Microbiologic Staining Method

顏 慕 庸

高 雄 榮 總

Microbiological Staining

Gram stain

Acid fast stain

KOH stain

Wet mount

Indian ink

Gram Stain

• Christian Gram, 1884

• crystal violet

• iodine-KI

• alcohol wash

• safranin

Significance

• Culture

• Buy time for golden period

• Diagnosis

Interpretation

• Morphological

• Associate environment

• Site-specific

• Immunity-oriented

Morphologic interpretation

• cocci vs bacilli, cocco-bacilli

• Gram positive vs Gram negative

Morphologic interpretation

• cocci vs bacilli, cocco-bacilli

• Gram positive vs Gram negative

Gram-Positive Cocci

• GPC in group• GPC in chain• GPC w/c capsule

• GPC in pair• lancet shape• kidney shape• sarcinae

Gram-Positive Cocci

• GPC in group• GPC in chain• GPC w/c capsule

• GPC in pair• lancet shape• kidney shape• sarcinae

Gram Negative Cocci

Cocco-bacilli, Kidney-shape

G (+) vs G (-)

Bacilli

• Gram positive bacilli– spore– spindle– box car– palisade– beaded– branching

• Gram negative bacilli– pleomorphic– cocco-bacilli– capsule– curved

Bacilli

• Gram positive bacilli– spore– spindle– box car– palisade– beaded– branching

• Gram negative bacilli– pleomorphic– cocco-bacilli– capsule– curved

Presumptive Diagnosis

• Staphylococcus aureus

• Streptococcus pneumoniae

• Hemophilus influenzae

• Klebsiella pneumoniae

• Nocardia spp

• Candida spp

Associate environment

• Neutrophils, epithelial cells

• Aid for judgement of G(+), G(-)

• Intracellular vs extracellular

• Mixed flora

• w/o PMN, w/o bacteria

• Non-bacteria organisms

Associate environment

• Neutrophils, epithelial cells

• Aid for judgement of G(+), G(-)

• Intracellular vs extracellular

• Mixed flora

• w/o PMN, w/o bacteria

• Non-bacteria organisms

Associate environment

• Neutrophils, epithelial cells

• Aid for judgement of G(+), G(-)

• Intracellular vs extracellular

• Mixed flora

• w/o PMN, w/o bacteria

• Non-bacteria organisms

Site specific

• Gram negative cocci– urine:– CSF:– sputum:

• Gram positive bacilli– CSF:– sputum:

• Gram positive cocci– urine:

Site specific

• Gram negative diplococci

– urine: N. gonorrheae– CSF: N. meningitidis– sputum: M. catarrhalis

Site specific

• Gram positive bacilli– CSF: L. monocytogenes– sputum: Corynebaterium straitum

• Gram positive cocci– urine: Enterococcus , Viridans strep.

Immunity - oriented

• Community-acquired

– GNB in CSF– cocco-bacilli in

sputum

• T-cell immunity– GNB in joint

• Nosocomial infection

– GNB in CSF– cocco-bacilli in

sputum

• T-cell immunity– GPB in CSF

Immunity - oriented

• Community-acquired

– GNB in CSF

• K. pneumoniae

– cocco-bacilli sputum

• H. influenzae

• Nosocomial infection

– GNB in CSF• P. aeruginosa• Enterobactericeae

– cocco-bacilli in sputum• A. baumannii

Immunity - oriented

• T-cell immunity– GNB in joint

– Salmonella spp.

• T-cell immunity– GPB in CSF

– L. monocytogenes

Acid Fast Stain

• Ziehl-Neelsen stain

• Kinyoun modification

– Mycobacteria

– Nocardia, partial stain

– NOT for actinomycosis