1. MLAB 2434 – MICROBIOLOGY KERI BROPHY-MARTINEZ Microscopic Examination of Infected Materials

2. Microscopic Examination of Infected Materials (cont’d) Purposes for direct gram stain from patient specimens Confirm that specimen submitted is representative (ex. Sputum vs. spit) Determine probability of infection by identifying debris of inflammation Identify specific infectious agents Guide initial antibiotic therapy Use for epidemiologic data

3. Microscopic Examination of Infected Materials (cont’d) Preparing smears Using swabs Smears should not be made using swab that has been used to streak cultures Ideally, if culture AND gram stain are to be done, two swabs of the specimen should be collected and sent to the lab Swab is NOT rubbed on the slide, but rolled back and forth

4. Microscopic Examination of Infected Materials (cont’d) Smears from thick liquids or semisolids (ex: stool)

5. Microscopic Examination of Infected Materials (cont’d) Smears from thick or mucoid material “Pull” or “fish”slides

6. Microscopic Examination of Infected Materials (cont’d) Smears from thin fluid (ex: CSF) Mark back of slide with marking pen to show location of specimen on the slide for ease in finding under microscope Cytocentrifuge Prepares nonviscous fluids, like CSF, by depositing cellular elements and microorganisms onto the surface of the slide as a monolayer

7. Microscopic Examination of Infected Materials (cont’d) Stains Simple Differential Microscopes Bright-field

8. Terminology for Direct Examination

9. Checklist of Material Examination Is there evidence of contamination by normal flora? Epithelial cells, bacteria without inflammatory cells, other debris Are amorphous debris in the background? Evidence of destruction and remains of tissue Are unexpected elements present?

10. Search for Microorganisms Examine more than one area of the smear Should find more than one organism What kind of site? Sterile? Do not overinterpret the findings Strict criteria for microbial morphologies should be applied Wait for additional test or staining if necessary

11. Microscopic Examination of Infected Materials (cont’d) Clinical Specimens SPUTUM First evaluate the smear to determine if it is REALLY sputum Unacceptable = >25 squamous epithelial cells/LPF Acceptable = <10 PMN/LPF Evaluate smear first under low power to look for background material, such as WBC (pus), epithelial cells, etc. Search for microorganims under 100X

12. Microscopic Examination of Infected Materials (cont’d) Other Clinical specimens Epithelial’s not significant Pure Culture Goal is to verify organism morphology WBC’s and Epithelial’s should not be present nor quantitated

13. References Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology. Baltimore, MD: Lippincott Williams and Wilkins. http://www.brickschools.org/staff/sdudek/Images1/Forms/ DispForm.aspx?ID=67&RootFolder=%2Fstaff%2Fsdudek%2 FImages1 Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.