[2S] UNIT 4 Smear Prep & Staining Tech (Exp 6) Flashcards by JULIA CASSANDRA RECCION

Principle: Distinguish if the isolate is Gr (+) or Gr (-)

Gram staining

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2 TYPES OF SMEAR

Prepared from colonies growing from plated media

Indirect Smear

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GRAM STAINING

Gr (+) bacteria : stained?

Purple

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GRAM STAINING

Gr (-) bacteria is stained?

Red

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Based on the reaction of bacterial cell wall on staining solutions

Gram Staining

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2 TYPES OF SMEAR

Prepared from clinical specimen

Direct Smear

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Gram Stain Procedure
Stain:
Mordant:
Decolorizer:
Counterstain:

VIAS

Stain: Crystal Violet, 1 min
Mordant: Gram’s Iodine, 1 min
Decolorizer: Acetone-alcohol / 95% Ethanol, 10 secs
Counterstain: Safranin, 30 secs

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DIRECT GRAM SMEAR

Select the ________ or ___________ of the sputum specimen using an applicator stick, pasteur
pipette, or inoculation loop

purulent or blood-tinged portion

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What to do when the smear is too thick?

Pull apart method of smear preparation can be performed

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Direct gram smear must be a _____ size or ____ to form a thin film of smear

thumb; 2x3 cm

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How many times should you pass your direct gram smear through the flame?

4-5 times

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Alternative for heat fixing in direct gram smear

Flooding with absolute methanol for 1 minute

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T/F: Gram staining smear should have pink/red or clear background and cells are overlapping

F; cells should not be overlapping

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Adding 1-2 drops of NSS on the slide

Indirect Gram Smear

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Shape of the smear

oval

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INDIRECT GRAM SMEAR (+NSS)

Fish out ___ similar looking colonies emulsify with NSS

1-2

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INDIRECT GRAM SMEAR

Air dry → Pass through the flame ____

3 - 4 x

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GRAM STAINING

Examine in ______ / ______ scanning

Horizontal / Vertical

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MICROSCOPIC EXAMINATION : DIRECT STAINING

Microscopic structures present

HERP

Host cellular material
Epithelial cells
RBCs
PMNs

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MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Cells Under LPO = 1+

1+ = </LPF

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MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Cells Under LPO = 4+

4+ = >25/LPF

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MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Cells Under LPO = 2+

2+ = 1-9/LPF

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MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Cells Under LPO = 3+

3+ = 10-25/LPF

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MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Bacteria Under OIO = 1+

1+ (rare or occasional) = <1/OIF

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MICROSCOPIC EXAMINATION : DIRECT STAINING Enumeration of Bacteria Under OIO = 2+

2+ (few) = 1-5/OIF

MICROSCOPIC EXAMINATION : DIRECT STAINING Enumeration of Bacteria Under OIO = 3+

3+ (moderate) = 6-30/OIF

Principle: For the detection of Tubercle bacilli in sputum specimen

Acid Fast Staining

MICROSCOPIC EXAMINATION : DIRECT STAINING Enumeration of Bacteria Under OIO = 4+

4+ (heavy) = >30/OIF

AFS Causative agent of Mycobacterium Tuberculosis (PTB & EPTB)

Tubercle bacilli

Resist decolorization w/ acid-alcohol and appear red on a blue / green background after decolorization

Acid Fast Staining: Tubercle bacilli

AFS Alternative for Direct Sputum Smear Microscopy

GeneXpert (Nucleic Acid Amplification Test)

AFS Primary diagnostic and monitoring tool for TB

Direct Sputum Smear Microscopy

AFS specimen collection and time

Collected preferably early morning

AFS A sputum should be?

A sputum should be thick, cloudy, and sticky

AFS SMEAR PREPARATION Using the flattened end of an applicator stick or a flamesterilized loop, pick the _______ portion of sputum sample and place it on the slide

purulent

AFS SMEAR PREPARATION Spread until it forms an oval-shaped smear with _____ dimension

2x3 cm

AFS SMEAR PREPARATION Smear should be spread properly showing ______ patterns

concentric / coil-like

AFS SMEAR PREPARATION Air dry → Pass over the flame ____ for ____ seconds

2 - 3 x, 2 - 3 seconds

AFS STAINING PROCEDURE Placed the fixed smears on at least ___ apart

1 cm

AFS STAINING PROCEDURE Stain used

0.3% Ziehl’s Carbol Fuchsin

AFS STAINING PROCEDURE After __ mins, remove filter paper and rinse with __________

10; distilled water

AFS STAINING PROCEDURE Decolorizer

Acid-alcohol, 3 mins / until red disappears

AFS STAINING PROCEDURE Counterstain

0.3% Methylene blue / Malachite Green, 1 min

MICROSCOPIC EXAMINATION : AFS Read using the ___

OIO

MICROSCOPIC EXAMINATION : AFS Manner of scanning

From left to right go downward a bit and then right to left

MICROSCOPIC EXAMINATION : AFS Microscopic structures present

● Squamous Cells ● Leukocytes ● Mucus Threads ● Bacterial cells aside from AFB ● AFB = red or pink bacilli; thin, filamentous or beaded

MICROSCOPIC EXAMINATION : AFS Enumeration of AFB under OIO = +n

+n : 1 - 9 AFB seen in 100 OIF

MICROSCOPIC EXAMINATION : AFS Enumeration of AFB under OIO = 1+

1+ : 10-99 AFB seen in 100 OIF

MICROSCOPIC EXAMINATION : AFS Enumeration of AFB under OIO = 2+

2+ : 1-10 AFB/OIF in at least 50 fields

MICROSCOPIC EXAMINATION : AFS Enumeration of AFB under OIO = 3+

3+ : >10 AFB/OIF in at least 20 fields

AFS Color of bacterial cells aside from AFB

Blue & Green

SPECIAL STAINING : CAPSULE Pathogenic organisms : protected with a covering made up of (2)?

polysaccharide polymer and polypeptide

AFS Color and morphology of AFB

red or pink bacilli; thin, filamentous or beaded

SPECIAL STAINING Stain used for capsules

India ink / Nigrosin

SPECIAL STAINING Appears halo-like against stained amorphous background

Capsule

SPECIAL STAINING A virulence factor which inhibits phagocytosis

Capsule

SPECIAL STAINING: CAPSULE Specimen used

Klebsiella pneumoniae on a nutrient agar slant or plate

CAPSULE STAINING Place __ drop of India Ink

one

CAPSULE STAINING Place the end of another clean microscope slide at a _______ angle on the slide containing

30 degree

CAPSULE STAINING T/F: Air-dry → Heat fix & blot dry

F; DO NOT HEAT FIX AND BLOT DRY

SPECIAL STAINING Multilayered ovoid or spherical structures present inside Gr (+) bacteria

Endospores

SPECIAL STAINING Seen among Bacillus and Clostridium

Endospores

SPECIAL STAINING Endospores can be found?

centrally, subterminal, or terminal

SPECIAL STAINING Main component of spore

Calcium dipicolinate or dipocolonic acid calcium complex

SPECIAL STAINING Highly resistant to heat, desiccation, and chemical disinfectant

Endospores

SPECIAL STAINING : SPORE Technique used

Schaeffer-Fulton Method

SPECIAL STAINING : SPORE Specimen used

Pure colonies of Bacillus subtilis in a nutrient slant or plate

SPECIAL STAINING : SPORE T/F: NSS is used to emulsify the fished out colonies

SPECIAL STAINING : SPORE T/F: After heat fix, do not cover the smear with a strip of filter paper

F; Cover the smear with a strip of filter paper

SPECIAL STAINING : SPORE Air-dry → Heat fix, pass through the flame ___

3 - 5x

SPECIAL STAINING : SPORE Alternative for spore staining

o Stream the slide over a container of boiling water for 5-10 mins o Make sure the smear does not dry out o Add stain if it's getting dry o Stain for 10 minutes

SPECIAL STAINING : SPORE Stain used + Heat slides underneath using an alcohol lamp or cotton dipped in alcohol

6.5% Malachite green

SPECIAL STAINING : SPORE Counterstain & Time

Safranin O, 30 secs

SPECIAL STAINING : SPORE Endospores stained ____

green

SPECIAL STAINING : SPORE Vegetative cells stained _____

red/pink

SPECIAL STAINING : FLAGELLAR Technique

Leifson staining technique

SPECIAL STAINING : FLAGELLAR LST Primary stain

Basic fuchsin

SPECIAL STAINING : FLAGELLAR LST Mordant

Tannic acid

SPECIAL STAINING : FLAGELLAR BACTERIAL SUSPENSION Emulsify the picked portion of an isolated colony in a clean microcentrifuge tube containing ____ distilled water by gently mixing it or using a vortex

10 mL

SPECIAL STAINING : FLAGELLAR LST Counterstain

Methylene blue

SPECIAL STAINING : FLAGELLAR BACTERIAL SUSPENSION Properly prepared bacterial suspension = (appearance)

slightly cloudy

SPECIAL STAINING : FLAGELLAR SMEAR PREPARATION Using a serological pipette or automatic pipettor, place approximately ____ of the bacterial suspension on the slide and spread evenly onto the slide

10 uL

SPECIAL STAINING : FLAGELLAR STAINING PROCEDURE Flood smear with Leifson dye solution for ___ minutes

7 - 15

SPECIAL STAINING : FLAGELLAR SMEAR PREPARATION T/F: Air-dry then heat fix

F; don't heat fix

SPECIAL STAINING : FLAGELLAR STAINING PROCEDURE Wash the smear with distilled water when?

1. Film of golden ppt is formed on the dye surface 2. Precipitates appear on the smear

SPECIAL STAINING : FLAGELLAR Color of bacterial cells and flagella

Red

SPECIAL STAINING : FLAGELLAR devoid of flagella

Atrichous

SPECIAL STAINING : FLAGELLAR flagella on one pole/end

Monotrichous

SPECIAL STAINING : FLAGELLAR a tuft/group of flagella on one pole/end

Lophotrichous

SPECIAL STAINING : FLAGELLAR flagella on both poles/ends

Amphitrichous

SPECIAL STAINING : FLAGELLAR surrounded with flagella

Peritrichous