Methods Of Identification Flashcards by Kyra Catedral

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

Living state
Fixed state

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• Visualize size, shape & arrangement
• Check motility

Examples:
WET MOUNTS
HANGING DROP METHODS

LIVING STATE

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FIXED STATE

METHODS OF FIXATION

HEAT FIXATION
METHANOL FIXATION

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PURPOSE of FIXED STATE

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

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STAINING
MICROBIAL STAINING METHODS:

Direct
Indirect

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Types of Direct staining

Differential
Simple
Special

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Bg is stained

Indirect staining

India ink

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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

Direct staining

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• 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

SIMPLE STAINING

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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

SPECIAL STAINING

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

INDIA INK

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

DIFFERENTIAL STAINING

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DIFFERENTIAL STAINING

This includes…

gram staining, and acid-fast staining method

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MOST COMMON staining technique employed in the microbiology lab

Differential staining

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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

INDIRECT STAINING

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Preserves the overall morphology but
NOT the internal structures

HEAT FIXATION

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• 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

METHANOL FIXATION

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Capsule
(WHAT-GIM?)

Welch
Hiss
Anthony’s Stain
Tvler
Gin
India ink
Muir

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Cell wall

Dyar

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Metachromatic Granules (ALL-N)

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

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Flagella (GLF)

Gray
Leifson’s
Fischer-Conn

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Endospore (DoS-WiG)

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

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

Negative staining method

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

Gram positive
Gram negative

Developed gram staining

Hans Christian Gram

Gran staining reagents and stains

Crystal violet Grams Iodine Acetone alcohol Safranin

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

Crystal Violet - Primary Stain

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

Gram's lodine - Mordant

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.

Acetone Alcohol - Decolorizer

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

Safranin - Counterstain

Hans orig reagent/ stain

Methyl violet Acetone Dilute carbolfuchsin

Modifications of Gram staining

HUCKER'S MODIFICATION BURKE'S MODIFICATION

HUCKER'S MODIFICATION

CV Gram Iodine Acetone Alcohol Safranin

BURKE'S MODIFICATION

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

All cocci are gram (+) EXCEPT

Neisseria Moraxella Veilonella Branhamella

• All bacilli are gram (-) EXCEPT BCCMLLNE?

Bacillus Corynebacterium Clostridium Mycobacterium Lactobacillus Listeria Nocardia Erysipelothrix

All spiral bacteria are ______when stained.

gram (-)

THEORIES OF GRAM STAINING

MgRNA Theory Benian Theory Stearn & Stearn Theory Lipid content

• 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.

MgRNA Theory

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

Benian Theory

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.

Stearn & Stearn Theory

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.

Lipid Theory

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

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

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

Inadequate decolorization Thick smears

Non-Stain System to Determine True Gram Stain Reaction

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

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

yellow if G(-) Aminopeptidase activity

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

string--like material indicates G(-) organisn

ACID FAST STAINING - responsible for the acid fastness of mycobacteria

• MYCOLIC ACID

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

Mycobacterium Nocardia spp. Corynebacterium spp.

ACID FAST STAINING PAUL EHRLICH in 1882 & later improved by…

ZIEHL and NEELSEN.

AFS Chemicals

Carbolfuchsin Heat Acid alcohol Methylene blue

Ways To Facilitate Acid Fast staining:

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

TYPES OF ACID FAST STAINING

1. ZIEHL - NEELSEN METHOD 2. KINYOUN METHOD 3. PAPPENHEIM'S 4. BAUMGARTEN'S

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

ZIEHL - NEELSEN METHOD

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

KINYOUN METHOD

• resolic acid in alcohol = decolorizer • Differentiates M. tb from M. lacticala and M. smegmatis • M. lacticala and M. smegmatis (blue) • M. tb (red)

PAPPENHEIM'S

PAPPENHEIM’S METHOD •________= decolorizer • Differentiates M. tb from____ and _____ • M. lacticala and M. smegmatis (color) • M. tb (color)

resolic acid in alcohol M. lacticala and M. smegmatis blue red

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

BAUMGARTEN'S

BAIGARTEN’s •_______ = primary stain • Differentiates blue_____& red_____

diluted alcoholic fuchsin M. tb M. leprae