Adhi
Kristianto Sugianli
Office: Dept. of Clinical Pathology and Laboratory Medicine, Faculty of Medicine Universitas Padjadjaran
Dr. Hasan Sadikin General Hospital Bandung
Email: [email protected]
Academic Qualification
• M.D, Faculty of Medicine Universitas Kristen Maranatha, 2007
• Master, Faculty of Medicine Universitas Padjadjaran, 2011
• Residency, Clinical Pathology Dr. Hasan Sadikin General Hospital, 2011
• Fellowship, Infection and Tropical Disease, Dr. Hasan Sadikin General Hospital, 2016
• PhD candidate, Epidemiology and Clinical Microbiology, University of Amsterdam, Present
Professional Experience
• Senior lecturer, Faculty of Medicine Universitas Padjadjaran, 2011 – present
• Laboratory staff, Dept. of Clinical Pathology Dr. Hasan Sadikin General Hospital, 2011 - present
• Laboratory manager, Women & Children Melinda Hospital, 2014 – present
• Consultant/Laboratory staff, Santo Borromeus, 2016 – present
Organization
• INAEQAS for microbiology
Universitas Padjadjaran
How to Read the Gram Panel
Adhi Kristianto Sugianli
Dept. of Clinical Pathology Faculty of Medicine Universitas Padjadjaran /
dr. Hasan Sadikin General Hospital Bandung Indonesia
Reference
1. Thairu, Y. Abdulhamid, IA. Usman, Y. Laboratory perspective of gram staining and its
significance in investigations of infectious diseases. Sub-saharan African Journal of
Medicine.2014:1(4): 168-174.
2. Hashimoto, Shime. Evaluation of semi-quantitative scoring of Gram staining or semi-
quantitative culture for the diagnosis of ventilator-associated pneumonia: a retrospective
comparison with quantitative culture. Journal of Intensive Care, 2013:1(2):1-5
3. Tille, PM et all.Bailey & Scott’s Diagnostic Microbiology. 13ed. St.Louis, Elsevier. Mosby. 2014
4. Ayers LW. Microscopic Examination of Infected Materials. In: Mahon CR, Lehman DC,
Manuselis G, editors. Textbook of Diagnostic Microbiology. 4 ed. Maryland, Missouri: WB
Saunders, Elsevier, 2011
5. Sharp, S. E., A. Robinson, M. Saubolle, M. Santa Cruz, K. Carroll, and V. Baselski. 2004.
Cumitech 7B, Lower Respiratory Tract Infections. Coordinating ed., S. E. Sharp. ASM Press,
Washington, D.C.
Outline
• Gram stain mechanism – basic knowledge
• Reading Gram panel INAEQAS
• Pitfall / Issue in reading Gram panel
• Tutorial website INAEQAS Mikrobiologi
Introduction
• Gram stain:
• Developed in 1884 by Hans Christian Gram
• Widely known as preliminary tools in bacterial identification
• Old technique
• INAEQAS:
• Gram panel quality assurance
• Goal: improve the capability of reading Gram smear
Gram Stain
• Old staining technique used to differentiate bacteria species
into two group: Gram-positive and Gram-negative
• Preliminary morphologic identification of any specimen
• Specimen eligibility screening, before culture:
• Particularly in Sputum
• To differentiate between saliva and sputum
• Bartlett’s-, Murray-Grading System
• Number of Epithelial cells < 10
• Number of WBC > 25
Reagent & Mechanism
Reagent Stain Application Mechanism Cell Color
Crystal Violet (CV+) Primary stain CV+ ion will bind to the Purple
negative charged
component of bacterial
cell
Iodine (I-) Trapping agent Complex CV and Iodine Purple
prevent removal of CV
Alcohol Decolorizer Interact with the lipid GPB: Purple
of membrane cell. GNB: colorless
Safranin (S+) Counter stain Positive charged will GPB: Purple
interact with the cell of GNB: Pink
GPB / GNB
* GPB: Gram-positive Bacteria; GNB: Gram-negative Bacteria
Step # 1
• Evaluate the general appearance of the smear (10x /lpf)
Observation Hallmark
Proper decolorized Clear background or Gram negative (reddish)
Cell appearance WBC : light pink cells with dark pink/red nucleus
RBC : faint color (if present, rarely)
Appropriate thickness No overlapping cell
Evidence of Number of PMN, MN, RBC
inflammation Number of EC
General appearance of microorganism (often difficult to see)
What do we see in
Gram stain?
No overlapping
Clear background/Reddish
Epithelial
WBC
Gram Stain Sputum,
100x- low power field
What do we see in Gram stain?
Clear background or Gram negative (reddish)
Gram stain Sputum Gram stain Sputum Gram stain Sputum
1000x – oil immersion field 1000x – oil immersion field 1000x – oil immersion field
Epithelial cell WBC Bacteria
Light pink cells with dark pink / Light pink cells with dark Coccus or bacilli
red nucleus pink / red nucleus; PMN / Morphology arrangement
MN
Step # 2
• Evaluate the present of microorganism (100x /oif)
• 20 – 40 field (average)1
• Report:
• No microorganism: ‘no microorganism seen’ / Negative
• Microorganism seen:
• Report relative number (=quantitative)
• Describe morphology
1. Thairu, Y. Abdulhamid, IA. Usman, Y. Laboratory perspective of gram staining and its significance in investigations of infectious diseases. Sub-saharan African
Journal of Medicine.2014:1(4): 168-174.
Step # 3
• Examine the characteristic morphology and arrangement of
microorganism1-3
Item Report
Predominant shapes* coccus, bacilli
Appearance of structure diplo-, tetrad, cluster, chain,
etc.
*Predominant = the most organism seen in microscopic field
1. Thairu, Y. Abdulhamid, IA. Usman, Y. Laboratory perspective of gram staining and its significance in investigations of infectious diseases. Sub-saharan African
Journal of Medicine.2014:1(4): 168-174.
2. Tille, PM et all.Bailey & Scott’s Diagnostic Microbiology. 13ed. St.Louis, Elsevier. Mosby. 2014
3. Ayers LW. Microscopic Examination of Infected Materials. In: Mahon CR, Lehman DC, Manuselis G, editors. Textbook of Diagnostic Microbiology. 4 ed. Maryland,
Missouri: WB Saunders, Elsevier, 2011
What do we see in Gram stain? SIFAT GRAM SIF
A DIBANDINGKANDIB
DI PREPARAT
DI
A
?
YANG SAMA
A
C
B YA
Figure 1
Gram stain Sputum,
B Figure 2
Gram stain Sputum,
Figure 3
Gram stain Sputum
?D B
1000x – oil immersion field 1000x – oil immersion field 1000x – oil immersion field
Fig.1: (A) diplococci Gram-positive bacteria, Fig.2: (A) cluster and (B) chain– Fig.3: (A) cluster and (B) chain–shaped arrangement
(B) diplococci Gram-negative bacteria shaped arrangement Gram-positive Gram-positive bacteria; (C) diplococci Gram-positive
bacteria bacteria; (D) budding cell
What do we see in Gram stain?
A. Epithelial cell
B. WBC
C. Single-bacilli Gram-negative
bacteria
D. Diplococci Gram-positive bacteria
D (Over-decolorization/old aged)
A
B
A
Gram stain Sputum,
1000x – oil immersion field
Step # 4
• Quantitate the organism, WBC and EC 1-3
Bacteria Grade Description WBC, EC Grade Description
Many 4+ More than 30 bacteria per field Many 4+ 25 or greater per lpf
Moderate 3+ 6 - 30 bacteria per field Moderate 3+ 10 - 25 per lpf
Few 2+ 1 - 5 bacteria per field Few 2+ 2 to 10 per lpf
Rare 1+ Less than 1 bacterium per field Rare 1+ Fewer than 2 per lpf
None 0 No bacteria None 0 None
1. Hashimoto, Shime. Evaluation of semi-quantitative scoring of Gram staining or semi-quantitative culture for the diagnosis of ventilator-associated pneumonia: a retrospective comparison
with quantitative culture. Journal of Intensive Care, 2013:1(2):1-5
2. Tille, PM et all.Bailey & Scott’s Diagnostic Microbiology. 13ed. St.Louis, Elsevier. Mosby. 2014
3. Ayers LW. Microscopic Examination of Infected Materials. In: Mahon CR, Lehman DC, Manuselis G, editors. Textbook of Diagnostic Microbiology. 4 ed. Maryland, Missouri: WB Saunders,
Elsevier, 2011
What do you see from the picture?
Can you quantitate the cell?
Clear background/reddish
No overlapping
Epithelial cell: 3+ (10 – 25/lpf)
WBC: 4+ ( 25 or > /lpf)
Microorganism seen
Gram Stain Sputum,
100x – low power field
What do you see from the
picture?
Can you quantitate the cell?
A. Epithelial cell
B. WBC
C. Coccus, Gram-positive
bacteria, 3+ (6 – 30 A
bacteria per field)
D. Bacilli, Gram-negative C D
bacteria, 4+ ( >30
bacteria per field)
E. Coccus, Gram-negative
bacteria, 3+ (6 – 30
B
bacteria per field)
Arrangement:
C. Coccus, Gram-positive
bacteria: cluster, diplococci, E
chain
D. Bacilli, Gram-negative
bacteria: single
Gram Stain Sputum,
1000x – oil immersion field
Pitfalls of Gram Stain
Pitfalls Effect
Over-decolorization False Gram-negative result
Under-decolorization False Gram-positive result
Too thick Retain staining
‘Old age’ culture Cell dead will not retain stain
Antimicrobial effect Altered Gram stain reactivity
Stain precipitation False reading
Quality Control
Internal Quality Control External Quality Control
Using Well-known Strain Intra-activity Monitoring Direct specimen
(Gram-staining)
Staphylococcus Routine submitted
aureus specimen
Escherichia coli Observe and report
Gram-staining
Make suspension
Gram-staining GNB GPB GNB GPB
Reviewed by Blind-
observer
Correlation with culture
GNB GPB result identification
*GNB, Gram-negative bacteria
GPB, Gram-positive bacteria
Tutorial website INAEQAS Mikrobiologi
• Access the website using cellular phone / PC
• Open the website: bit.ly/inaeqasmikro
• Document:
• Petunjuk Teknis Pelaksanaan
• Petunjuk Pengisian Hasil
