Chapter 7: Microscopic Examination

Question 1

Rapid response on direct examination:

Answer 1

– 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

Question 2

Preparation of Samples Brightfield Microscopy:

Answer 2

1) Should be examined grossly to determine best sample preparation approach

2) Thick, not opaque, and thin smear areas should be produced

Question 3

Preparation of Samples: Swabs

Answer 3

– 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)

Question 5

Smears from thick, granular, or mucoid
materials:

Answer 5

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

Question 6

Smears from Thin Fluids:

Answer 6

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

Question 7

Cytocentrifuge Preparations:

Answer 7

– 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

Question 8

How to use cytocentrifuge:

Answer 8

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

Question 9

Purpose of fixing slides before staining:

Answer 9

– Stick cells to slide
– Kill cells
– Preserve cell morphology
– Prevent lysis

Methods: heat and chemicals

Question 10

Simple stains:

Answer 10

– Color forms and shapes
– Wright-Giemsa
– Methylene blue

Question 11

Differential stains:

Answer 11

– Coloring specific components
– Gram; acid fast, calcofluor white

Question 12

Probe-mediated stains:

Answer 12

– Directed at specific organism identification
– Antibody or DNA probe stains

Question 13

Bright-field compound light microscope:

Answer 13

primary scope used

Question 14

Darkfield microscope:

Answer 14

Primarily for spirochetes

Question 15

Dissecting microscope:

Answer 15

Large parasites

Question 16

Fluorescent microscope:

Answer 16

Special applications and fluorescent stains

Question 17

Electron microscope:

Answer 17

Very specialized, usually for nonculturable viruses

Question 18

Microorganism description includes:

Answer 18

– 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)

Question 19

Fastest and least expensive methods for presumptive diagnosis in clinical settings:

Answer 19

gram stain and acid-fast stain

Question 20

In clinically evident infections, ___

Answer 20

10^5 colony-forming units (CFUs) are common

Question 21

Polymicrobial infections most common in :

Answer 21

surgical wounds, aspiration pneumonias, perirectal
abscesses, tubo-ovarian abscesses

Question 22

Characterization of Background Material:

Answer 22

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

Question 23

Checklist of Material Examination – Low Power

Answer 23

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?

Question 24

Contaminating Materials:

Answer 24

not supposed to be there

Question 25

Local Materials:

Answer 25

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

Question 26

Purulence:

Answer 26

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

Question 27

Mixed Materials:

Answer 27

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

Question 28

Most common contaminated specimen:

Answer 28

Sputum

Question 29

Criteria for rejection:

Answer 29

* Less than 25 PMN leukocytes/low power field (LPF) * Greater than 10 epithelial cells or mixed bacteria/LPF

Question 30

Gram smear report:

Answer 30

Quantitate contaminating materials: 1+ = light; 2+ = moderate; 3+ = moderately heavy; 4+ = heavy

Question 31

Local material example:

Answer 31

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

Question 32

Reports of direct examinations should be:

Answer 32

– 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

Question 33

Points to Remember:

Answer 33

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