DIPLOMA IN MEDICAL LABORATORY TECHNOLOGY

LEARNING OUTCOME At the end of this lecture, students will be able to: 1.State the workflow at the Respiratory bench in Medical Microbiology Laboratory 2.Describe the standard operating procedure for Respiratory bench in Medical Microbiology Laboratory

Specimen types
The following samples are processed at the respiratory bench: Sputum Tracheal secretions/aspirates Nasopharyngeal secretions Throat swab Nasal swab Pernasal swab Bronchiol alveolar lavage Pleural fluid Tissues lung, tonsils Sinus aspirates Tracheostomy swab

Specimen processing
Routine sputum specimens Microscopic examination Smear is made from a purulent portion of the sputum, allowed to dry, fixed and gram stained. This is examined under low power field. The number of pus cell and epithelial cells are recorded per low power field(lpf) Under oil immersion predominant organism are noted.

Specimen processing (cont)

Epithelial cells (EC) < 10/lpf 10-25/lpf <25/lpf >25/lpf Pus cells (PC) >25/lpf >25/lpf <25/lpf comments Good specimen. Accepted for full processing Process predominant pathogens only Lecturer to decide whether to fully process pathogen Reject specimen as inadequate for culture and make a request for another specimen

Specimen processing (cont) Routine sputum specimens (cont) Culture A purulent portion of sputum is inoculated onto plate of BA, CA, Mac All the plates are incubated at 35oC for 24-48 hours.

Specimen processing (cont)

Tracheal secretions / aspirates and nasopharyngeal secretions Procedure is similar to that for sputum except: Specimen is not rejected based on the number of epithelial cells seen Smears are gram stained and the amount of pus cells and epithelial cells are noted (per lpf) and also the predominant type organism

Specimen processing (cont) Throat / nasal swabs Microscopic examination: This is not done for routine specimens Direct smear is done in cases of Vincents angina

Specimen processing (cont) Throat / nasal swabs (cont) Culture: Inoculate swab onto plate of BA, CA, Mac All plates are incubated at 35oC for 24-48 hours

Specimen processing (cont)

Pleural fluid Microscopic examination: Smear is made and gram stained Examine under oil immersion lens and note pus cells and organisms Inform ward if organism are seen Direct antibiotic susceptibility test if organisms noted microscopically Check with blood culture if necessary

Specimen processing (cont)

Pleural fluid (cont) Culture: Inoculate swab onto BA, CA, Mac FAA Thioglycollate broth incubated at 35oC for 18-24 hours. For anaerobic cultures incubate for 48 hours Subculture from thioglycollate broth after overnight incubation onto BA, CA, Mac, FAA+Metronidazole (MZ) disc

Specimen processing (cont) Tissues and other fluids Microscopic examination: Smear is made and gram stained Examine under oil immersion lens and note pus cells and organisms Culture: Same as above

Specimen processing (cont) Bronchial aspiration/washing/lavage Microscopic examination as in tissues and other fluids Smear is made and gram stained Examine under oil immersion lens and note pus cells and organisms

Specimen processing (cont)

Bronchial aspiration/washing/lavage (cont) Culture: Using a standard loop technique, inoculate onto: BA, CA, Mac FAA Thioglycollate broth incubated at 35oC for 18-24 hours. For anaerobic cultures incubate for 48 hours Subculture from thioglycollate broth after overnight incubation onto FAA+MZ disc

Specimen processing (cont)

Bronchial aspiration/washing/lavage (cont) Interpretation: CFU/ml <50 colonies 50 200 colonies 200 colonies or more Bacteria density/ml <104 104 - 105 >105

Specimen processing (cont) Pernasal swab culture: Inoculate onto Charcoal BA Charcoal BA with cephalexin Incubate at 35oC aerobically in a moist chamber for 7 days and examine daily for any growth

Processing of pathogens General Plates are examine after 18-24 hours and the pathogens are picked out from the normal flora and identified Inoculate pathogen/s onto appropriate plates (e.g. MH,MHBA,CA) for antibiotic susceptibility tests Perform necessary test (biochemical, bile, DNAse) to identify pathogen/s

Processing of pathogens (cont) General (cont) Record antibiotic susceptibility test result If three or more morphologixal types of GNR are isolated, report as mixed growth of coliforms Plates with no growth are kept for a further 18-24 hours

Processing of pathogens (cont) Throat / nasal swab For routine tonsilitis/pharyngitis cases, only haemolytic streptococci are processed. Inoculate pathogen/s onto appropriate plates for susceptibility tests Plates with no growth are kept for a further 18-24 hours

Processing of pathogens (cont) Pleural fluid Plates are examined after 18-24 hours Any growth is gram stain and correlated with direct smear Inoculate pathogen/s onto appropriate plates (MH,MHBA,CA) for antibiotic susceptibility test

Processing of pathogens (cont) Pleural fluid (cont) Perform necessary test (biocemical test, bile, DNAse) to identify pathogen/s Record antibiotic susceptibility test result Plates with no growth are kept for a further 18-24 hours After 48 hours read anaerobic plates

Identification of Bordetella pertussis Specimen type pernasal swab Specimen collection Pernasal swab should be collected using wire swab B.pertussis is very susceptible to drying Therefore, specimen should be transported in Amies transport medium immediately after collection

Identification of Bordetella pertussis (cont) Culture Charcoal BA Charcoal BA with cephalexin Incubate at 35oC aerobically in a moist chamber for 7 days

Identification of Bordetella pertussis (cont)

Colonial morphology B. pertussis Convex, grey, smooth, very shiny(mercury drop) Variable in size and may run together. They require 3 or more days to appear B. parapertussis Similar to B. pertussis, but greyer and less domed, grow more rapidly than B. pertussis colonies Gram stain Gram negative coccobacilli

Bordetella pertussis

Identification of Bordetella pertussis (cont) Agglutination test Slide agglutination test with antisera B. pertussis and B. parapertussis Antibiotic susceptibility testing Not necessary. Erythromycin is the drug of choice

Identification of Corynebacterium diptheriae Specimen type Throat swab Nasal swab Culture BA, CA, Mac, (5% CO2) Loefflers medium and Potasium tellurite BA (aerobically in 35O for 5 days)

Identification of Corynebacterium diptheriae (cont)

Staining Gram staining Pleomorphic gram positive rods in angular arrangements (like chinese characters) Alberts stain To demonstrate metachromatic granules The rods have beaded appearance that consists of polyphosphate granules

Corynebacterium diptheriae

Identification of Corynebacterium diptheriae (cont)

Colonial morphology PTBA Gunmetal gray colony and 1 to 3mm in size Biotype intermidius appear small and flat Biotype mitis and gravis will appear larger, convex, smooth or wrinkled BA Biotype intermidius smaller, flat, creamy, no hemolysis Biotype mitis and gravis larger, convex with weak beta hemolysis

throat culture on blood agar and tellurite medium. black colonies on tellurite medium are Corynebacterium diphtheriae

Identification of Corynebacterium diptheriae (cont)

In-vitro toxicity test (Elekss test) A strip of sterile paper saturated with 1ml of diphtheria antitoxin (100 U/ml) is placed in the center of melted agar base before it sets completely Streak the plate with the test strains as well as control positive and negative at right angles to the strip Incubate at 37OC and examine at 24 and 48 hours Positive reaction shows toxin antitoxin lines at 45O angles to the line of inoculum

Elekss test

REFERENCES Monica Cheesbrough. District Lab Practice in Tropical Countries Part 2. Cambridge, 2007. Irving, Boswell & Alaaldeen. Medical Microbiology. Taylor & Francis, 2005. Standard Operating procedure for Medical Microbiology Laboratory University Malaya Medical Centre