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[MT 6320] Clinical Bacteriology Laboratory > [2S] UNIT 4 Smear Prep & Staining Tech (Exp 6) > Flashcards

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

1
Q

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

A

Gram staining

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2
Q

2 TYPES OF SMEAR

Prepared from colonies growing from plated media

A

Indirect Smear

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2
Q

GRAM STAINING

Gr (+) bacteria : stained?

A

Purple

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3
Q

GRAM STAINING

Gr (-) bacteria is stained?

A

Red

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4
Q

Based on the reaction of bacterial cell wall on staining solutions

A

Gram Staining

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4
Q

2 TYPES OF SMEAR

Prepared from clinical specimen

A

Direct Smear

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5
Q

Gram Stain Procedure
Stain:
Mordant:
Decolorizer:
Counterstain:

A

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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6
Q

DIRECT GRAM SMEAR

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

A

purulent or blood-tinged portion

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7
Q

What to do when the smear is too thick?

A

Pull apart method of smear preparation can be performed

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7
Q

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

A

thumb; 2x3 cm

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8
Q

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

A

4-5 times

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9
Q

Alternative for heat fixing in direct gram smear

A

Flooding with absolute methanol for 1 minute

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10
Q

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

A

F; cells should not be overlapping

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10
Q

Adding 1-2 drops of NSS on the slide

A

Indirect Gram Smear

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11
Q

Shape of the smear

A

oval

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12
Q

INDIRECT GRAM SMEAR (+NSS)

Fish out ___ similar looking colonies emulsify with NSS

A

1-2

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13
Q

INDIRECT GRAM SMEAR

Air dry → Pass through the flame ____

A

3 - 4 x

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14
Q

GRAM STAINING

Examine in ______ / ______ scanning

A

Horizontal / Vertical

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15
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Microscopic structures present

A

HERP

Host cellular material
Epithelial cells
RBCs
PMNs

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16
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Cells Under LPO = 1+

A

1+ = </LPF

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17
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Cells Under LPO = 4+

A

4+ = >25/LPF

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17
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Cells Under LPO = 2+

A

2+ = 1-9/LPF

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17
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Cells Under LPO = 3+

A

3+ = 10-25/LPF

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18
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Bacteria Under OIO = 1+

A

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

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19
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Bacteria Under OIO = 2+

A

2+ (few) = 1-5/OIF

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20
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Bacteria Under OIO = 3+

A

3+ (moderate) = 6-30/OIF

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21
Q

Principle: For the detection of Tubercle bacilli in sputum specimen

A

Acid Fast Staining

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22
Q

MICROSCOPIC EXAMINATION : DIRECT STAINING

Enumeration of Bacteria Under OIO = 4+

A

4+ (heavy) = >30/OIF

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23
Q

AFS

Causative agent of Mycobacterium Tuberculosis (PTB & EPTB)

A

Tubercle bacilli

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24
Q

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

A

Acid Fast Staining: Tubercle bacilli

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25
Q

AFS

Alternative for Direct Sputum Smear Microscopy

A

GeneXpert (Nucleic Acid Amplification Test)

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26
Q

AFS

Primary diagnostic and monitoring tool for TB

A

Direct Sputum Smear Microscopy

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27
Q

AFS specimen collection and time

A

Collected preferably early
morning

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28
Q

AFS

A sputum should be?

A

A sputum should be thick, cloudy, and sticky

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29
Q

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

A

purulent

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30
Q

AFS SMEAR PREPARATION

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

A

2x3 cm

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31
Q

AFS SMEAR PREPARATION

Smear should be spread properly showing ______ patterns

A

concentric / coil-like

32
Q

AFS SMEAR PREPARATION

Air dry → Pass over the flame ____ for ____ seconds

A

2 - 3 x, 2 - 3 seconds

33
Q

AFS STAINING PROCEDURE

Placed the fixed smears on at least ___ apart

34
Q

AFS STAINING PROCEDURE

Stain used

A

0.3% Ziehl’s Carbol Fuchsin

35
Q

AFS STAINING PROCEDURE

After __ mins, remove filter paper and rinse with __________

A

10; distilled water

36
Q

AFS STAINING PROCEDURE

Decolorizer

A

Acid-alcohol, 3 mins / until red disappears

36
Q

AFS STAINING PROCEDURE

Counterstain

A

0.3% Methylene blue / Malachite Green, 1 min

37
Q

MICROSCOPIC EXAMINATION : AFS

Read using the ___

38
Q

MICROSCOPIC EXAMINATION : AFS

Manner of scanning

A

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

39
Q

MICROSCOPIC EXAMINATION : AFS

Microscopic structures present

A

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

40
Q

MICROSCOPIC EXAMINATION : AFS

Enumeration of AFB under OIO = +n

A

+n : 1 - 9 AFB seen in 100 OIF

41
Q

MICROSCOPIC EXAMINATION : AFS

Enumeration of AFB under OIO = 1+

A

1+ : 10-99 AFB seen in 100 OIF

42
Q

MICROSCOPIC EXAMINATION : AFS

Enumeration of AFB under OIO = 2+

A

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

43
Q

MICROSCOPIC EXAMINATION : AFS

Enumeration of AFB under OIO = 3+

A

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

44
Q

AFS

Color of bacterial cells aside from AFB

A

Blue & Green

45
Q

SPECIAL STAINING : CAPSULE

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

A

polysaccharide polymer and
polypeptide

45
Q

AFS

Color and morphology of AFB

A

red or pink bacilli; thin, filamentous or beaded

46
Q

SPECIAL STAINING

Stain used for capsules

A

India ink / Nigrosin

46
Q

SPECIAL STAINING

Appears halo-like against stained amorphous background

47
Q

SPECIAL STAINING

A virulence factor which inhibits phagocytosis

48
Q

SPECIAL STAINING: CAPSULE

Specimen used

A

Klebsiella pneumoniae on a nutrient agar slant or plate

49
Q

CAPSULE STAINING

Place __ drop of India Ink

50
Q

CAPSULE STAINING

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

51
Q

CAPSULE STAINING

T/F: Air-dry → Heat fix & blot dry

A

F; DO NOT HEAT FIX AND BLOT DRY

52
Q

SPECIAL STAINING

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

A

Endospores

53
Q

SPECIAL STAINING

Seen among Bacillus and Clostridium

A

Endospores

54
Q

SPECIAL STAINING

Endospores can be found?

A

centrally, subterminal, or terminal

55
Q

SPECIAL STAINING

Main component of spore

A

Calcium dipicolinate or
dipocolonic acid calcium complex

56
Q

SPECIAL STAINING

Highly resistant to heat, desiccation, and chemical disinfectant

A

Endospores

57
Q

SPECIAL STAINING : SPORE

Technique used

A

Schaeffer-Fulton Method

58
Q

SPECIAL STAINING : SPORE

Specimen used

A

Pure colonies of Bacillus subtilis in a nutrient slant or plate

59
Q

SPECIAL STAINING : SPORE

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

60
Q

SPECIAL STAINING : SPORE

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

A

F; Cover the smear with a strip of filter paper

60
Q

SPECIAL STAINING : SPORE

Air-dry → Heat fix, pass through the flame ___

61
Q

SPECIAL STAINING : SPORE

Alternative for spore staining

A

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

61
Q

SPECIAL STAINING : SPORE

Stain used + Heat slides underneath using an alcohol lamp or cotton dipped in alcohol

A

6.5% Malachite green

62
Q

SPECIAL STAINING : SPORE

Counterstain & Time

A

Safranin O, 30 secs

63
Q

SPECIAL STAINING : SPORE

Endospores stained ____

64
Q

SPECIAL STAINING : SPORE

Vegetative cells stained _____

64
Q

SPECIAL STAINING : FLAGELLAR

Technique

A

Leifson staining technique

65
Q

SPECIAL STAINING : FLAGELLAR

LST Primary stain

A

Basic fuchsin

66
Q

SPECIAL STAINING : FLAGELLAR

LST Mordant

A

Tannic acid

67
Q

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

67
Q

SPECIAL STAINING : FLAGELLAR

LST Counterstain

A

Methylene blue

68
Q

SPECIAL STAINING : FLAGELLAR BACTERIAL SUSPENSION

Properly prepared bacterial suspension = (appearance)

A

slightly cloudy

69
Q

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

70
Q

SPECIAL STAINING : FLAGELLAR STAINING PROCEDURE

Flood smear with Leifson dye solution for ___ minutes

71
Q

SPECIAL STAINING : FLAGELLAR SMEAR PREPARATION

T/F: Air-dry then heat fix

A

F; don’t heat fix

72
Q

SPECIAL STAINING : FLAGELLAR STAINING PROCEDURE

Wash the smear with distilled water when?

A
  1. Film of golden ppt is formed on the dye surface
  2. Precipitates appear on the smear
73
Q

SPECIAL STAINING : FLAGELLAR

Color of bacterial cells and flagella

74
Q

SPECIAL STAINING : FLAGELLAR

devoid of flagella

75
Q

SPECIAL STAINING : FLAGELLAR

flagella on one pole/end

A

Monotrichous

76
Q

SPECIAL STAINING : FLAGELLAR

a tuft/group of flagella on one pole/end

A

Lophotrichous

77
Q

SPECIAL STAINING : FLAGELLAR

flagella on both poles/ends

A

Amphitrichous

78
Q

SPECIAL STAINING : FLAGELLAR

surrounded with flagella

A

Peritrichous

[MT 6320] Clinical Bacteriology Laboratory (15 decks)

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