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Bacte LEC > Methods Of Identification > Flashcards

Methods Of Identification Flashcards

1
Q

SMEAR PREPARATION
Microbes can be viewed in the ff. states:

A

Living state
Fixed state

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2
Q

• Visualize size, shape & arrangement
• Check motility

Examples:
WET MOUNTS
HANGING DROP METHODS

A

LIVING STATE

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3
Q

FIXED STATE

METHODS OF FIXATION

A

HEAT FIXATION
METHANOL FIXATION

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4
Q

PURPOSE of FIXED STATE

A

• Kills & preserves the organism
• Anchors the smear to the slide

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5
Q

STAINING
MICROBIAL STAINING METHODS:

A

Direct
Indirect

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6
Q

Types of Direct staining

A

Differential
Simple
Special

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7
Q

Bg is stained

A

Indirect staining

India ink

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8
Q

are techniques which allow the stain to come in contact with the organism, to be identified or objectively viewed.

Directly color the organism and leave the background colorless

A

Direct staining

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9
Q

• Employ the use of ONE COLOR or one stain like crystal violet or methylene blue to be able to achieve the colored appearance of a fixed smear

• However, it is not widely used in the laboratory

A

SIMPLE STAINING

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10
Q

Can either be DIRECT or INDIRECT

Used to highlight or showcase or demonstrate the presence of special structures that cannot be ordinarily seen or regularly seen in gram-stained slides

A

SPECIAL STAINING

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11
Q

It is a special staining process for the identification of capsules.

A

INDIA INK

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12
Q

Employ the use of several chemicals, several stains to SHOW CONTRAST and DIFFERENTIATION of organisms that may be found in a single smear

A

DIFFERENTIAL STAINING

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13
Q

DIFFERENTIAL STAINING

This includes…

A

gram staining, and acid-fast staining method

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14
Q

MOST COMMON staining technique employed in the microbiology lab

A

Differential staining

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15
Q

BACKGROUND IS STAINED leaving the organism to be identified as colorless

In effect, It is actually helpful because the colorless organism is highlighted against the dark background

A

INDIRECT STAINING

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16
Q

Preserves the overall morphology but
NOT the internal structures

A

HEAT FIXATION

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17
Q

• Prevent lysis of RBCs

Useful in avoiding damage to all host cells and it results in a cleaner background compared to when we do heat fixation

A

METHANOL FIXATION

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18
Q

Capsule
(WHAT-GIM?)

A

Welch
Hiss
Anthony’s Stain
Tvler
Gin
India ink
Muir

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19
Q

Cell wall

A

Dyar

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20
Q

Metachromatic Granules (ALL-N)

A

Albert’s
Ljubinsky
Loeffler Alkaline Methylene Blue
(LAMB)
Neisser’s

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21
Q

Flagella (GLF)

A

Gray
Leifson’s
Fischer-Conn

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22
Q

Endospore (DoS-WiG)

A

Dorner’s
Schaeffer & Fulton
Wirtz-Conklin
Glacial Acetic Acid

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23
Q
  • to demonstrate the capsules. You can see that the background is fully colored, while the organisms is colorless
A

Negative staining method

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24
Q

GRAM STAINING
Based on the thickness & chemical composition of the cell wall
• It can differentiate bacterial agents into two general group

A

Gram positive
Gram negative

25
Q

Developed gram staining

A

Hans Christian Gram

26
Q

Gran staining reagents and stains

A

Crystal violet
Grams Iodine
Acetone alcohol
Safranin

27
Q

Stains both gram positive and negative cells will purple because the dye enters the wall of both cell types.

A

Crystal Violet - Primary Stain

28
Q

Forms large crystals with a dye that are too large to escape out through the cell wall.

Locked together with crystal violet, teichoic acid, MgRNA

A

Gram’s lodine - Mordant

29
Q

Dehydrates the peptidoglycan layer of gram positive cells, making the pores close.

The crystal violet-iodine complex becomes trapped in the peptidoglycan layer.

In gram negative bacteria, it would create pores on the lipopolysaccharide layer, allowing the acetone alcohol to enter into the thin peptidoglycan layer.

A

Acetone Alcohol - Decolorizer

30
Q

Gram negative bacteria will absorb the color of the counterstain which is red.

A

Safranin - Counterstain

31
Q

Hans orig reagent/ stain

A

Methyl violet
Acetone
Dilute carbolfuchsin

32
Q

Modifications of Gram staining

A

HUCKER’S MODIFICATION
BURKE’S MODIFICATION

33
Q

HUCKER’S MODIFICATION

A

CV
Gram Iodine
Acetone Alcohol
Safranin

34
Q

BURKE’S MODIFICATION

A

Crystal violet
Gram’s iodine
Sodium bicarbomate + ether acetone
Safranin

35
Q

All cocci are gram (+) EXCEPT

A

Neisseria
Moraxella
Veilonella
Branhamella

36
Q

• All bacilli are gram (-) EXCEPT

BCCMLLNE?

A

Bacillus
Corynebacterium
Clostridium
Mycobacterium
Lactobacillus
Listeria

Nocardia
Erysipelothrix

37
Q

All spiral bacteria are ______when stained.

A

gram (-)

38
Q

THEORIES OF GRAM STAINING

A

MgRNA Theory

Benian Theory

Stearn & Stearn Theory

Lipid content

39
Q

• A compound of MgRNA and a basic protein, concentrated at the cell wall, helps gram-positivenbacteria to retain the primary dye.
• However, this material is not found in the cell membrane of gram-negative organisms.

• If MgRNA is present, it binds well with the CV lodine complex, creating an insoluble compound.

A

MgRNA Theory

40
Q

Cell walls of gram-positive organisms are less permeable, so some substances are not easily absorbed into the thick peptidoglycan layer especially when another substance is trapped (e.g., crystal violet).

However, the lipopolysaccharide layer of gram negative organisms is sensitive to acetone

A

Benian Theory

41
Q

This has something to do with the isoelectric points and pH.

Gram-positive organisms - low isoelectric point making them acidic. When a material is acidic, it binds perfectly with basic dyes (e.g., CV)

Gram negative organisms - their cell walls have increased isoelectric points, making them basic. Hence, the basic dye does not bind well.

A

Stearn & Stearn Theory

42
Q

The cell wall of gram-negative bacteria contains lipopolysaccharide (outer membrane) which is lipid.

But it does NOT HAVE TEICHOIC ACID, so lipids are easily penetrated by the action of acetone alcohol, allowing materials to come in and out of the cell walls.

A

Lipid Theory

43
Q

ERRORS IN GRAM STAINING
Gram (+) becomes Gram (-)

A

Using acidic Gram’s iodine

Aging, dying, autolysis, overheating

Removal of MgRNA w/ precipitation from bile salts media

Low concentration of crystal violet

Over decolorization

Excessive washing, excessive counterstaining

44
Q

ERRORS IN GRAM STAINING

Gram (-) becomes Gram (+)

A

Inadequate decolorization

Thick smears

45
Q

Non-Stain System to Determine True
Gram Stain Reaction

A

• L- alanine, 4 - nitroanilite (LANA)
• 3% KOH String Test

46
Q

L- alanine, 4 - nitroanilite (LANA)
• Turns______
•_______-> cause release of 4 nitroaniline from the reagent - yellow

A

yellow if G(-)

Aminopeptidase activity

47
Q

• 3% KOH String Test
• Formation of______
• Dilute alkali - Lysis -> release of cellular DNA - viscous (suspension)

A

string–like material indicates G(-) organisn

48
Q

ACID FAST STAINING
- responsible for the acid fastness of mycobacteria

A

• MYCOLIC ACID

49
Q

GENERAL RULE:
All organisms are non-acid fast EXCEPT:
• - longest chain of mycolic acid
• - short cell wall bound mycolic acid
• - shortest chain of mycolic acid

A

Mycobacterium

Nocardia spp.

Corynebacterium spp.

50
Q

ACID FAST STAINING

PAUL EHRLICH in 1882 & later improved by…

A

ZIEHL and NEELSEN.

51
Q

AFS Chemicals

A

Carbolfuchsin
Heat
Acid alcohol
Methylene blue

52
Q

Ways To Facilitate Acid Fast staining:

A

• Using steam
• Increase concentration of phenol & basic fuchsin
• Prolonged contact time
• Adding wetting agent (tergitol)

53
Q

TYPES OF ACID FAST STAINING

A
  1. ZIEHL - NEELSEN METHOD
  2. KINYOUN METHOD
  3. PAPPENHEIM’S
  4. BAUMGARTEN’S
54
Q

• uses Heat (hot stain)
• red AFB
• blue Non- AFB

A

ZIEHL - NEELSEN METHOD

55
Q

• Uses wetting agents
• red AFB
• green / blue Non - AFB
(Malachite green/ Methylene blue)

A

KINYOUN METHOD

56
Q

• resolic acid in alcohol = decolorizer

• Differentiates M. tb from M. lacticala and M. smegmatis

• M. lacticala and M. smegmatis (blue)

• M. tb (red)

A

PAPPENHEIM’S

57
Q

PAPPENHEIM’S METHOD

•________= decolorizer

• Differentiates M. tb from____ and _____

• M. lacticala and M. smegmatis (color)

• M. tb (color)

A

resolic acid in alcohol

M. lacticala and M. smegmatis

blue

red

58
Q

• diluted alcoholic fuchsin = primary stain
• Differentiates blue M. tb & red M. leprae

A

BAUMGARTEN’S

59
Q

BAIGARTEN’s

•_______ = primary stain
• Differentiates blue_____& red_____

A

diluted alcoholic fuchsin

M. tb

M. leprae

Bacte LEC (29 decks)

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