Methods Of Identification Flashcards
SMEAR PREPARATION
Microbes can be viewed in the ff. states:
Living state
Fixed state
• Visualize size, shape & arrangement
• Check motility
Examples:
WET MOUNTS
HANGING DROP METHODS
LIVING STATE
FIXED STATE
METHODS OF FIXATION
HEAT FIXATION
METHANOL FIXATION
PURPOSE of FIXED STATE
• Kills & preserves the organism
• Anchors the smear to the slide
STAINING
MICROBIAL STAINING METHODS:
Direct
Indirect
Types of Direct staining
Differential
Simple
Special
Bg is stained
Indirect staining
India ink
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
• 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
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
It is a special staining process for the identification of capsules.
INDIA INK
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
DIFFERENTIAL STAINING
This includes…
gram staining, and acid-fast staining method
MOST COMMON staining technique employed in the microbiology lab
Differential staining
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
Preserves the overall morphology but
NOT the internal structures
HEAT FIXATION
• 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
Capsule
(WHAT-GIM?)
Welch
Hiss
Anthony’s Stain
Tvler
Gin
India ink
Muir
Cell wall
Dyar
Metachromatic Granules (ALL-N)
Albert’s
Ljubinsky
Loeffler Alkaline Methylene Blue
(LAMB)
Neisser’s
Flagella (GLF)
Gray
Leifson’s
Fischer-Conn
Endospore (DoS-WiG)
Dorner’s
Schaeffer & Fulton
Wirtz-Conklin
Glacial Acetic Acid
- to demonstrate the capsules. You can see that the background is fully colored, while the organisms is colorless
Negative staining method
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
- ZIEHL - NEELSEN METHOD
- KINYOUN METHOD
- PAPPENHEIM’S
- 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
