Different Staining ProceduresAquitania

Staining Preparation

A. Unstained/unfixed preparation•WET MOUNT & HANGING DROP•Natural condition suspended in fluid•BF, DF & PC microscopes•Motility: Brownian movement & true

motility•Morphology

B. Fixed/Stained Preparation

•SMEAR•HEAT FIXED – Cell’s molecules change

shape•CHEMICAL FIXATION – No destruction of

structure

•STAINING•Increase visibility•Reveal additional information about the

bacteria

•DYE (salt)▫Chromophore▫Color of basic dye positive ion▫Color of acidic dye negative ion

▫Bacteria: slightly negatively charged at pH 7

▫The colored positive ion in a basic dye is attracted to the negatively charged bacterial cell

Staining Procedure

Type of Staining

•SIMPLE

•DIFFERENTIAL

Type of Staining

•SIMPLE •Single dye•Cells & structures stain the same color•Characteristics of size, shape & cell

arrangement

Type of Staining

•DIFFERENTIAL•More than one dye•Distinguish between structures within a

cell; diff. cell types

•React with specific microbial structures

Gram’s Staining

•Most important staining•Hans Christian Joachim Gram•Fundamental step in diagnosis &

treatment of diseases•Determine the most effective antibiotic

for critically ill patients

Gra

m’s S

tain

ing

Steps Reagent RemarksInitial stain Crystal violet Colors cytoplasm

purple regardless of cell type

Mordant Iodine Combines w/ crystal violet to form an

insoluble complex inside the cell

Decolorizer Alcohol or Acetone alcohol

Purple dye is retained by Gram

positive cells but is readily removed from Gram negative cells

Counter stain Safranin Stains the colorless Gram negative

bacteria red while Gram positive cells

remain purple

Gram positive – PurpleGram negative - Red

Gra

m’s S

tain

ingFigure (1). Gram’s staining procedure. Gram positive bacteria is

E. coli and Gram negative short bacilli is B. subtilis.

Gram’s Staining InterpretationBacteria are qualified as follows:

<5 bacteria cell/OIF Occasional

5 – 10 bacteria cell/OIF +1

16-25 bacteria cell/OIF +2

26-35 bacteria cell/OIF +3

>35 bacteria cell/OIF +4

Respiratory Sputum Evaluation

<10 squamous epithelial cell/LPF

Acceptable

>10 squamous epithelial cell/LPF

Unaceptable

Gram’s StainingGram positive Gram negative

Gram reaction Purple color Red color

Peptidoglycan layer thick thin

Teichoic acid present absent

LPS content absent present

Periplasmic space absent present

Outer membrane absent present

Lipid & lipoprotein content

low High

Flagellar structure 2 rings in basal body 4 rings in basal body

Toxins produced exotoxin endotoxin

Gram’s Staining

Gram positive Gram negativeResistance to physical disruption

high low

Cell wall disruption by lysozyme

high low

Susceptibility to penicillin & sulfonamide

high low

Susceptibility to streptomycin, chlorampenicol & tetracycline

low high

Inhibition by basic dyes high low

Susceptibility to anionic detergents

high low

Resistance to sodium azide

high low

Resistance to drying high low

Acid Fast Bacilli Staining

•Ziehl-Neelsen•Franz Ziehl & Friedrich Neelsen •ID: genus Mycobacterium•Mycobacteria contain high levels of lipid

material (mycolic acid) that repel water-soluble dyes that are difficult to stain by standard procedures

•All mycobacteria are acid fast

Acid

Fast B

acilli S

tain

ing

Steps Reagent RemarksInitial stain Carbolfuchsin All cells are

colored red

MordantPhysicalChemical

Steam/heatPhenol

Fixes the stain & facilitates entrance

of the dye

Decolorizer Acid-alcohol Nonacid fast are decolorized; acid

fast remains colored red

Counter stain Methylene blue Stains the colorless non acid fast blue

while acid fast remains colored

red

Nonacid fast bacilli - blueAcid fast bacilli - red

Acid

Fast B

acilli S

tain

ingFigure (2). Ziehl-Neelsen stain of the cid-fast bacilli Mycobacterium

tuberculosis. Placental thrombus. Media from CDC Public Health Image Library

AFB Staining InterpretationNational Standard Reporting Scale

0 No AFB seen in 300 visual fields

+n 1 – 9 AFB seen in 100 visual fields

1+ 10 – 99 AFB seen in 100 visual fields

2+ 1 – 10 AFB/OIF in at least 50 visual fields

3+ More than 10 AFB/OIF in at lest 20 visual fields

Respiratory Sputum Evaluation

<10 squamous epithelial cell/LPF

Acceptable

>10 squamous epithelial cell/LPF

Unaceptable

KOH Staining• Rapidly diagnose fungal infections

of the hair, skin, or nails

• Differentiate infections produced by dermatophytes and Candida albicans from other skin disorders.

• A sample of the infected area is analyzed under a microscope following the addition of a few drops of potassium hydroxide

KOH Staining

Figure (3). A potassium hydroxide preparation demonstrating branching fungal hyphae typical of a dermatophyte fungus. The arrows point to fungal elements.

KOH Staining

•Additional Tests▫WHIFF TES for Bacterial vaginosis

Anaerobic bacteria = Volatile amines (Fishy odor)

lysine to cadaverine arginine to putrescine trimethylamin oxide to trimethylamine.

KOH Staining

•Additional Tests▫GRAM (+) VS GRAM (-) ID - culture

3% KOH dissolves cell wall of Gram (-) but does not affect Gram (+) [not lysed]

DNA: viscous and has large enough cell mass that it can be seen sticking to or dragging from a loop when touched.

Requires large amt. of visible clumps of cells