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Laboratory diagnosis of_infectious_diseases


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Laboratory diagnosis of_infectious_diseases

  1. 1. Laboratory Diagnosis of Infectious diseases: An overview of Diagnostic cycle. Shilpa.K Microbiology Tutor AIMSRC
  2. 2. Preanalytical Phase Analytical phase Post analytical phase Diagnostic cycle
  3. 3. THE DIAGNOSTIC CYCLE PRE-ANALYTICAL PHASE Visit Examination & Provisional diagnosis Collection of sample IdentityRequisitionTransportRecords
  4. 4. ANALYTICAL PHASE Macroscopy Microscopy Culture Biochemical identification Serology Molecular Analysis
  5. 5. POST-ANALYTICAL PHASE Reporting of Identification & Antibiotic sensitivity Treatment
  6. 6. Specimen Collection & Transport Collection from actual site with minimum contamination Optimal time of collection Sufficient quantity of specimen Appropriate collection device, container & culture media Proper labelling of containers Obtain samples before starting antibiotics
  7. 7. BLOOD CULTURES • One blood culture usually consists of blood from a single venepuncture inoculated into two separate bottles (one aerobic, one anaerobic) • Optimal blood-to-broth ratios are 1:5 to 1:10. • Sodium polyanethol sulfonate (SPS)- 0.025 to 0.05%- is added as an anticoagulant, an antiphagocytic agent that inactivates complement, and a neutralizing agent to inhibit effects if many antimicrobial and antibacterial factors in blood.
  8. 8. • Recommended total volume and numbers of blood cultures Syringe needed Aerobic bottle Anaerobic bottle Adult 20 ml 10 ml 10 ml Pediatric 20 ml 2.5 -10 ml 2.5 -10 ml Infant 3 ml 0.5 -1 ml 0.5-1 ml Adult (low volume) All None • Timing of blood cultures  Before or during fever spike  Acute sepsis or another condition (osteomyelitis, meningitis, pneumonia or pyelonephritis): two blood cultures of maximum volume consecutively from different anatomical sites before starting therapy
  9. 9.  Pyrexia of Unknown Origin, Subacute Bacterial Endocarditis or other continuous bacteraemia or fungemia: maximum of three blood cultures with maximum volume  Patients on antimicrobial therapy : Sample is drawn when antimicrobial agents are at their lowest concentration. • Skin antisepsis and collection of blood from venepuncture  A different venepuncture site for each blood culture is selected.  If blood for culture is required to be drawn through a port in an indwelling catheter, the second culture must be from a peripheral site.  Vigrous cleaning is done with 70% isopropyl or ethyl alcohol. Allowed to dry.  Swab or wipe concentric circles of 1 to 2% tincture of iodine, moving outward from centre of the site.  Allow the iodine to dry and avoid touching the site.
  10. 10.  First the aerobic bottle is inoculated and then the anaerobic bottle. • Collection of blood from intravascular catheters  Comparison of cultures that are drawn through an indwelling intravenous catheter and through a peripheral site may be useful for diagnosis of catheter related sepsis.  Bottles are labelled with patient name, site, date and time of draw.  Using two separate alcohol preps, catheter hub connection is scrubbed for 15 sec with 70% alcohol. Air dry.  Wear gloves, disconnect tubing or cap of catheter and attach syringe to collect discard blood ( 3ml for adults and 0.2ml for pediatric patients), which is not used for culture.  Using a new syringe, blood for culture is collected through the hub. Quickly reconnect tubing.
  11. 11. • Specimen Transport  Blood cultures should not be refrigerated. They are generally held at room temperature until processed, for a maximum of 4 h. • Rejection Criteria  Blood cultures that are received unlabelled.  If the tube or bottle is cracked or broken.  Labelled blood cultures are not rejected even if medium is expired, volume or number of bottles is insufficient, or bottles are received >12 h after collection.
  12. 12. Lower Respiratory Tract Cultures • Expectorated sputum • Induced sputum  Used for Pneumocystis carinii and Mycobacterium tuberculosis  Patient’s mouth is rinsed thoroughly with sterile water or saline.  Using and ultrasonic nebulizer, patient is made to inhale approx. 20 to 30ml of 30% NaCl.
  13. 13. • Tracheostomy and endotracheal aspirates  The specimen is aspirated into a sterile sputum trap or leak proof cup.  Tracheostomy aspirate should not be cultured unless clinical pneumonia is present (fever and infiltrates). Tracheostomy is followed by colonization within 24h of insertion, and results may not correlate with disease. • Bronchoscopy specimens  Include BAL samples, bronchial washings, transbronchial biopsy specimens.  Culture BAL samples quantitatively or semiquantitatively for bacterial pathogens Mucus extractor for ET aspirates
  14. 14. • Specimen Transport  Specimens are collected in leakproof cups and labelled with source of material.  Specimens are labelled with demographic information, date and time of collection, and site of collection.  Provisional diagnosis should be mentioned for proper evaluation of the cultures.  Appropriate tests should be requested. Anaerobic cultures should be limited to specimens that are not contaminated with upper respiratory microbiota (eg. Biopsy samples) and only when aspiration pneumonia or similar disease is being considered.  Specimens should be stored at 2 to 80C until cultures can be submitted or processed. Sputum sample container
  15. 15. • Rejection Criteria  Repeat cultures at intervals of less than every 48 h.  24 h sputum collection  Contaminated sputum and endotracheal specimens per Gram stain rejection criteria  Saliva  Nasal washes and aspirates or swabs of nares  Throat specimens  Specimens for anaerobic culture, except transtracheal aspirates, biopsy samples, pleural fluid or other uncontaminated specimens.  For specimens delayed in transit time more than 2 h without refrigeration.
  16. 16. URINE CULTURES • Clean voided midstream urine collection  Females- labia are held apart, vulva is washed from front to back  Circumcised males- no preparation.  Uncircumcised males- foreskin is retracted, glans penis is washed  Patient is asked to collect voided urine directly into a disposable leak proof container, instructing the patient to not halt and restart the urine stream for a “midstream” collection but preferably move the container into the path of already voiding urine. • Catheter urine  Using a needle and syringe, urine is collected through the catheter port, after cleaning with alcohol.  Urine obtained from a catheter bag should not be send. • Timing of specimen collection  Early morning specimens should be obtained whenever possible.  Fluids should not be forced in order to have the patient void urine.
  17. 17. • Specimen Transport  Urine should be transported to the lab after collection or, if urine cannot be delivered within 2 h after collection, can be refrigerated for up to 24 h. • Rejection Criteria  No evidence of refrigeration and the specimen is >2 h old.  Collection time and method of collection have not been provided.  24- h urine collections  Foley catheter tips  Urine from the bag of a catheterized patient  Specimens that arrive in leaky containers  Except for suprapubic bladder aspirates, reject specimen requests for anaerobic culture. Urine sample container
  18. 18. Wound and Soft tissue cultures • Specimen should be collected prior to initiation of therapy and only from wounds that are clinically infected or deteriorating or that fail to heal over a long period. • Skin or mucosal surfaces should be cleansed.  Closed wounds and aspirates- disinfected with 2% chlorhexidine or 70% alcohol followed by iodine solution (1 to 2% tincture of iodine or a 10% solution of povidine-iodine)  Open wounds should be debrided, and thoroughly rinsed with sterile saline prior to collection. • Viable infected tissue should be sampled, rather than superficial debris. • Swab collection should be avoided if aspirates or biopsy samples can be obtained.
  19. 19. • Containers:  Anaerobe transport vial for small tissues.  Sterile cup for large tissues with nonbacteriostatic saline on a gauze pad to keep moist.  Syringes  Broth culture medium in small sterile snap –top microcentrifuge tubes for FNA  Swabs (ideally, submit two, one for Gram stain and one for culture) • Specimen collection  Cold abscesses- infected material is aspirated with needle and syringe.  Open wounds- superficial area is cleansed with sterile saline. Overlying debris is removed with scalpel and swabs or sponges. Biopsy or curette sample is collected from base or advancing margin of lesion. Anaerobic specimen collector Swabs
  20. 20.  Pus- deepest portion of the lesion or exudate is aspirated with a syringe and needle.  Tissue and biopsy samples- 3- to 4-mm biopsy samples are collected, avoiding necrotic areas. • Label specimen and requisition  Demographic information of the patient.  Type of specimen (deep tissue, superficial tissue, decubitus, catheter site, boil, abscess, cellulitis, aspirate, pus, drainage, surgical incision site, etc.) should be described. Anatomic location (arm, leg, etc.) should be mentioned  Collection time and date should be recorded  Diagnosis, including cause and clinical signs of infection should be mentioned.  Antimicrobial therapy prior to specimen collection should be mentioned Aspiration of pus using needle & syringe
  21. 21. • Specimen transport  Aspirates and tissues should be delivered to the lab within 30 min  Tissues should be kept moist to preserve organism viability  Specimen should not be refrigerated or incubated • Rejection Criteria  Specimens in container with formalin  If numerous squamous epithelial cells are present on Gram stain  Swabs that have been delayed in transit more than 1 h if they are not in some transport system.  For multiple requests (AFB, fungal, bacterial and viral) but little specimen.
  22. 22. Faecal Culture for Aerobic Pathogens • Patient should pass stool into a clean, dry pan. • Transfer at least 5 ml of diarrhoeal stool, 1 g of material or a walnut sized portion of stool. • Container:  Clean, leak proof container with a tight fitting lid or  Buffered glycerol saline  Modified Cary-Blair medium  Stool enrichment broths  Anaerobic transport tube for C. difficile culture • Rectal swabs  Pass the tip of a sterile swab approx. 1 in. beyond the anal sphincter  Carefully rotate the swab to sample the anal crypts, and withdraw the swab • Timing  Submit specimen during acute stage of infection (usually 5 to 7 days)
  23. 23. Anaerobic Culture • The best specimen is obtained by using a needle and syringe. • Tissue samples and biopsy samples are also very good specimens. • The least desirable specimen is collected by swab • Specimen Transport  Tissue samples, biopsy samples, or curettings should be placed into an anaerobic transport device or a sterile tube or petri dish. All this is placed into a sealable plastic bag that generates an anaerobic atmosphere.  If specimens must be collected by swab, swabs should be transported in a tube containing anaerobic transport medium.  Transport containers – Syringe or needle for aspiration, Tube or vial, Swab/plastic jacket system, Bio-bag or plastic pouch.
  24. 24. Fungal Culture • Respiratory specimens- Sputum, tracheal secretions and BAL fluid. • CSF • Blood • Tissue, Bone Marrow and Body Fluids • Skin, Hair and Nail-  Skin scraping.  Nail clippings should be taken from discoloured, dystrophic or brittle parts of nails.  The hairs are removed by plucking them with forceps • Urine • Vaginal secretions • Stool • Eye-  corneal scrapings are taken from base and margins of ulcer aseptically using Kimura’s spatula under 4% xylocaine  Aspiration of hypopyon in keratomycosis.
  25. 25. Collection of Faecal samples for Parasitology • All faecal specimens should be collected prior to the administration of antibiotics or antidiarrhoeal agents. • Use of mineral oil, bismuth, and barium prior to faecal collection should be avoided • Faecal specimen is collected in a clean, wide mouthed container and transfer it to a container with a tight-fitted lid. • Specimen transport  Specimen is transported to the lab as soon as possible, or kept refrigerated until transport is possible. • Rejection criteria  Dried specimens (diarrhoeic, semiformed, or formed). Stool sample container
  26. 26. ANALYTICAL PHASE Blood Culture • Macroscopic signs of growth caused by organisms commonly encountered in blood culture  Haemolysis- Streptococci, Staphylococci, Listeria spp, Clostridia, Bacillus  Turbidity- Aerobic GNB, Staphylococci, Bacteroides.  Gas formation- Aerobic GNB, anaerobes  Pellicle formation- Pseudomonas spp., Bacillus spp., yeast cells  Clotting- Staphylococcus aureus  Visible colonies (“puffballs”)- staphylococcus, streptococci • Microscopy- A thin smear is Gram stained from the broth or agar immediately when suggestive of growth 24h for optimal pt. care. • Subcultured to agar media and biochemical tests based on Gram stain results. Bac T Alert
  27. 27. Lower Respiratory Tract Cultures • Inoculation  Most purulent or most blood–tinged portion of the specimen is selected  In case of bronchoscopy, quantitative cultures are performed on BAL samples  Gram stain is prepared  Using a sterile swab, stick or pipette, inoculate specimen on blood agar, chocolate agar, and MacConckey agar • Incubation  Plates are incubated at 35 to 370C in 5% CO2 for a minimum of 48 h.
  28. 28. • Direct Tests  Gram stain: note cells and bacteria • Culture examination  Plates are observed at 24 h  Plates are incubated for an additional 24 to 48 h, which is useful to detect moulds and slow growing, fastidious gram negative rods  Gram stain result is used as a guide to interpreting the culture.  Presence of inflammatory cells and bacteria is used in deciding on the extent of processing the culture  If the culture does not match the smear results, the smear is reviewed a second time.
  29. 29.  Organisms present in significant amounts are identified, defined as colony types that are not considered part of normal respiratory microbiota and are present in following amounts: Large numbers in the second or greater quadrant of the plate >104 in a BAL sample Any amount of selected pathogens in a patient with cystic fibrosis  Small amount of selected pathogens in the culture that are consistent with an etiological agent seen in the Gram stain associated with inflammatory cells  Colonies in the first quadrant of the plate, only if there is little or no other microbiota on the plate and the smear suggests inflammation  Subcultured on blood agar &/or chocolate agar to obtain isolated colonies for accurate identifications from mixed cultures • Biochemical tests and AST
  30. 30. Urine Cultures • Microscopic and other methods  Gram stain is useful in rapidly determining the type and count of bacteria and cells in urine and should be performed on request.  Detection of pyuria- Detect by either Gram stain or urinalysis, from examination of freshly collected uncentrifuged urine. Pyuria with a WBC count of >10/µl (or >5 per high-power field in conventional urinalysis) has a specificity of 90% for predicting CA-UTI with >105 CFU/ml but a sensitivity of only 37%. • Culture methods  Only streak the blood plate for colony count. Other plates should be streaked in quadrants for isolation of colonies, rather than for colony count, to minimize delays in obtaining isolated colonies and false-negative culture results due to antimicrobial inhibition.  Inoculation methods: Calibrated-loop method, Pipette inoculation method
  31. 31. • Incubation  In ambient air overnight at 35 to 370C  If convenient, blood agar and CNA is incubated in 5% CO2 to enhance growth of gram- positive organisms. • Examination of culture media  Cultures that have been incubated overnight are examined but a final reading is made at 18 h.  For positive cultures, culture media is examined for quantity and morphological type of organisms present  Normal urogenital flora is not identified to the genus or species level  Identification is done always to the species level if oxidase positive and indole positive, since such organisms are pathogens regardless of the count (eg. Aeromonas and Vibrio spp.)
  32. 32. Wound and Soft tissue culture • Inoculation  Anaerobic culture is performed first, preferably in an anaerobic chamber. Incubated immediately.  Smear is always prepared after the culture has been inoculated. • Gram stain  Relative numbers of WBCs, epithelial cells, and bacterial and fungal morphotypes are recorded • Culture workup  Plates and broths are read daily
  33. 33.  Generally up to three microorganisms are identified if any of the following is true:  PMNs are present on direct smear  Specimen was collected from a normally sterile site The specimen was of good quality (eg. No or few epithelial cells present) The organism was seen on direct smear  Any number of microorganisms that only grow on chocolate agar, and not on blood agar ( N. gonorrhoea, Haemophilus, and Francisella) are identified • Microorganism is identified and AST is performed
  34. 34. Faecal Culture for Aerobic Pathogens • Wet preparations are performed for faecal leukocytes from fresh stools. • Culture Methods  Inoculation of media- Swab is rolled over one small area of blood agar and Mac Conckey media and streaked in quadrants for isolated colonies. Use larger amounts of specimen for HEK, XLD and SS agars. • Biochemical tests performed • Salmonella and Shigella somatic (O) antigens are tested for agglutination when the screening biochemical tests fit.
  35. 35. Anaerobic Culture • Macroscopic Examination  Presence of blood, purulence, necrotic tissue, foul odour, and sulphur granules. • Specimen preparation  Purulent specimens are vortexed grossly in the anaerobic transport vial to ensure even distribution of microorganisms.  Bone or tissue are grounded with approximately 1ml of liquid medium to make a thick paste.  Swabs are wringed out in 0.5ml of liquid medium and then treated as a liquid specimen  Large volume of nonpurulent material is centrifuged. Sediment is used to inoculate the media and to prepare the Gram stain • Inoculation of media  Media for anaerobic culture:
  36. 36. Brucella agar with 5% sheep blood supplemented with vitamin K and hemin for isolation of most organisms Phenylethyl alcohol (PEA)- sheep blood agar for the inhibition of enteric and other facultatively anaerobic gram-negative bacilli that may overgrow the anaerobes. PEA also reduces the spreading or swarming characteristic of some anaerobes. Kanamycin-Vancomycin-laked blood agar for selection of pigmented Prevotella and other Bacteroides spp. Bacteroides bile esculin agar Chopped meat broth or THIO (supplemented with Vit K & hemin) Prereduced anaerobically sterilized (PRAS) media
  37. 37. • Inoculation procedure  Prepared specimen is transferred onto the appropriate aerobic and anaerobic media, liquid medium and a slide for Gram stain. • Incubation • Microscopic Examination  Gram staining: Reveals the types and relative numbers of microorganisms and host cells present and serves as a QC measure for the adequacy of anaerobic techniques. Anaerobic Chamber
  38. 38. Fungal Culture • Inoculation of media  The number of media inoculated may be dependent on the specimen type and amount.  Both non-selective (SDA and BHI) and selective media should be included, and specialized media may be required for specimens in which difficult-to-grow etiological agents are suspected. • Incubation  Fungal cultures are incubated at 300C for a minimum of 4weeks.  Plates should be examined daily for first 7 days and at least twice per week thereafter.
  39. 39. Faecal sample examination for Parasites • Macroscopic Examination  Consistency  Surface of faecal specimen for the presence of blood and mucus.  Areas of blood or mucus are sampled for examination of trophic amoeba • Microscopic examination  Saline and iodine wet mounts are prepared  Examined under 10x. Use 40x objective for more detailed study. • Floatation and concentration methods are used for the recovery of all protozoa, eggs, and larvae present. Iodine and Saline mounts
  40. 40. Molecular Techniques Potentially infected individual Sampling: Tissue, blood, urine, respiratory secretion Microbe detection procedures Direct target nucleic acid detection Target nucleic acid amplification and detection In-situ nucleic Nucleic acid In vivo amplification acid detection purification In vitro amplification DNA, RNA 1. PCR Target detection by 2. LCR, NASBA, SDA 1. Dot or slot blot hybridisation Amplified target DNA 2. Restriction fragment length 1. Gel electrophoresis polymorphism (RFLP) analysis 2. Dot or slot blot hybridisation 3. Pulsed-field electrophoresis 3. Southern procedure 4. Solid phase capture &
  41. 41. Antibiotic susceptibility testing • Only isolates producing an infection should be tested. • In most situations a disk diffusion is adequate to guide clinical therapy • Choice of antimicrobial agents and the susceptibility pattern is guided by recommendations of CLSI • Only antimicrobial agents appropriate for the infection should be included in a report. • Antimicrobial agents that do not penetrate into a site should not be reported for organisms isolated from that site • Commercial systems include Vitek which uses a computer assisted analysis of growth in plastic cards to calculate MIC. Kirby-Bauer disk diffusion method
  42. 42. Post-analytical Phase Blood culture • Reporting results  For “No growth cultures”, length of incubation is indicated: “No growth after x days of incubation” for both preliminary and final reports.  Positive cultures: Gram stain results of all positive cultures are reported immediately Number of positive cultures compared with total number of specimens collected for specific patient. Date and time of collection and receipt. Date and time of positive result is reported and whether it was from a catheter draw or a peripheral draw.
  43. 43. For single positive cultures with microorganisms generally considered skin contaminants, only minimal identification is performed and AST is not done. Reported as “one set of two positive. Isolation does not necessarily mean infection. No susceptibility tests performed. Contact laboratory for further information.” • Interpretation  The report of a positive culture generally means that the patient is bacteremic. However, skin microbiota may infect the culture, causing a false-positive result or pseudobacteremia.  Mixed cultures can be present and account for a significant number of bacteremias.  Performance and reporting of AST are critical for timely patient care and increase the chance of appropriate therapy and cure.
  44. 44. Lower Respiratory Tract Cultures • Reporting results  Gram stained smear is reported. Rare (<1 in 20 fields) numbers of bacteria seen in the smear are not reported.  Reported as: “No growth of pathogens or normal upper respiratory tract microorganisms” if there is no growth on any plates  Positive reporting Preliminary and final results are reported as “Isolates consistent with microorganisms encountered in the upper respiratory tract” if no pathogens isolated All pathogens are reported and AST is performed.
  45. 45. • Interpretation  A positive culture with S. pneumoniae or H. influenzae generally indicates infection with that organism, although carriage may lead to false positive results.  A positive culture with a predominant gram-negative rod or S. aureus generally indicates infection with that organism if smear suggests an infectious process involving the corresponding morphology.  A negative culture cannot rule out infection.
  46. 46. Urine Culture • Reporting results  Gram stain results for bacteria & cells per Gram stain are reported.  Negative results If no growth is observed on all media, report “ No growth after 48hrs “  Positive results If only urogenital or skin microbiota is observed, report as such. Mixed cultures are reported with the count in CFU per millilitre, followed by “Multiple bacterial morphotypes present; possible contamination; suggest appropriate recollection, with timely delivery to the laboratory, if clinically indicated”
  47. 47. • Interpretation  A mixed culture in an uncomplicated outpatient population likely indicates contamination.  Low levels (<104/ml) of organisms commonly found on the skin and external and internal genitalia are considered to be contaminants, but in selected circumstances, a count of Enterobacteriacae of 102 CFU/ml or more, especially for Salmonella, can be considered significant.  Performance of AST directly from the urine specimen is not recommended.
  48. 48. Wound and soft tissue infections • Reporting results  Report gram stain results as soon as possible, generally within 1 h for specimens from critical sites.  Report all negative cultures as “ no growth in-------days”  Report individually those organisms that are always considered pathogenic with enumeration, using preliminary identification initially and the genus and species as the final identification.  Due to their known virulence factors, indicate the presence of the following species: Beta-haemolytic Streptococci, S. aureus, P. aeruginosa, Clostridium perfringens, Pigmented anaerobes, Bacteroides spp., Mixed anaerobes.  Report AST on gram-negative rods, enterococci, or S. aureus  When multiple morphologies are present, report with minimal identification  Additionally, if mixed microbiota are cultured with no predominant microorganism, report as GI, oronasal, skin, or genital microbiota.
  49. 49. • Interpretation  Performance of AST is not indicated in cases of mixed microbiota indicative of infection of the abdominal cavity with bowel contents. Treatment should include broad-spectrum coverage for normal intestinal microbiota.  Use of the Gram stain can improve the accuracy of evaluating the importance of each potential pathogen. Organism present in the Gram stain of an appropriately collected specimen correlate with ≥105 organisms per g of tissue.  Microbial load in an acute wound can predict delayed healing or infection.  Many wound infections are polymicrobic, and the isolation of an organism in culture may or may not correlate with infection of the wound.
  50. 50. Faecal culture for aerobic pathogens • Reporting results  If gram negative enteric microbiota are not present in the cultures, add a comment “no normal enteric gram-negative rods isolated”.  Positive cultures: report presumptive presence of any enteric pathogens with or without quantitation. Report the pathogen with the preliminary designation as “probable” until the identification is confirmed by both biochemical testing and serology.  Report AST on Shigella, Aeromonas, Plesiomonas, Edwardsiella, Vibrio, Yersinia and selected cases of Salmonella.  Generally report only ampicillin, cotrimoxazole, and a quinolone. • Interpretation  The isolation of a stool pathogen may not identify the cause of the disease. Eg. Salmonella is present in the carrier state, without disease.
  51. 51. Fungal culture • Reporting and interpretation of yeast: Nitrate assimilation test for yeast speciation Sporulation Carbohydrate fermentation tests • Reporting and interpretation of mould: Reporting the growth of a mould as soon as such growth is evident but before identification structures are formed is important where immunocompromised patients are concerned but is not normally necessary in cases of subcutaneous infection of immunocompetent patients.
  52. 52. Parasitic infestations • To ensure the recovery of parasitic organisms that are passed intermittently and in fluctuating numbers, the examination of a minimum of three specimens collected over a 7- to 10- day period is recommended. • Report the presence of adult helminths .Morphology and size are usually adequate for identification of pinworm and Ascaris adults and tapeworm proglottids. • Report the presence of blood on or in the faecal sample
  53. 53. References • Clinical Microbiology Procedure Handbook by Henry D. Isenberg, 2nd edition • Koneman’s Colour Atlas & Textbook of Diagnostic Microbiology, 6th edition • Mackie Mc Cartney Practical Medical Microbiology, 14th edition