L16 microscopy in ID

Question 1

Bright field (light) good for

Answer 1

Bacteria, fungi, parasites

Question 2

Fluorescence good for

Answer 2

Bacteria, fungi, parasites, viruses

Question 3

Dark field good for

Answer 3

Spirochetes

Question 4

Electron microscopy (EM) good for

Answer 4

Viruses, microsporidia

Question 5

Gram Stain

Answer 5

Invented by Hans Christian Gram over 100 years ago
Classify organisms into two groups
Gram positive = retain dye complex
Gram negative = loses purple color after decolorization
Based on the composition of the cell wall
Observe presence of inflammatory cells

Question 6

Gram Stain Procedure

Answer 6

1. cells on the slide
2. primary stain Crystal violet
3. mordant - grams iodine
4. decolorizer - alcohol or acetone
5. counterstain - safranin

Question 7

uses for gram stain

Answer 7

Direct Examination of clinical material to make an initial diagnosis
Determine the quality of the specimen
Suggest an unusual organism or provide early presumptive bacterial identification
Verify the clinical relevance of the culture

Question 8

example Determine the quality of the specimen with gram stain

Answer 8

sputum vs spit - sputum is rejected if there are more than 10 squamous cells present

Question 9

Clostridium tetani

Answer 9

Small rod with round terminal spores giving a drumstick appearance. Often isolated from deep wounds (e.g., nail puncture) gram +

Question 10

give an example of verifying the clinical relevance of a culture using gram staining

Answer 10

Pathogenic and non-pathogenic staphylocooci look the same however the presence of inflammatory cells indicates if it is pathogenic

Question 11

Acid Fast Stains

Answer 11

Designed to stain bacteria with cell walls containing long chain fatty acids
Use heat or detergent to drive stain into cell, once in if the cell resists decolorization = acid fast bacilli
Methods: Ziehl-Neelsen or Kinyoun (use a stronger decolorizer than gram stain)

Question 12

Acid fast procedure

Answer 12

1. cells on slide
2. primary stain (carbolfuchsin red)
3. Decolorizer (HCL, alcohol)
4. Counterstain (methylene blue)
   positive = red; negative =blue

Question 13

What organisms stain in acid fast stain

Answer 13

mycobacteria, nocardia, cryptosporidia, Cyclospora

Question 14

Lactophenol cotton blue

Answer 14

fungal stain for Histoplasma (kills the organism and stains blue)

Question 15

Gomori methenamine silver (GMS)

Answer 15

fugal stain for Yeast cells and hyphae (turns a grey/purple color)
Pneumocystis carinii
Aspergillus

Question 16

Periodic acid-Schiff (PAS)

Answer 16

fugal stain for Yeast cells and hyphae (Pink)

Aspergillus

Question 17

Mucicarmine (mucin)

Answer 17

Cryptococcus capsule

Question 18

Warthin-Starry or Steiner

Answer 18

bacteria and spirochetes (too tiny to be seen with gram)
Silver impregnated stain for formalin fixed tissue
Demonstrate spirochetes, cat scratch disease (B. henselae)

Question 19

Wright’s Giemsa

Answer 19

parasite stain
Used to examine peripheral blood smear
Detect Histoplasma, Leishmania, Trypanosomas, malaria
parasites are purple cells greyish

Question 20

Wet mounts

Answer 20

parasite stains (Giardia)

Question 21

Trichrome

Answer 21

parasites stains

Giardia

Question 22

Modified acid fast

Answer 22

parasite stains

Question 23

Staphylococcus aureus in gram stain

Answer 23

gram-positive coccal bacterium typically found in clusters

Question 24

Neisseria meningitides in gram stain

Answer 24

gram negative diplococci in CNS

Question 25

Streptococcus pneumoniae in gram stain

Answer 25

Gram-positive, diplococci

Question 26

E. coli in gram stain

Answer 26

gram negative, rod shaped bacteria

Question 27

Actinomyces species in gram stain

Answer 27

gram postitive purple- branch like organisms

Question 28

Campylobacter species
 in gram stain

Answer 28

small gram negative pink organisms with s and w curved shapes

Question 29

Nocardia species in gram stain

Answer 29

threads of gram + bacteria, rod shaped, beaded branching filaments