Different Staining Procedures

Aquitania

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 Cells 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

Grams Staining
Most important staining Hans Christian Joachim Gram Fundamental step in diagnosis & treatment of diseases Determine the most effective antibiotic for critically ill patients

Steps
Initial stain Mordant Decolorizer Counter stain Gram positive Purple Gram negative - Red

Reagent
Crystal violet Iodine Alcohol or Acetone alcohol Safranin

Remarks
Colors cytoplasm purple regardless w/ cell type Combines of crystal violet to formretained by Purple dye is an insoluble complex insidecells cell is Gram positive the but Stains the colorless Gram readily removed from negative bacteria red Gram Gram positive cells while negative cells remain purple

Grams Staining

Grams Staining

Figure (1). Grams staining procedure. Gram positive bacteria is E. coli and Gram negative short bacilli is B. subtilis.

Grams Staining Interpretation

Bacteria are qualified as follows: <5 bacteria cell/OIF 5 10 bacteria cell/OIF 16-25 bacteria cell/OIF 26-35 bacteria cell/OIF >35 bacteria cell/OIF Respiratory Sputum Evaluation <10 squamous epithelial cell/LPF >10 squamous epithelial cell/LPF Occasional +1 +2 +3 +4

Acceptable Unaceptable

Grams Staining
Gram positive
Gram reaction Peptidoglycan layer Teichoic acid LPS content Periplasmic space Outer membrane Lipid & lipoprotein content structure Flagellar Toxins produced Purple color thick present absent absent absent low 2 rings in basal body exotoxin

Gram negative
Red color thin absent present present present High 4 rings in basal body endotoxin

Gram positive

Gram negative
low low low high low low low low

Grams Staining

Resistance to physical high disruptiondisruption by Cell wall high lysozyme Susceptibility to penicillin high & sulfonamide Susceptibility to low streptomycin,basic dyes high Inhibition by chlorampenicol & Susceptibility to anionic high tetracycline detergents to sodium Resistance high azide Resistance to drying high

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

Steps

Initial stain Carbolfuchsin Mordant Physical Steam/heat Decolorizer Acid-alcohol Chemical Phenol Counter stain Methylene blue Nonacid fast bacilli - blue Acid fast bacilli - red

Reagent

Remarks

All cells are colored red Fixes the stain & facilitates entrance of Nonacid fast are the dye decolorized; acid fast Stains the colorless non remains colored red acid fast blue while acid fast remains colored red

Acid Fast Bacilli Staining

Figure (2). Ziehl-Neelsen stain of the cid-fast bacilli Mycobacterium tuberculosis. Placental thrombus. Media from CDC Public Health Image Library

Acid Fast Bacilli Staining

AFB Staining Interpretation

National 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 >10 squamous epithelial cell/LPF

Acceptable 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