MEDT 477 - Clinical Microbiology Practicum

Updated Nov, 2007

 
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SPECIMEN PROCESSING/SAFETY
The first step in the accurate diagnosis of infectious diseases is the proper collection and handling of specimens. Specimen handling involves the following steps: proper specimen collection, appropriate specimen containers, accurate labeling of forms and containers, timeliness of transport, special procedures based on suspected pathogens, proper handling in laboratory with respect to selection of growth media, stains, incubation times and temperatures and reporting results. Thorough knowledge of safety procedures is essential before performing any duties in the clinical laboratory which might be hazardous to personnel.

Upon completion of this course, the student will be able to:

  1. Explain the importance of safe and proper collection and transport of specimens.
  2. Describe criteria for evaluating specimen quality and corrective actions to be taken to resolve problems.
  3. Log in and label specimens accurately.
  4. Fill in daily worksheets according to laboratory protocol.
  5. Report and call results according to laboratory protocol.
  6. Identify which specimen is appropriate for the culture requested, e.g. anaerobic cultures are not performed on stool specimens.
  7. Enter patient information and culture results into Laboratory Information System (LIS) if applicable.
  8. Demonstrate proper technique in preparing smears for direct microscopic examination according to laboratory protocol.
  9. Plate specimens on appropriate media, selecting proper atmospheric conditions, incubation times and temperatures according to laboratory protocol.
  10. Explain the rationale for use of the biological safety hood.
  11. Demonstrate safe techniques in handling and disposal of infectious materials according to laboratory protocol.
  12. List and describe the action of some common disinfectants and evaluate which disinfectant would be appropriate in any given situation.
  13. Explain the function of an autoclave and state the pressure (p.s.i.), temperature, and time most commonly used for sterilization of media and contaminated laboratory waste.
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QUALITY CONTROL
The purpose of quality control in the microbiology department is to ensure that the final product has an acceptable degree of conformity within previously established tolerance limits. It is only by constant self evaluation of the laboratory's performance by thorough monitoring of reagents, equipment, culture media, and proficiency that a high level of expertise and accuracy can be attained.

Upon completion of this course, the student will be able to:

  1. Explain the rationale for a quality control program in the clinical microbiology laboratory.
  2. List specific areas which require surveillance.
  3. Discuss how the performance of specific media is evaluated and how often.
  4. Describe the components of the media allowing for the selective and differential characteristics.
  5. Evaluate reactivity of media using proper quality control procedures.
  6. Discuss the necessity of keeping written records of all quality control, of periodic review of surveillance records, and of corrective action documentation.
  7. Discuss accreditation requirements with regard to thermometers, and CO2 incubators and sterilization procedures (i.e. autoclaving).
  8. Discuss use of proficiency testing.
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BLOOD CULTURES
Blood is one of the most important specimens received in the microbiology laboratory. Quick and accurate reporting of preliminary and final results can have lifesaving effects for the patient. The blood specimen must be handled correctly from collection to final subculturing for the results to be valid.

Upon completion of this course, the student will be able to:

  1. Explain the proper procedures for collection of blood cultures, including recommended number of cultures and volume of blood collected.
  2. Select proper incubation temperatures, times, and growth requirements for blood cultures.
  3. List the major components of the blood culture media and explain the rationale for their use.
  4. Explain the rationale for blind subcultures.
  5. Recognize visual signs of a positive blood culture.
  6. Explain the procedure and principle of automated blood culture detection systems used in the affiliate’s laboratory.
  7. Select and perform procedures for the handling of positive cultures.
  8. List those organisms most frequently isolated from blood cultures.
  9. Differentiate possible contaminants from pathogens isolated from blood cultures.
  10. Discuss the significance of a positive blood culture.
  11. Perform and interpret blood culture Gram stains.
  12. Work up a positive blood culture, from proper subculturing to biochemical identification, and susceptibility testing.
  13. Explain the affiliate's procedure for reporting positive blood cultures.
  14. Correlate positive blood cultures with positive cultures from other sites for the same patient.
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RESPIRATORY CULTURES
Respiratory cultures include all routine cultures of mouth, throat, nose, nasopharynx, ear, sputum, endotracheal tube aspirates and transtracheal aspirates. These cultures are difficult to interpret, but with the help of a Gram stain, potential pathogens and contaminants may be recognized and differentiated from those organisms representing "normal flora."

Upon completion of this course, the student will be able to:

  1. List examples of respiratory specimens.
  2. Determine acceptability of sputum for culture by evaluating the Gram stain.
  3. Perform, interpret, and evaluate direct Gram stains of respiratory specimens.
  4. List organisms which may be considered normal oropharyngeal flora.
  5. List common pathogens from each respiratory source.
  6. Explain the rationale for selection and use of the primary plating media for each respiratory specimen.
  7. Plate all types of respiratory specimens to appropriate media.
  8. Select incubation atmosphere, temperature, and time for each culture.
  9. Describe colony morphology and growth characteristics of normal flora and pathogens.
  10. Differentiate pathogens from normal flora in respiratory cultures.
  11. Quantitate potential pathogens in respiratory cultures.
  12. Correlate direct specimen Gram stains with culture results.
  13. Determine appropriate biochemical tests or adjunct procedures required for identification of significant isolates, e.g. X and V factor requirements for Haemophilus spp.
  14. Perform antimicrobial susceptibility tests as required and evaluate their appropriateness with regard to organism isolation.
  15. Explain the rationale for use of the transtracheal aspiration for collection of respiratory specimens.
  16. Explain the principle of the agglutination test for Streptococcus pyogenes and other beta-hemolytic streptococci.
  17. Correlate culture results with clinical history and presentation.
  18. Evaluate respiratory cultures for epidemiological problems.
  19. Discuss the special procedures required for the isolation of Nocardia, Actinomyces, Bordetella, fungi, Mycoplasma, Legionella, and viruses from the respiratory tract.
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STOOL CULTURES
In most clinical laboratories, a stool specimen submitted for routine culture is examined for Salmonella, Shigella, and Campylobacter. Proper media selection and a rapid identification procedure are important tools for the microbiologist. The physician must alert the laboratory to look for other suspected pathogens. These pathogens might include Yersinia, Vibrio, Aeromonas hydrophila, Pleisiomonas shigelloides, staphylococci, Clostridium difficile, enterohemorrhagic E. coli, and yeast.

The student will complete assigned readings in procedure manuals, handouts, and reference books. For satisfactory completion of this unit, the student must achieve each of the following objectives to the satisfaction of the instructor:

  1. List the major bacterial pathogens found in stools.
  2. Explain the rationale for the differential media used to plate stools.
  3. Explain the growth requirements and selective and differential media necessary for the isolation of:
  4. Prepare, stain, and interpret direct stool smears for leukocytes.
  5. Recognize suspicious colonies of each of the possible enteric pathogens on all differential and selective media.
  6. Recognize colonies of normal fecal organisms on all types of media.
  7. Select and inoculate the proper biochemicals to identify all pathogens, including serotyping.
  8. Perform slide agglutination tests for serogrouping Salmonella and Shigella.
  9. Explain what should be done if the organism does not type because of the presence of Vi antigen.
  10. Explain the rationale for Clostridium difficile culture and/or toxin assay.
  11. Explain the rationale for screening stools for non-lactose fermenters.
  12. Explain the rationale for screening stools for oxidase positive gram-negative rods.
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URINE CULTURES
Urinary tract infection is one of the most common bacterial diseases. The urine specimen is easy to obtain and can be collected in several different ways. A quantitative culture result can help diagnose significant bacteriuria and is performed by most laboratories.

Upon completion of this course, the student will be able to:

  1. List methods for obtaining urine specimens suitable for microbiological analysis.
  2. Perform and interpret direct Gram stains from urine.
  3. Plate urine specimens on the appropriate media.
  4. Explain the rationale for the use of each primary plating media.
  5. Select proper incubation temperatures, times, and atmosphere conditions.
  6. Quantitate urine cultures plated with calibrated loops, e.g. 0.01 and 0.001 μL.
  7. Perform quality control on calibration loop.
  8. Discuss the significance of colony counts as related to the methods of urine collection.
  9. List organisms that are often urine contaminants.
  10. Differentiate grossly contaminated specimens from those which should be considered to likely contain pathogens.
  11. Select media to isolate overgrown organisms on subculture.
  12. Determine biochemical tests for the identification of isolates.
  13. Perform antimicrobial susceptibility testing as required.
  14. Evaluate colony counts that do not correspond on both plates from the same urine, and explain possible discrepancies.
  15. Discuss urine screening methods available.
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ANAEROBE CULTURES
Anaerobic bacteria are found in a variety of clinical specimens and often are involved in clinically significant infections. Because antimicrobial therapy is often dependent on the species involved, it is important for the laboratory to isolate and identify these etiologic agents.

Upon completion of this course, the student will be able to:

  1. Characterize the following groups of bacteria in relation to their oxygen requirements:
  2. List anaerobes found as normal flora in:
  3. List the types of infectious diseases commonly caused by anaerobes or their toxins.
  4. List the types of specimens that are appropriate for the diagnosis of specific anaerobic infections.
  5. List specimens that are usually inappropriate for anaerobic culture.
  6. Explain the techniques and importance of proper collection and handling of anaerobic specimens.
  7. Discuss the rationale for the use of:
  8. List the key components of primary plating media and the rationale for their use in culturing anaerobic bacteria.
  9. Discuss the rationale for the aerotolerance test on all anaerobic isolates.
  10. Select incubation temperature and time for each specimen.
  11. Describe the anaerobic incubation systems in use at the affiliate microbiology laboratory:
  12. Describe the cultural and microscopic morphologies of selected anaerobes such as:
  13. Correlate Gram stains of direct smears or enrichment broths with culture results.
  14. List medically important anaerobic genera of:
  15. Give rationale for differentiating the Bacteroides fragilis group from other anaerobic gram-negative rods.
  16. Perform identification of anaerobic isolates with one or more of the following systems:
  17. Explain the principles of gas-liquid chromatography.
  18. Discuss problems encountered in performing anaerobic antimicrobial susceptibilities.
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MISCELLANEOUS
Miscellaneous cultures include all routine cultures of tissue, bone, cerebrospinal fluid and other fluids, wounds, genital tract, catheter tips, etc. Miscellaneous cultures are all routine cultures other than respiratory specimens, stools, anaerobic cultures, urines and blood cultures, which have individual unit objectives.

Upon completion of this course, the student will be able to:

  1. List examples of miscellaneous specimens.
  2. List common isolates and pathogens from each miscellaneous source.
  3. List sites normally considered sterile vs. areas that may have normal flora.
  4. Discuss the predisposing factors that allow normal flora to cause disease.
  5. Explain the rationale for use of each of the primary plating media for each miscellaneous specimen.
  6. Plate specimens to appropriate media.
  7. Select proper incubation temperatures and times for each specimen.
  8. Perform and interpret direct Gram stains of miscellaneous specimens.
  9. Interpret colony morphology and growth characteristics of organisms commonly isolated from miscellaneous specimens.
  10. Determine biochemical tests for identification of isolates.
  11. Perform antimicrobial susceptibility testing as required.
  12. Determine appropriate sensitivity patterns.
  13. Select proper media for subculture.
  14. Differentiate commensals from potential pathogens.
  15. Evaluate culture results with respect to type of specimen submitted.
  16. Correlate direct specimen Gram stains with culture results.
  17. Correlate culture results with clinical history and presentation.
  18. Evaluate miscellaneous cultures for epidemiological problems.
  19. Perform and interpret a wet preparation for trichomonads.
  20. Explain the procedure for darkfield examination for spirochetes of syphilis.
  21. Observe and explain molecular testing methodss used for the diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae infections.
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SUSCEPTIBILITY TESTING
One of the important services offered by the microbiology laboratory to the attending physician is the determination of the antimicrobial susceptibility pattern of bacterial pathogens by standardized methods.

Upon completion of this course, the student will be able to:

  1. Name the general test methods available in antimicrobial susceptibility testing.
  2. For the Kirby-Bauer (K-B) method:
  3.  Discuss the effects of the following factors on the K-B method.
  4. Select the appropriate antimicrobial agents for testing:
  5. Describe the storage requirements for antimicrobial disks.
  6. List the major groups of antimicrobial agents and give examples of each.
  7. Define beta-lactam and penicillinase resistant antibiotics and give example of each.
  8. List antimicrobial agents which act on the bacterial cell wall.
  9. List antimicrobial agents which inhibit protein synthesis.
  10. List antimicrobial agents which are effective against Mycobacterium sp.
  11. List antimicrobial agents which penetrate the blood brain barrier.
  12. List antimicrobial agents used primarily for urinary tract infections.
  13. Explain the procedure for the serum bactericidal (Schlichter) test.
  14. List the attributes of an ideal antimicrobial agent.
  15. Discuss the indications for direct susceptibility tests on clinical materials.
  16. For the agar dilution minimal inhibitory concentration (MIC) method:
  17. For the brothmicrodilution MIC method:
  18. For quality control of susceptibility testing:
  19. Correlate MIC to blood levels and explain therapeutic considerations in the selection of appropriate antimicrobial therapy.
  20. Explain the principle and rationale of the beta-lactamase test.
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MYCOLOGY
Since symptoms of fungal infection are often nonspecific and appear similar to other infections, it is especially important for the physician to gather all pertinent information regarding the patient's history, background, and whereabouts. This information must be communicated to the microbiology lab so that proper specimens will be collected and the correct media inoculated. Complete identification of yeasts and fungi is especially important today due to the increased number of immunocompromised patients. It has become harder for the physician and microbiologist to distinguish between "pathogen" and "contaminant" as many organisms formerly termed "contaminants" have now been shown to be opportunistic pathogens.

Upon completion of this course, the student will be able to:

  1. Describe safety precautions for handling fungal cultures.
  2. Explain the rationale for use of selective fungal media.
  3. Select proper incubation temperatures and times for fungus cultures.
  4. Properly inoculate fungus specimens.
  5. Describe the principles and perform and interpret the procedures for direct microscopic detection of fungi in specimens and the identification of fungi in cultures.
  6. List fungi frequently isolated as contaminants.
  7. List the important dimorphic fungi and associated diseases.
  8. Explain the rationale for complete identification of yeasts and fungi.
  9. Describe methods for identifying yeasts, including commercial methods.
  10. Describe the pathogenic Actinomycetes, including related organisms and diseases.
  11. Describe methods for identifying moulds.
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MYCOBACTERIOLOGY
Mycobacteria are acid-fast aerobic bacilli which grow very slowly compared to other aerobic bacteria. For this reason, specimens which may contain other bacteria or normal flora must be "decontaminated" prior to plating so that overgrowth doesn't occur. Direct and concentrated smears for acid fast bacilli, therefore, may provide for a rapid presumptive diagnosis of Mycobacterium tuberculosis. Actual colonies of Mycobacterium tuberculosis may take six to eight weeks for growth and identification.

Upon completion of this course, the student will be able to:

  1. List safety measures that must be employed in processing acid-fast bacilli (AFB) cultures and explain why they are so important.
  2. Discuss the appropriate digestion/decontamination procedure as outlined in the procedure manual for contaminated specimens.
  3. Select and perform the appropriate digestion/decontamination procedure as outlined in the procedure manual for contaminated specimens.
  4. Explain the action of each component in the digestion/decontamination procedure.
  5. Identify specimens which do not require decontamination.
  6. Perform direct plating of cultures, selecting appropriate media, incubation times and temperatures.
  7. Compare the media, incubation times, incubation temperatures, and growth conditions for the growth of the most commonly isolated mycobacteria.
  8. Discuss the necessity for a gastric lavage and explain the procedure for processing a lavage for AFB.
  9. Perform both the Ziehl-Neelsen (or Kinyoun's) and fluorochrome stains for mycobacteria, on direct and concentrated smears.
  10. Define Runyoun's classification of mycobacteria and give examples of species in each Runyon group.
  11. Describe DNA probe technology for identification of mycobacteria.
  12. Examine AFB cultures and select potential colonies of mycobacteria.
  13. Correctly identify Mycobacterium tuberculosis by accurately interpreting the following test results:
  14. Explain the principle of using high pressure liquid chromotography (HPLC) for the identification of Mycobacterium sp.
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PARASITOLOGY
The accurate identification of ova and parasites requires expertise. It is important for the clinical laboratory scientists and the physician to communicate patient's history (i.e. immunosuppression) or travel to areas of the world where parasites are endermic.

Upon completion of this course, the student will be able to:

  1. Describe appropriate collection techniques for stool specimens including timing, number of specimens and special procedures.
  2. List specimens other than stool, which may be submitted and parasites which may be present in these specimens.
  3. List substances which would interfere with a fecal exam for parasites.
  4. List several methods of stool preservation and the advantages and disadvantages of each.
  5. Explain the flotation and formalin-ethyl acetate sedimentation procedures for examining stools.
  6. Perform the formalin-ethyl acetate concentration.
  7. Examine direct saline wet mounts of watery stools for parasites.
  8. Examine iodine wet mounts of stool concentrates for parasites.
  9. Recognize microscopically the common human parasites.
  10. Explain the need for the ocular micrometer when doing parasitology.
  11. Calibrate and use the ocular micrometer.
  12. Explain and describe common methods of staining parasites.
  13. Recognize by morphology each stage of the life cycle of Plasmodium vivax, P. falciparum, P. malariae and P. ovale.
  14. Explain methods of examining specimens for Cryptosporidium, including the acid-fast stain and fluorescent stain.
  15. Perform and interpret an acid fast stain for Cryptosporidium.
  16. Prepare, stain, and interpret stool smears using the trichrome stain.
  17. Describe the methods used to prepare and examine blood smears for parasites.
  18. Describe the morphology of Babesia sp.
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VIROLOGY
The commercial availability of rapid detection systems for the diagnosis of viral infections makes this aspect of microbiology increasingly accessible to most clinical laboratories. A laboratory may elect to limit its services to the isolation of only herpes simplex virus by utilizing one of the several available rapid systems, or may elect to give a slightly more extensive service by employing a limited but select number of cell lines to detect the presence of viruses due to their cytopathic effect (CPE) and then submitting only those cultures showing positive CPE to reference laboratories for complete identification. Chlamydia trachomatis, an obligate intracellular bacterium, requires cell culture for growth and is therefore included in this unit. These objectives can only be completed at affiliate locations that offer virology services.

Upon completion of this course, the student will be able to:

  1. Define and differentiate primary, semi-continuous (finite), and continuous cell lines.
  2. List tissue cultures used for the rapid isolation of herpes simplex virus.
  3. Give a general description of the media and key supplements used to propagate tissue cultures.
  4. List the types of specimens suitable for viral cultures.
  5. Explain the procedures for the proper handling, transport and storage of viral specimens.
  6. Describe the cytopathic effects produced by different viruses.
  7. List molecular diagnostic methods currently used in the affiliate's laboratory.
  8. Discuss methods used for the diagnosis of Chlamydia trachomatis infection.
  9. List the cell culture line most commonly used for the isolation of Chlamydia trachomatis.
  10. Discuss the staining methods for the detection of Chlamydia in cell cultures.
  11. Describe the appearance of Chlamydia trachomatis in

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MOLECULAR DIAGNOSTIC and IMMUNOLOGIC ASSAYS

Upon completion of this unit, the student will be able to:
  1. At each affiliate laboratory, identify the molecular diagnostic assays utilized.
  2. List the assays and identify the analyte in the affiliate's laboratory that utilize diagnostic immunologic techniques.


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