Department of Laboratory Medicine and Pathology

Clinical Laboratory Scientist (CLS)/ Medical Technologist Program(MT)

Pre-Clinical Microbiology Competencies

INTRODUCTION
The clinical microbiology rotation at HCMC consists of a total of 8 weeks of self-instructional and practical laboratory experiences. The various disciplines studied, including clinical microbiology, are: anaerobic microbiology, virology, mycology, mycobacteriology, and parasitology.

During the course of the rotation the student will be directly involved in the daily procedures of a large urban hospital's clinical microbiology department. Our personnel routinely evaluate an average of 7,500 specimens per month from a variety of body sites and a diverse patient population. We also perform identification in specialized areas of clinical microbiology, such as parasitology, mycology, anaerobic bacteriology, virology, and mycobacteriology.
In order for the students to attain career-entry competencies, we ask that they come to us with specific "pre-clinical" competencies.

Following is an outline of major academic areas we wish to be covered before entering the 9 month program.

Attached is an expansion of that outline that includes listings of specific objectives to be met for each category before the beginning of the 9 month clinical study and references that we feel will be of value in teaching and review.

NOTE: Each student is required to write out answers for all of the objectives. If the objective involves the performance of some activity, describe what you did in your college courses in order to meet that objective. These answers must be turned in to the HCMC program director two weeks before the start of the internship. They will be evaluated for completeness by the HCMC education techs. Students will be required to review and know this material when they enter each of the rotations.

Diane K. Berke, MT (ASCP)
Medical Technologist Specialist
Clinical Microbiology/Parasitology
Reviewed 2-01, 1-02, 12-02, 1-04, 1-07, 1-08

I. MEDIA
The isolation of bacteria from clinical material as etiologic agents of infection is dependent upon selecting media suitable for bacterial growth.

1. Define enriched, enrichment, selective, and differential.

2. List the following media and identify if it is enriched, enrichment, selective or differential, and name at least one organism that will grow on this media.

II. TECHNIQUE
1. Describe aseptic technique.

2. Draw the standard isolation streaking pattern for inoculating an agar plate.

3. Describe "safe" techniques (Universal Blood and Body Fluid Precautions) when working with microbiologic materials.

4. Describe appropriate temperature and atmospheric conditions to incubate bacteriologic

cultures.

5. Describe the difference between disinfection and sterilization.

III. STAINS
The most important stain used in the clinical microbiology lab is the Gram stain. The

acid-fast stain is also an important tool in alerting physicians to the possible presence of Mycobacterium sp. in patient specimens.

1. Write a short discussion of bacterial cell wall content in relation to the Gram stain reaction of the bacterial cell.

2. Write a description of how to perform a Gram stain.

3. Describe basic cellular components that may be found in a Gram stain of clinical material, such as PMN's, mononuclear WBC's, epithelial cells, tissue cells.

4. Describe the steps in a Ziehl-Neelson acid fast stain and an auramine-rhodamine fluorescent stain.

IV. BIOCHEMICAL TESTS
Write a short explanation of each of the following tests (principles, reactions, media used, reagents) and list an organism or group of organisms that the test is used to identify. The test listings are common and should be practical at the university level.

V. NORMAL AND PATHOGENIC FLORA
List at least 2 organisms classified as pathogens from the following sites. If the site is normally "sterile" you will indicate it as "sterile."

VI. ORGANISMS
A. Staphylococci

1. List the growth and media requirements of Staphylococcus aureus, epidermidis, and saprophyticus. 2. Discuss the identification of Staphylococcus on the basis of:   a) Gram stain morphology b) glucose utilization c) hemolytic activity d) catalase reaction e) coagulase test or rapid latex test 3. List a test that will separate and identify Staph aureus from Staph epidermidis. 4. Write a short discussion of the pathogenicity of Staph aureus in relation to specific toxin and enzyme production. 5. Write an explanation of the relationship of coagulase, capsular polysaccharide and beta lactamase production to the virulence of Staph. 6. List at least 5 diseases caused by Staphylococci and a drug of choice used in treating staphylococcal infections. 7. List at least 1 biochemical characteristic that separates the Staph from the Micrococci and Aerococcus. 8. List at least 3 clinical sites and the species of Staph that can be found as "normal flora." 9. Discuss the pathogenicity of Staphylococcus saprophyticus and a test to confirm its identification. 10. Discuss the pathogenicity of Staphylococci in terms of cell wall composition and toxin and enzyme production. In your discussion you should be able to define: exotoxin enterotoxin exfoliative toxin coagulase proteinase TSS toxin leukocidin penicillinase 11. Discuss the mechanism of antibiotic resistance in staphylococci, ie: MRSA (methicillin resistant Staph aureus), VISA (vancomycin intermediate Staph aureus) and how these organisms are detected in the Microbiology Lab.

B. Streptococci

1. List the growth and media requirements of Streptococci. 2. Define the differences between alpha, beta, gamma, and alpha-prime hemolysis. 3. Discuss the criteria used in categorizing streptococcus by Lancefield grouping. 4. Discuss the use of the following media and tests and how they are used to identify streptococci (also see section IV): FA Bile solubility 6.5% NaCl broth CAMP test Optochin Esculin medium PYR test LAP test 5. Discuss the pathogenicity and host immunity of the following Strep, identifying at least one disease process with each: S. pneumoniae S. viridans S. agalactiae Enterococcus group S. pyogenes 6. Describe the streptococci on the basis of: Gram stain Colony morphology Hemolytic activity Catalase reaction 7. List at least one test that will separate the strep from the staph. 8. Discuss the virulence of certain streptococci in relationship to the presence of antigenic substances and enzymes or toxins. You should use and define the following terms in your discussion: C carbohydrate M protein Erythrogenic toxins Hemolysins Hyaluronidase 9. Define streptolysin O and S. 10. List 2 post-Streptococcal diseases. 11. Define VRE and VSE as related to the enterococci.

C. Neisseria

1. Discuss media, growth requirements and common site of isolation of the Neisseria/Moraxella. 2. Discuss the characteristic morphology and oxidase reaction of the Neisseria species. 3. Discuss the function and composition of Thayer-Martin agar. 4. Discuss the 4 main antigenic groups of Neisseria meningitidis and how they are classified. 5. Discuss test used to speciate Neisseria and Moraxella. 6. Discuss host immunity to N. gonorrhoeae and N. meningitidis. 7. List the species of Neisseria implicated in meningitis and a venereal disease and the specimens of choice to collect for isolation. 8. Define the term "carrier" as associated with N. meningitidis. 9. Discuss the significance of beta-lactamase testing of Neisseria in determining drug therapy. 10. List at least one "non-cultural" method of identifying an infection with Neisseria gonorrhoeae.

D. Yeasts

1. Describe yeast and/or arthrospores on a Gram stain. 2. List a yeast commonly associated with meningitis in the compromised host. 3. Describe the germ tube test and its use. 4. Describe the cryptococcal antigen test and its use. 5. List at least 3 common laboratory media (from section I) that support the growth of yeast. 6. List at least 1 mycologic media that supports the growth of yeast. 7. List at least 3 sites where C. albicans is endogenous and 3 sites where it may be pathogenic.

E. Enterobacteriaceae

1. Define the Enterobacteriaceae on the basis of: Gram stain Oxidase reaction Growth requirements Motility Nitrate reduction Glucose utilization Gas production 2. List important biochemical characteristics and implicated pathogenicity of the following: E. coli 0157:H-7 Enterobacter Escherichia Klebsiella Morganella Proteus Providencia Salmonella Serratia Shigella Yersinia 3. Define K, Vi, O, and H antigens. 4. Describe the composition of gram negative bacterial endotoxins and their effects on the host. 5. List one genus-species of Enterobacteriaceae that produce endotoxins and two that produce enterotoxins. 6. Discuss enteric serotyping and why it is used. 7. Define the term ESBL (extended spectrum beta lactamase production). Name one genera of enterobacteriaceae where this enzyme is found.

F. Miscellaneous Gram Negative Coccobacillary Bacteria including:

Pasturella Francisella Bordetella

Brucella Haemophilus Yersinia

Campylobacter Legionella Helicobacter pylori

1. Discuss the pathogenicity of Pasturella multocida and how it is transmitted to man. 2. Name the etiologic agent of tularemia and describe how it is transmitted. How is it isolated in the laboratory? 3. Name the bacteria that causes whooping cough and why Regan-Lowe media is used to isolate this organism. 4. Discuss the value of serologic procedures in confirming cases of tularemia and molecular procedures in confirming whooping cough. 5. Name the bacteria that causes "plague" and how it is transmitted to man. 6. Name the bacteria that causes brucellosis. How does man acquire this disease? How would you isolate the bacteria? 7. Name the Gram negative coccobacillary bacteria of the above group that is an "intracellular" obligate parasite. 8. Discuss the special growth and media requirements of the Haemophilus, defining the terms X and V factor in the discussion, and ALA. 9. Describe "satelliting." 10. List the species and serologic type of Haemophilus associated with meningitis. 11. Discuss the role of beta lactamase testing in the treatment of Haemophilus infections. 12. Name the species of Haemophilus associated with an ulcerative venereal disease. Where is this disease endemic? 13. Describe the Gram's stain, oxidase reaction, media, and growth temperature for isolating Campylobacter sp. vs. Helicobacter pylori. 14. Describe the Gram's stain reaction and morphology of Legionella species, the bacteria associated with Legionnaire's disease. List its growth requirements. 15. List the media and growth requirements of Yersinia sp.

G. Aerobic and Anaerobic Spore Forming and Non-spore Forming Gram Positive Bacilli including:

Cornebacterium sp. Listeria

Nocardia Clostridium

JK-like organisms Erysipelothrix

Bacillus Anthracis Bacillus Cereus

1. Discuss the mechanisms of toxin, antitoxin, and toxoid production of Corynebacterium diptheriae. 2. Describe the colonial morphology and gram staining characteristics of Listeria monocytogenes. 3. Describe the Gram stain and colonial morphology of Erysipelothrix rhusiopathiae. 4. Describe the acid fast stain reaction of Nocardia sp. 5. Discuss the mechanisms of toxin production in Clostridium perfringens, botulinum, difficile, and tetani. 6. Discuss the cellular antigen and exotoxin components of Bacillus anthracis.

H. Mycobacteria

1. Define the term "acid fast," and discuss the cell wall composition of mycobacteria that contributes to their acid fast properties. 2. List common body sites for isolation of Mycobacterium tuberculosis and Mycobacterium leprae. 3. Name a stain of choice used to detect the presence of acid-fast bacteria in clinical material. 4. Name the usual causative agent of tuberculosis and leprosy. 5. List a group of tests used to separate M. tuberculosisfrom other Mycobacterium sp. 6. Name a mycobacteria that is a "rapid grower" and also a marker for HIV. 7. List at least one species of saprophytic mycobacteria, not associated with clinical disease.

I. Non-Enterobacteriaceae and Other Unusual Gram Neg Rods

1. Define the term "non-fermentor" using the terms glucose utilization, oxidase reaction, Gram stain reaction and growth on MacConkey agar. 2. Describe the reaction of non-fermentors on TSI. 3. Describe the colonial and biochemical characteristics of Ps. aeruginosa using the following terms: Odor Pyocyanin Pyoverdin Fluorescein Oxidase reaction Pyorubin Pyomelanin 4. Define the term "opportunist."

J. Chlamydia

1. List the characteristics that make chlamydia different from viruses and bacteria. 2. List the species of chlamydia that causes a common sexually transmitted disease. 3. Discuss a cultural and non-culture method to identify chlamydia.

VII. SUSCEPTIBILITY TESTING
In a short paragraph, describe the Kirby-Bauer procedure, the MIC procedure and the
E-Test and the advantages of each in testing.

VIII. ANAEROBES

IX. PARASITOLOGY

X. MYCOLOGY
A college level course in medical mycology is highly recommended. At HCMC, a 5 session workshop will be provided to enhance skills acquired at the college level.

XI. VIROLOGY

XII. LABORATORY SAFETY
You should be able to discuss and practice laboratory safety and universal blood and body fluid precautions standards.

XIII. MOLECULAR BIOLOGY
Write a short discussion on principles of DNA probe methods, LCR (ligase chain reaction) and PCR (polymerase chain reaction).

XIV. REFERENCES

Brown, Harold W.:Basic Clinical Parasitology, 5th Ed., New York City, 1975, Appleton- Century-Crofts.

** Forbes, Betty A., et al: Bailey and Scott's Diagnostic Microbiology, 12th Ed., St Louis, 1998, C.V. Mosby Company.

Garcia, et al.: Diagnostic Medical Parasitology, 5th ed., 2007. ASM, Washington DC.

Koneman, E.W., Allen, S.D., Dowell, V.R., Jr. Sommers, H.J.: Color Atlas and Text booK of Diagnostic Microbiology, 5th Ed., Philadelphia, 1997, J.B. Lippincott Co.

Balows, A., Hausler, Wm. J., et al: Manual of Clinical Microbiology, 7th Ed., Washington, D.C., 1999, ASM

McFaddin, Jean F.: Biochemical Tests for Identification of Medical Bacteria, 2nd Ed., Baltimore, 1980, Williams and Wilkins.

** Leventhal and Cheadle, Medical Parasitology Self-Instructional Unit, 5th Ed., 2002, F.A. Davis Co.

** Kern, Martha E., Medical Mycology, A Self-Instructional Unit, 2nd Ed., 1997, F.A. Davis Co.

* Recommended for student to purchase
** Required for student to purchase

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