its da final

Differential Stain

detects differences between organisms through the use of many dyes

and reagents

Example of Differential Stain

Gram Stain

Peptidoglycan

Differentiates bacteria into two groups based on amount of cell wall

Gram Staining: Primary Dye

Crystal Violet

Gram Staining Mordant

Gram’s Iodine

Gram Staining Decolorizer

95% Ethanol

Gram Staining Secondary Dye or Counterstain

Safranin

Gram Negative

Gram Positive

Cationic Dyes Definition

Basic dyes, positively charged chromophores

Cationic Dyes Examples

Methylene Blue or Crystal Violet

Anionic Dyes Definition

Acidic dyes, negatively charged chromophores

Anionic Dyes Examples

Acid Fuchsin, Congo Red, Nigrosin

Nigrosin

Black Anionic Dye

Dye For Simple Stain

Crystal Violet (Cationic)

Illumination of Microscope

Light Source

Condensor

Directs light towards the objective lens in bright field microscopy

Iris diaphragm

Adjusts the diameter of the cone of light so that it just fills the objective lens

Ocular

10x

Objectives

4X, 10X, 40X and 100X

Ocular x Objective

Total Magnification

Resolution

Smallest distance between two objects which can be seen as separate

Oil Immersion

100x

d(Resolution)

λ/(2NA)

Illumination / λ

Wavelength

NA

Numerical Aperture

Numerical Aperture Definition

Light Gathering Capacity of Lens

Morphology

Bacterial Shapes

Staphylococcus

Streptococcus

Bacillus (Rods)

Streptobacillus

Comma Shaped

Vibrio (Cholerae)

Rigid & Spiral

Spirillum Volutans

Pleomorphic

Club Shaped

Acid Fast Staining Type

Differential Stain

Endospore Stain Staining Type

Structural Stain

Structural Stain

Confirm structural characteristics of cells

Acid-Fast/Ziehl-Neelsen Primary Dye

Carbol Fuschin

Acid-Fast/Ziehl-Neelsen Mordant

Steam

Acid-Fast/Ziehl-Neelsen Decolorizer

Acid Alcohol

Acid-Fast/Ziehl-Neelsen Secondary Dye / Counterstain

Methylene Blue

Acid Fast Genera

Mycobacterium

Spore Staining/Schaeffer-Fulton Primary Dye

Malachite Green

Spore Staining/Schaeffer-Fulton Mordant

Steam

Spore Staining/Schaeffer-Fulton Decolorizer

Water

Spore Staining/Schaeffer-Fulton Secondary/Counterstain

Safranin

Green

Free/Endospores

Pink

Vegetative Cells

Gram Positive Rods:
Bacillus (Anthracis)
Clostridium (Botulinum, Tetani, Difficile)

Spore Forming Bacteria

Cerise/Pink/Violet

Acid-Fast Cells (High Mycolic Acid)

Blue

Acid-Fast Negative

Pure Culture

Single Microbial Species

What Streaking is This

Three-Zone

Three Zone Dilutes From

Primary Streak

Subculture from Primary Streak

Secondary Streak

When streaking is considered a pure culture

Secondary Streak

Three-Zone Streak Zones

Support growth of variety of microorganisms

General Purpose Media

Nutrient Agar (NA), Tryptic Soy Agar (TSA), Brain Heart Infusion (BHI)

Example General Purpose Media

Use inhibitors to prevent growth of certain organisms

Selective Media

Phenyl Ethyl Agar (PEA), Eosin Methylene Blue (EMB), MacConkey Agar

Example Selective Media

Allow growth of many microbes, but differentiation is seen by indicators that detect changes that have occurred

Differential Media

EMB, Blood agar, MacConkey Agar

Example Differential Media

Both Selective & Differential

Combination Media

EMB & MacConkey Agar

Example Combination Media

Undefined, exact chemical composition not known

Complex Media

Example Complex Media

BHI, TSA

Known chemical composition for each component

Defined Media

Organisms that have flagella

Motile

Motility Detected by Using

Soft Agar

Organisms that can swim through the soft agar and spread away from the inoculation stab line

Motile

Organisms that remain confined to the path of inoculation by the soft gel of the medium

Non-Motile

Tube Dilution x Plate Dilution

Final Dilution Equation

A / A + B

Dilution Equation

Strong chemical agents used on inanimate surfaces

Disinfectants

Chemical agents that may be safely applied to living tissue

Antiseptics

Compounds that may be administered for systemic circulation

Antibiotics

Kirby Bauer

Standardized antibiotic sensitivity testing method

Zone is measured to determine if an organism is susceptible, resistant, or intermediate to a certain antibiotic

Zone of inhibition

Growth Medium for Kirby-Bauer

Agar Muller-Hinton (MH)

Must be maintained within a narrow range (7.2 to 7.4), pH outside this range can affect the stability and function of the antimicrobials

pH Muller-Hinton (MH)

Some media have factors that can interfere with antimicrobial function

Nutrient Composition Muller-Hinton (MH)

–4 mm, can affect lateral diffusion rate of antibiotics

Agar Depth Muller-Hinton (MH)

Overestimation of bacterial sensitivity

LOW Initial concentration of bacteria inoculated

“Swamp” the antimicrobial, or quickly deplete nutrients, which can affect the growth of the bacteria

HIGH Initial concentration of bacteria inoculated

≤14mm

Resistant

15-17 nm

Intermediate

≥18mm

Susceptible

Antimicrobial in the disk start to diffuse into the agar

Diffusion Area

Highest concentration closest to the disk and lower concentrations farther from the disk

Concentration Gradient

Litmus Turns Pink

Acidic pH

Litmus Result

WEAK Lactose Fermentation Acid

Litmus Result

STRONG Lactose Fermentation Acid

Litmus Result

Alkaline Peptone Deamination

Litmus Result: Color Retention

O2 ReOxidation