Chapter 7: Microscopic Examination Flashcards
Rapid response on direct examination:
– Identify cellular components and debris of inflammation to estimate the probability of infection
– Identify specific infectious agents
– Guide physicians to early treatment with
antibiotics
– Develop epidemiologic data
Preparation of Samples Brightfield Microscopy:
1) Should be examined grossly to determine best sample preparation approach
2) Thick, not opaque, and thin smear areas should be produced
Preparation of Samples: Swabs
– Do not use swabs used to inoculate media, must do smear from separate swab
– Always collect 2 swabs
– Prepare by rolling swab back and forth over slide (DO NOT RUB)
Smears from thick, granular, or mucoid
materials:
1) Thick and thin areas, crush granules
2) Use 2-slide technique
3) Place portion of sample on labeled slide
4) Press second slide label side down on top
5) Rotate glass surfaces against each other
6) Pull slides smoothly away from each other
Smears from Thin Fluids:
For urine, CSF, other fluids
1) Drop on slide with mark reverse of slide (do not spread unless too turbid)
2) Stain slide
3) Cytocentrifuge preparations and preferred method
Cytocentrifuge Preparations:
– Deposits cellular elements and microorganisms
as a monolayer on slides using centrifugal force
(type of centrifuge) – concentrates cells
– Clears protein using a filter pad to clear background
– Enhances morphology and concentrates sample, making viewing faster
How to use cytocentrifuge:
1) Few drops of fluid placed in funnel attached to microscopic slide
2) Fluid spun into column using cytospin
3) Cells deposited onto a glass slide
Purpose of fixing slides before staining:
– Stick cells to slide
– Kill cells
– Preserve cell morphology
– Prevent lysis
Methods: heat and chemicals
Simple stains:
– Color forms and shapes
– Wright-Giemsa
– Methylene blue
Differential stains:
– Coloring specific components
– Gram; acid fast, calcofluor white
Probe-mediated stains:
– Directed at specific organism identification
– Antibody or DNA probe stains
Bright-field compound light microscope:
primary scope used
Darkfield microscope:
Primarily for spirochetes
Dissecting microscope:
Large parasites
Fluorescent microscope:
Special applications and fluorescent stains
Electron microscope:
Very specialized, usually for nonculturable viruses
Microorganism description includes:
– Where specimen was collected / Type of specimen
– Type of smear
– Stain (pathogen if present: outcome (Gram+ or Gram-), morphology, size)
– Type of microscopy and magnification
– Describe background when present (purulence, local materials, contaminating materials, mixed materials)
Fastest and least expensive methods for presumptive diagnosis in clinical settings:
gram stain and acid-fast stain
In clinically evident infections, ___
10^5 colony-forming units (CFUs) are common
Polymicrobial infections most common in :
surgical wounds, aspiration pneumonias, perirectal
abscesses, tubo-ovarian abscesses
Characterization of Background Material:
1) Examine material without microscope first
2) Scan under low power (×2.5 to ×10 objective magnification [obj])
Homogeneous vs heterogeneous
Distributed vs in one field
Checklist of Material Examination – Low Power
1) Is there evidence of contamination by normal
(resident) microbiota?
2) Is necrotic (amorphous) debris in the background?
3) Is there evidence of purulence (pus)?
4) Are unexpected structures present?
Contaminating Materials:
not supposed to be there
Local Materials:
There, but not the infectious material
Will likely yield:
– Less than 25 PMN/LPF
– Less than 10 contaminating epithelial cells/LPF
– Cellular and fluid elements
Purulence:
Sample component consisting of neutrophils, protein, and necrotic debris - Pus
Criteria
– Greater than 25 PMNs/LPF
– Fewer than 10 epithelial cells / LPF
– Mucus and/or heavy proteinaceous material present
– Foul odor
Mixed Materials:
Purulent exudate, contaminating materials, and local materials in one smear
Criteria
– Greater than 25 PMN/LPF
– Less than 10 epithelial cells or contaminating bacteria/LPF
– Local secretions
Most common contaminated specimen:
Sputum
Criteria for rejection:
- Less than 25 PMN leukocytes/low power field (LPF)
- Greater than 10 epithelial cells or mixed bacteria/LPF
Gram smear report:
Quantitate contaminating materials:
1+ = light; 2+ = moderate; 3+ = moderately heavy; 4+ = heavy
Local material example:
Respiratory secretions: mucus, macrophages, goblet cells, ciliated columnar cells
CSF: cellular elements
Cavity fluid: macrophages, mixed white blood cells (WBCs), mesothelial cells
Wounds: blood and proteinaceous fluid
Amniotic fluid: anucleate squamous cells and havy
proteinaceous fluid
Cervix: mucus, columnar epithelial cells, goblet cells,
leukocytes
Prostatic secretions or semen: spermatozoa and mucus
Reports of direct examinations should be:
– Made and sent as soon as direct examination
results are completed.
– Simple and include all elements needed for others
to understand the report and
1) the type of material submitted.
2) whether the observed microbes are of significance
Points to Remember:
Direct microscopy of materials submitted to the
laboratory for identification of infectious organisms offers the first, last, and best opportunity to:
– Suspect probable infection.
– Confirm the diagnostic quality of specimen submitted.
– Recognize “classic” organism morphotypes associated with infection.
– Recognize pathogens that will not grow in culture or the culture type requested, thus requiring a different diagnostic approach.
– Provide a “stat” laboratory response to serious infection.
– Provide a pathway to confirm the suspected cause of infection
