspending colony in normal saline or inoculating into suitable broth & incubating at 37°C for 2 hrs
  46. 46. • Turbidity – matching with 0.5 McFarland standard(1.5 X 108CFU/ml) • Lawn culture done using swabs. • Antibiotic discs placed at least 24 mm apart(6 discs in 100 mm plate ) • Incubation – 37°C for 16 to 18 hrs.
  47. 47. • Interpretation :- • Antibiotics in the disk diffuses through the solid medium; conc. is highest near the site of application of antibiotic disk and decreases gradually away from it. • Susceptibility is determined by zone of inhibition of bacterial growth around the disk & is measured using vernier caliper. • Interpretation of zone into sensitive, intermediate or resistant is based on the standard zone size interpretation chart, provided by CLSI or EUCAST guidelines
  48. 48. • Automated Antimicrobial susceptibility testing:-  work by the principle of micro broth dilution.  Use commercially available panels.  Provide more rapid results. o VITEK 2 o Phoenix system o micro scan walk away system
  49. 49. SEROLOGY • Include detection of either antigen or antibody in the serum of patient by various immunological assays – precipitation, agglutination, ELISA & rapid test. • Various serological tests are: o widal test – enteric fever o Standard agglutination test – Brucellosis o Weil-felix test – Rickettsial infection o VDRL or RPR - Syphilis
  50. 50. MOLECULAR METHODS Amplification of nucleic acid based: • Polymerase chain reaction(PCR) • Real-time PCR (rt-PCR) • Biofire Film Assay • CBNAAT Non-amplification methods : • DNA hybridisation method • (line probe assay)
  51. 51. THANKYOU

Editor's Notes

  • Bact. Approach is by using microscopic observation, macroscopic identificatn, biochemical tests and molecular methods.
    Immunological by the detetction of antibodies against microorg. In patient serum.
  • A platinum or nichrome wire loop of 2-4 mm internal diameter is used .
  • Stroke culture done in tubes containing agar slope . Stab culture is used in maintaining stock culture.