MLAB 2434 –
MICROBIOLOGY
KERI BROPHY-MARTINEZ
Microscopic Examination of
Infected Materials
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
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
Microscopic Examination of Infected
Materials (cont’d)

Smears from thick liquids or semisolids
(ex: stool)
Microscopic Examination of Infected
Materials (cont’d)

Smears from thick or mucoid material
• “Pull” or “fish”slides
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
Microscopic Examination of Infected
Materials (cont’d)

Stains
Simple
 Differential


Microscopes

Bright-field
Terminology for Direct
Examination
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?
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

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
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
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.