Collection, Transport, Processing, and Staining of Specimens

PRINCIPLES OF SPECIMEN COLLECTION

• Major goal of microbiology laboratory – aid diagnosis of infectious disease
  
• Goal of specimen collector – maintain viability w/ minimal contamination
  

FUNDAMENTALS

• Collect in acute phase of infection and before antibiotics administration (or within 2-3 days for viral infection)
• Correct anatomic site for collection
• Proper technique and supplies w/ minimal contamination
• Appropriate quantity of specimen
• Container or transport medium designed to maintain viability of organism & avoid hazard from leakage.
• Accurate labeling of spx. With:
  
  • Specific anatomic site
  • Patient’s info
• Immediate transport of spx. to lab
• Provide environment that won’t degrade suspected organism
• Notify lab if unusual / agents of bioterrorism are suspected

COLLECTION PROCEDURE

• Spx. should be collected in sterile containers
  
  • Except for stool
• Swabs – not recommended
  
  • Don’t provide sufficient quantity
    
  • Easily contaminated
  • Can be dried out leading to organism loss
  • Often vortexed in 0.5 – 1ml of saline or broth for 10 – 20 secs. to
  • dislodge material from fibers.
  • Recommended for:
    
    • Upper respiratory tract
    • External ear
    • Eye
    • Genital tract
• “wound” is not appropriate specimen label (exact site must be provided)

PATIENT-COLLECTED SPECIMEN

• Most effective method for instruction – verbal and written instruction
  

PRESERVATION, STORAGE, AND TRANSPORT

• Primary Goal
  
  • Maintain specimen as near to its original state as possible w/ minimal deterioration
  • Prevent risk to specimen handler
• Specmin should be transported to lab ideally within 30 minutes of collection, preferably within 2 hours

STORAGE

Specimens that can be maintained @ 4C for 24 hours 

• Urine
• Stool
• Sputum
• Swabs (not for anaerobes)
• Catheters
• Viral specimens

CSF – if not processed, stored @ 35C for 6 hours

PRESERVATION
Specimens that can be preserved by preservatives

• Stool
  
• Urine
• Boric acid – used to maintain accurate urine colony count
  
• Stool – can be refrigerated
  
  • If delayed for 2 hours – can be added to Cary-Blair Transport media
    
  • Stool for Clostridium difficile toxin assay
    
    • Can be refrigerated
    • If delayed >48 hours: Frozen @ -700C
      
• Polyvinyl Alcohol / buffered formalin – for ova and parasite (O&P) exam

ANTICOAGULANT

• Sodium Polyanethol Sulfonate (SPS) 
  
  • Most common anticoagulant used for microbiology spx. 
  • must not exceed 0.025% (wt/vol) 
    
    • Neisseria spp. & certain anaerobes are inhibited by higher concentration
• Heparin – used for viral culture and isolation of Mycobacterium spp. from blood

HOLDING OR TRANSPORT MEDIA 

• Usually contains substances that do not promote multiplication of microorganisms but ensure their preservation 
• Available in swab collection system
• Stuart’s and Amie’s Transport Medium – commonly used
• JEMBEC (James E. Martin Biological Environmental Chamber) System
  
  • Used for specimens for N. gonorrhoeae

Level 1 – critical; represent potentially life-threatening illness; from invasive source
Level 2 – unprotected; may quickly degrade or have overgrowth of contaminating flora.
Level 3 – require quantitation; may affect accuracy of quantitation if delayed.
Level 4 – spx. in holding or transport media

UNACCEPTABLE SPECIMENS

• Info on request form doesn’t match to spx. 
• Not submitted in appropriate transport container or leaking container 
• Inadequate quantity of spx. 
• Spx. transported >2 hours; not preserved 
• Received in formalin 
• Requesting anaerobic culture in spx. in w/c anaerobes are indigenous 
• Dried up spx. 
• More than 1 source was submitted from the same specimen. Blood culture are exception 
• One swab was submitted with multiple requests for various organisms.

MACROSCOPIC EXAMINATION 

• Swab or Aspirate 
• Stool Consistency 
• Blood or Mucus present – part that is cultured and direct microscopic exam 
• Volume 
• Fluid – clear or cloudy
• If presence of gas, foul smell, sulfur granules – ANAEROBIC CULTURE

MICROSCOPIC EXAMINATION Purpose: 

• Determines quality of specimen 
• Gives indication of infectious process involved. 
• Guides routine culture workup based on the result of the smear 
• Dictate the need for non routine or additional testing.

ISOLATION TECHNIQUE 

• General purpose Isolation Streak 
  
  • Yields semiquantitative estimate of growth 
  • Useful for most specimen
  • Grading 
    
    • 1st quadrant – 1+ (light growth) 
    • 2nd and 3rd quadrant – 2+ (moderate growth) 
    • 4th quadrant – 4+ (heavy growth) 
• Quantitative isolation 
• For urine specimen & tissue from burn patients 
• Uses calibrate loops
  • 0.01
  • 0.001

INCUBATION

• 35 – 37°C – bacteria, AFBs, & viruses 
• 28 – 30°C – fungi 
• Most routine bacterial cultures are held for 48 – 72 hours 
• Anaerobes and broth cultures – held for 5 – 7 days
• Aerobes – grow in ambient air 
  
  • 21% O2 
  • 0.03% CO2
• Anaerobes – cannot grow in presence of O2
• Atmosphere in anaerobe jars
  
  • 5 – 10% H2
  • 5 – 10% CO2
  • 80 – 90% N2
  • 0% O2
• Capnophiles –requires
  
  •  5 – 10% CO2
  • 15% O2
• Candle Jar atmosphere 
  
  • 3% CO2
  • Examples: H. influenzae; N. gonorrhoeae
• Microaerophiles – grows in 
  
  • reduced O2 (5 – 10%) 
  • increased CO2 (8 – 10%)
  • Example: Campylobacter jejuni; Helicobacter pylori

NONROUTINE SPECIMENS 

• Implant soak solution 
  
  • Requires large volume and 
  • Concentration 
• Water sterility specimen 
  
  • Requires concentration 
    
    • Millipore Sampler – Uses 18ml of water 
• Intrauterine device 
  
  • Cultured for detection of Actinomyces spp. 
  • Inoculated into THIO 
• Vascular Catheter tips 
  
  • Use for catheter-related infection 
  • Uses Maki roll technique
    
    • Maki roll technique – 5-7cm segment of catheter is rolled of a blood agar plate 4 times

SMEARS

Smears for Swab 

• Should not be prepared from swab after used to inoculate culture media. (2 swabs are submitted) 
• Prepared by rolling the swab back and forth over contiguous areas of the glass slide to deposit a thin layer of sample material.

Smears from thick liquid 

• Swab method – swab is immersed in specimen for several seconds 
  
  • Used to prepare thin spread of material in the glass slide

Smears from thick, granular, mucoid materials 

• Opaque material must be thinly spread
• Most desirable to have both thick and thin areas 
• Granules must be crushed – to assess their makeup 
  
  • Too hard granules – probably don’t represent infectious material

Smears from Thin Fluids 

• Should be dropped but not spread on slide 
• Cytocentrifugation – preferred for this type of specimen.
  
  • Excellent for nonviscous fluids (CSF, BAL)
  • PROCEDURE
    1. Small aliquots of fluid (0.1 – 0.2 ml) are placed into cytocentrifuge
       holder
       
    2. Material is spun for 10 minutes
       
    3. Slide is removed. If deposit of cells is too heavy, a portion of cellular
       deposition can be smeared
       
    4. Fixed and decontaminated in 70% alcohol for 5 mins.

SMEARING

Reasons why organisms grow in culture that was not seen in Direct Smear 

• Slow-growing organism was present 
• Patient receiving antibiotic treatment – prevents growth of organism 
• Specimen was not appropriately processed 
• Organism is no longer viable 
• Organism requires special media for growth.

DIRECT SMEAR 

• preparation of primary clinical sample received in the laboratory for processing. 
• Provides mechanism to identify number & type of cells present in specimen.

INDIRECT SMEAR

• When
  
  • Primary sample has been processed in culture 
  • smear contains organisms following purification or growth on artificial media.

STAINS 

• Simple stains – directed toward coloring the forms and shapes present 
• Differential stains – directed toward coloring specific components present 
• Diagnostic antibody or DNA probe-mediated stain 
  
  • Specific at identification of organism
• Gram stain (by Hans Christian Gram, 1884) 
  
  • Fixative – heat / methanol 
  • Primary stain – crystal violet (hexamethyl-p-rosanaline chloride) – (30 secs.) 
  • Mordant – Gram’s iodine (no water rinse employed; 30 – 60 secs.) 
  • Decolorizer – alcohol-acetone (quick) 
  • Counterstain – Safranin (1 minute)

Precaution 

• If crystal violet rinsed too vigorously before complexed with iodine 
  
  • wash away and leave poor/no staining of gram-negative organism 
• If decolorizer is too vigorous or prolonged 
  
  • Gram-positive complex will be removed; gram-positive organism will not stain. 
• Decolorizer is insufficient 
  
  • False gram-positive organisms in thicker areas of sample 
• Presence of inflammatory cells – key indicator of infectious process.

ACID FAST STAINING

• Most common Acid-Fast staining methods 
  
  • Auramine-rhodamine 
  • Ziehl-Neelsen 
  • Kinyoun 

Fluorescent Stain 

• Primary stain – auramine-rhodamine T stain (25 mins.) 
• Decolorizer – 0.5% acid alcohol (2 mins) 
  
  • (0.5% HCl in 70% alcohol) 
• Counterstain – potassium permanganate (4 mins)
• POSITIVE RESULT – BRIGHT YELLOW/ ORANGE against GREENISH BACKGROUND

Ziehl-Neelsen Method (hot method)

• Heat – allows penetration of stain into waxy surface of microorganism
• Primary stain – carbolfuchsin (5 minutes)
• Decolorizer – acid-alcohol (3% HCl in 95% ethanol)
  
• Counterstain – methylene blue (1 min)

Kinyoun Method (cold method)

• Primary stain – carbolfuchsin (5 mins.)
• Decolorizer – acid-alcohol
• Counterstain – methylene blue (1 min)

Modified Kinyoun Method (for partial acid fast) 

• Primary stain – carbolfuchsin (5 mins.) 
• Decolorizer – 1% sulfuric acid 
• Counterstain – methylene blue (30 secs)

FUNGAL STAIN

Most common fungal stains are:

• KOH
• PAS 
• Grocott’s Methenamine Silver Stain
• Calcofluor White

Calcofluor White 

• colorless dye 
• binds to chitin and cellulose
• for fungal elements 
• fluoresce maximally at 440 nm
• Evans Blue – counterstain
  
  • RESULT: fungi appears bright apple-green / blue-white fluorescence