Workshop for the Generalist

Karen Stiles, SM(ASCP)CM State Training Coordinator Assistant Chemical Terrorism Coordinator Nebraska Public Health Laboratory 402-559-3590 [email protected]

1 GRAM STAIN OBJECTIVES:

Upon completion, the participant will be able to:

1. Explain the principle of the Gram stain procedure, including what elements can affect results

2. Correlate the most common with positive Gram stains from cultures and direct specimen sterile body fluid smears

3. Perform and interpret Grams stains

2 Purpose of Gram Stain

 Classify based on form, size, cellular morphology, Gram reaction  Assess quality of specimen  Identify specific infectious agent from morphology and Gram reaction  Correlation with culture growth  Correlation with culture-independent methodologist  Guide presumptive therapy 3 Principle of Gram Stain

composition  Gram positive – think layer with teichoic acid  Gram negative – high in content  Basic premise  – all cells take up primary stain  Gram’s – mordant to form complex  Decolorizer – mixture of and alcohol  Dehydrate in Gram negative cell walls, wash out complex  Gram positive cells resistant, retain stain complex  - 4  Gram negative cells take up counterstain Preparation of Samples

Specimen Type Preparation CSF/sterile body fluids Cyto/Centrifuge Broth Drop to slide Tissue Touch prep Tissue homogenate Drop to slide Swabbed material Roll to slide Sputum, exudates, stool Roll to slide

Appropriately label slide in pencil or use printed label۞ Perform under BSC or face shield 5۞ CSF & Sterile Body Fluids

 Centrifuge >1mL @ 1,500xg 15min, inoculate sediment  <1mL – inoculate directly to slide  Place 1 drop on slide but not spread  Cytocentrifugation – protein absorbed by filter pad, deposits cellular material within small drop-like area  Mark area on reserve side

6 Positive Blood Culture bottles:

 Work under BSC or with face shield  Aspirate positive* blood bottle with syringe  Avoid spray if pressure in bottle  Transfer 1-2 drops on slide  Spread to thin monolayer if needed

* Not performed on direct blood culture before incubation – concentrations too low to detect

7 Tissue & Homogenates:

 Touch Preps – preserve cellular characteristics  Cut tissue with sterile scissor or scalpel  Use sterile forceps to hold tissue, touch side to sterile glass slide  Homogenates – tissue grinder  Transfer 1-2 drops on slide  Spread to thin monolayer if needed

8 Smears from Swabs

 Should not be prepared from swab after inoculation of plate media  Two swabs should be submitted  If only 1 swab, roll onto sterile slide, next inoculate plates, then place in broth  Gently roll swab back and forth over contiguous area of glass slide to form monolayer  Do not rub swab back and forth, as cellular material can be broken or bacterial arrangements disrupted 9 Sputum, Exudates, Stool

 Immerse swab into specimen, specifically in more representative areas:  pus like or  bloody  Gently roll swab on glass slide  Dilute extremely thick specimens in drop of saline

10 Colonies from Culture Media

 Place drop of sterile saline onto slide  Touch top of colony with sterile applicator stick, wire needle or loop  Transfer and gently emulsify  Spread out to make thin smear

11 Smear Fixation

 Allow slides to air dry under BSC/shield  Heat fix on 60ºC slide warmer – 1minute  Overheating can cause over-decolorization  Under BSC/shield if possible  Methanol fix  Flood dried smear with methanol 60 seconds  Rise and dry  Cleaner background

12 Gram stain procedure

1. Place fixed slide on rack over sink 2. Avoid applying stains, water, decolorizer directly to specimen area, allow to flow over specimen 3. Flood with crystal violet – 30 seconds 4. Rinse gently with flowing distilled or tap water 5. Flood slide with Gram’s iodine – 30 seconds 6. Rinse gently with flowing distilled or tap water 7. Hold slide at an angle 8. Run gentle stream of decolorizer over smear, 1-5 seconds, until no more color is washed off 9. Quickly rinse with flowing distilled or tap water 10. Return to rack over sink, flood with safranin – 30 seconds 11. Rinse gently with flowing distilled or tap water 12. Drain slide, air dry or gently blot with paper towel or blotting paper 13. Dry completely before examining 13 Examination of Cellular Material

. Scan Low Power 10X . Background, WBC = pink . Specimen quality . Search for areas with WBC or RBC . Avoid thick areas, crystal violet precipitate or squamous cells if sputum . Quantitate average number of cells (Epi & WBC) . Rare/Occasional (1+) = <1 cell/lpf . Few (2+) = 1-10 cells/lpf . Moderate (3+) = 11-25 cells/lpf . Many (4+) = >25 cells/lpf 14 Examination of Bacteria

. Scan High Power 100X / Oil Immersion . Background, WBC and RBC = pink . Systematically scan good representative areas . Carefully observe 10-25 fields . Haemophilus & Nocardia can blend in . More than one organism should be found . Quantitate average number of bacteria & . Rare/Occasional (1+) = <1 cell/OIF . Few (2+) = 1-10 cells/OIF . Moderate (3+) = 10-25 cells/OIF . Many (4+) = >25 cells/OIF 15 Interpretation

Classify bacteria on: 1. Gram Reaction 2. Gram Morphology 3. Arrangement

16 Gram Reaction

. Gram positive – purple/blue . Gram negative – red/pink . Gram variable – irregularly stained, appears both blue & pink . Inherent to specific bacteria – S pneumoniae . Gram positives too old or treated with will decolorize . Excessive thick areas difficult to decolorize, portions remain blue 17 Gram Morphology Shape/Size varies greatly = rod like

Cocci = round or slightly oval

Spiral = rare, do not stain with Gram stain

18 Arrangements

Cocci  Single  Pairs  Chains – in a line  Clusters – grape like  Tetrads – packets of four  Kidney bean diplococci (Gram negative)

19 Arrangements – Clusters

20 Arrangements – Pairs

21 Arrangements – Chains

22 Arrangements – Tetrads

Pinterest.com https://s-media-cache- ak0.pinimg.com/736x/8c/20/e8/8c20e8969 23 5e165031bbd0ce4631839a9.jpg Arrangements – Diplococci

(Gram Negative)

24 Arrangements

Bacillus/Rods

o Rod

o Rod w spores

o Coccobacilli

o Palisade

o “Chinese Letter”

25 Gram negative rods

Gram negative rods not described in arrangements 26 Gram positive rods

27 Arrangements – Rods with Spores

28 Arrangements - Gram neg coccobacilli

29 Arrangements - Other

Gram positive rods

o Palisade

o “Chinese Letter”

30 Arrangements – Pleomorphic or Beading

31 Arrangements – Branching

32 Arrangements – GNR “gull winged”

33 Grading Materials

 Quantitative and qualitative analysis helpful in culture management  Interpretation of poor specimens can lead to delayed or inappropriate treatment  Basis for specimen rejection  Multiple grading systems:  Bartlett’s Score – report count WBC, SEC  Heineman’s Method – ration between WBC, SEC  Follow facility policy

34 Reporting of Direct Specimen Smears

 Turn-Around Time – physicians most important diagnostic tool  Report should be simple, clear  Significant elements only  Interpretation useful when certain bacteria recognized  ie) Gram positive diplococci consistent with pneumococci  Call critical results such as positive CSF

35 Common Pathogens in CSF

 Positive CSF – “critical value”  Must be communicated directly to physician or follow lab protocol  Derives presumptive diagnosis on:  Patient age  Physical exam  Local  Lab analysis  Radiologic tests

36 Bacterial related to age:

 Neonates (<1month)  Gram negative rods – E coli, Klebsiella, Enterobacter

agalactiae

monocytogenes

37 Bacterial meningitis related to age:

 Infant (1-23 months)   E coli 

meningitidis

38 Bacterial meningitis related to age:

 Bacterial meningitis related to age:  Children (>2 Years) and Adults  Streptococcus pneumoniae 

 Older Adults (>65 Years)  Streptococcus pneumoniae  Neisseria meningitidis

39 Pathogens in Blood

 Most common nosocomial bacteremias:  negative Staphylococci  S aureus  ssp   Klebsiella ssp   Most common blood contaminates:  Coagulase negative Staphylococci  Diphtheroids  Bacillus spp - **Send images or contact NPHL**  Alpha hemolytic Streptococci  P. acnes  GNDC, GNCB and GPR – continue all working BSC for avoid open bench exposure 40 Quality Control & Management

 Storage  Store at 15-30°C, decolorizer in flammable closet  Protect Gram iodine from light  Quality Control  Background material of clinical smears – pink/red  New lot and at least weekly  Prepare smear of known organism, fix and stain  E. coli ATCC 25922 –stains pink/red  S. aureus ATCC 25923 – stains blue/purple  Correlate with culture growth, both in type & amount of bacteria

41 Quality Control & Management

 Quality Improvement – corrective action  Poor specimen quality – maintain grading/specimen rejection policy  Poor stain quality – repeat from original specimen  Too thick or thin  Over-decolorized or under-decolorized  Precipitate in reagents – will be seen in multiple stains  Glass slides on pre-cleaned  Does not correlate with culture growth, in either type & amount of bacteria  Establish system to review gram stain reports  Correlate by bench personnel when reading out cultures  Supervisory review of final culture reports  Supervisory arbitrary review of CSF/Sterile sites  Investigate discrepancies – issue corrective report  Aid in correlating relevant clinical information  Determine training needs  Maintain set of reference slides for competency training  Multiple poor specimens consistently being submitted from same

location – Director should contact to determine plan of action 42 Morphology & Associated Organisms

 Gram positive - Implies possible:

 Cocci in clusters – spp

 Cocci in pairs – Streptococcus pneumoniae

 Cocci in chains – Streptococci or Peptostrep

 Rods, large – or Bacillus**

 ** Trigger point – work under  ** Notify Public Health Laboratory  ** Write into Blood Culture procedure!! 43 Morphology & Associated Organisms

 Gram negative - Implies possible:  Rods, medium – E coli  Rods, thin long – Pseudomonas spps  Coccobacilli – Haemohilus spp or Francisella**  Diplococci – Neisseria spp**  ** Trigger point – All work under Biosafety Cabinet  ** Notify Public Health Laboratory  ** Write into Blood Culture procedure!!

44 Triggers Points - BSC Required

 All manipulation of GNCB, GNDC or large box car shaped GPR’s seen in direct Gram stain.  Slow Growth @ 24-48hrs  Rapid Growth, non-hemolytic @12-16 hrs  All Rule Out/Refer workup  All Fungal or  Procedures that emit aerosols  Opening centrifuge rotors  Inoculation of all specimens to culture plates/slides, especially Blood/Sputum 45 Who Do You Call?

 Follow Laboratory Policy – notify supervisor immediately  Nebraska Public Health Laboratory  Attending Physician  Control  Infectious Disease Physicians  County Health Department  State Epidemiologist 46 How would you interpret?

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Gram stain of colony

60 Gram stain from blood culture How would you interpret?

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Compliments of CHI core labs!! 63 Resources

1. Garcia, L; Clinical Microbiology Procedures Handbook,3rd Edition 2. CLS 552 Human Microbiology & Immunology Laboratory, module 3 3. Mahon, Lehman, Manuselis; Textbook of 4. Images from NPHL STATPack™ unless otherwise marked 5. http://www.nphl.org

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