Specimen Processing & Microscopy

Question 1

Which tissue specimen is preferred over aspirate or pus?

Answer 1

Deep tissue specimen - especially good for anaerobic organisms

Question 2

Acute phase illness specimens are collected when?

Answer 2

Before antibiotic therapy

Question 3

How long should it take to transport specimens to the lab?

Answer 3

30 minutes

Question 4

How should specimens be transported?

Answer 4

Sealable and leak-proof, bags marked with biohazard label

Question 5

How should Shigella and N. meningitidis be transported?

Answer 5

Special media to ensure survival and recovery

Question 6

What is used as a preservative for urine samples and why?

Answer 6

Boric acid and it maintains colony count

of cfu/ml is critical in interpreting results for true infection

Question 7

What is used to maintain integrity of cysts and trophozoites in stool samples?

Answer 7

Polyvinyl alcohol (PVA) and buffered formalin

Question 8

Examples of holding media and why is charcoal added to some media?

Answer 8

Staurt’s medium, Amie’s medium - maintain organisms and charcoal is added to some media to absorb inhibitory fatty acids

Question 9

What temp is used to store urine, stool, viral specimens, sputum, swabs and catheters?

Should anaerobic cultures be placed in fridge?

Answer 9

4*C

Anaerobes are sensitive to cold temps, clinically significant grow at human body temp

Question 10

What temp is CSF samples stored?

Answer 10

37*C

Organisms that cause infection in CNS die at room temp or cooler

Question 11

Storage conditions of serum and tissue biopsies

Answer 11

-20*C serum up to 1 week

and tissue biopsies -70*C

Question 12

Reject specimens

Answer 12

• info on specimen does not match req form
• improper temp, medium and container for transport
• specimen is leaking, dried up or insufficient for testing
• specimen not preserved or transport takes over 2 hrs
• specimen for anaerobic culture taken from site that has normal or resident anaerobic flora (unable to distinguish normal flora and pathogenic)

Question 13

Gross examination of specimen

Answer 13

Note - presence of blood or mucus in specimen

Stool should be examined for barium

Question 14

Direct Microscopic Examination

Answer 14

Specimen work up based on what is grown in culture or seen in smear

Microscopy is usually not performed for throat, nasopharyngeal or stool specimens

Question 15

Nutritive Media

Answer 15

blood and chocolate agars support growth for wide variety of microbes

Question 16

Differential Media

Answer 16

differentiates between organisms on the basis of growth characteristics

*blood agar - hemolytic pattern

Question 17

Selective Media

Answer 17

Supports growth of one group over another by adding antimicrobials, dyes or alcohol (MacConkey agar and CNA)

Question 18

Selection media

Answer 18

Based on type or origin of specimen (CSF - meningitis causing organisms)

Question 19

Specimen Prep

Answer 19

Tissue - homogenization or grinding

Conc. by cent or filtration of large vol of pleural fluid

Swab specimens vortexed .5-1.0 ml saline/broth for 10-20 sec

Question 20

Inoculation of solid media

Answer 20

Burn victims urine/tissue cultures inoculated quant by dilution or quant loop

All other specimen inoculated using semiquant w/ ordinary loop

Question 21

Incubation conditions

Answer 21

• 28*C for fungi
• 35-37*C bacteria, viruses, AFB
• Aerobes grow 21% O2
• Anaerobes 80-90% N2 and 0% O2
• Microaerophiles (C. jejuni and H. pylori) O2 5-10% and CO2 8-10%
• Capnophiles (H. influenzae, N. gonorrhaea) CO2 5-10% and O2 15%

Question 22

Bacteria - Brightfield and Fluorescent Microscopy

Answer 22

Brightfield - gram stain, methylene blue, trichrome, ziehl neelsen and kinyoun

Fluorescent - auramine/rhodamine
acridine orange

Question 23

Gram Stain

Answer 23

CV forms insoluble complex with iodine and mordant, Gram (-) lose complex because of higher lipid content

Question 24

What are the different methods to fix the organism to the slide?

Answer 24

Air dry first!!

Fix to slide by heat or methanol

Question 25

Quality of a good specimen on a slide

Answer 25

High # of leucocytes and the absence or low # of epithelial cells

Question 26

Acid Fast Stains

Answer 26

Mycolic acids in cell walls or cystic forms of these organisms
Use - auramine-rhodamine for primary stain and potassium permanganate for counter stain
(orange to yellow organisms)
OR
Carbol fuchsin for primary stain and methylene blue for counter stain
(Red w/ blue background)

*mycobacteria

Question 27

Darkfield Microscopy

Answer 27

Light not transmitted directly, can see spirochetes

Question 28

Fluorescence Microscopy

Answer 28

Uses wavelength selection and Abs labeled w/ fluorescent dyes
Useful for T. pallidum identification

Question 29

Phase Contrast Microscopy

Answer 29

Unstained cells - urinanalysis

Question 30

Electron Microscopy

Answer 30

electron beam for illumination - ultra structure cells

Living cells can’t be used