4 Visualization

Magnification

-Apparent increase in size of an object and is indicated by a number and by “x”

-Results when a beam of radiation refracts as it passes through a lens

brightfield, darkfield, phase contrast

types of light microscopy

brightfield

-Background is illuminated

-Most elementary form of microscope illumination techniques and is generally used with compound microscopes

-It is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field

darkfield

Specimen is made to appear light against a dark background.

phase contrast

-Described by Frits Zernike

-Use the alignment or misalignment of light waves to achieve the desired contrast between a living specimen and its background

-Enhancing optical technique that can be utilized to produce high contrast images of transparent specimens

Resolution / Resolving Power of the Instrument

- Ability to distinguish between objects that are close together

- Ability of the objective lenses to separate image adjacent details that are present in the object

- Distance between 2 points in the object that result in the image

contrast

- Refers to the differences in intensity between two objects or between an object and its background

- Achieved by the use of stains

fluorescent

Use invisible UV light to cause specimens to radiate visible light, a phenomenon called _____nce

fluorochroming

reaction between the organism and the dye

Immunofluorescence or Fluorescent Antibody Technique (FAT)

Antibody is attached to the dye

electron microscopy

Uses a beam of electron instead of light to see the structure of the bacteria. Most of the bacterial structure inside can be visualized only with the use of electron microscopy.

Transmission (TEM)

● Can resolve particles with 0.001 micrometer in size - Ex: X-ray

● A technique in which a beam of electrons is transmitted through a specimen to form an image

Scanning (SEM)

Scans a focused electron beam over the surface to create an image can be 3d in view

direct wet mount preparation

(Using unstained living state)

used for the examination of motile protozoa and trophozoites

hanging drop preparation

(Using unstained living state)

using concave slides (almost the same as direct wet mount preparation)

intravital staining

(Using unstained living state)

combination of both direct wet mount preparation and hanging drop preparation. A non- toxic dye is introduced to a microorganism and selectively stains certain cells and tissues

smear preparation

(Fixed, Stained State)

use a clean glass slide

air drying

(Fixed, Stained State)

used to preserve the morphology of the organism

fixation

(Fixed, Stained State)

→ Heat, pass it 3 to 5 times under the flame

→ Chemical, immerse it in ethanol

staining

-using only one dye.

-If it is positive simple ___ the bacteria is stained but not the background

-It can also be negative or relief ____ the background is stained not the bacteria

Dilute Carbol fuchsin

example of positive simple staining

India ink method

example of negative staining

special staining

used for structures that are difficult to visualize under ordinary stain

differential staining

uses 2 dyes

gram staining

-Is a staining procedure discovered by Danish Scientist Hans Christian Joachim Gram in 1884

- It differentiates bacteria into two groups.

Neisseria GROUP, Moraxella (formerly Branhamella) catarrhalis (and Veillonella)

General rule for gram positive:

ALL COCCI ARE GRAM POSITIVE (+) EXCEPT THE

ACID FAST ORGANISMS (Mycobacterium, Nocardia), SPOREFORMERS (Bacillus, Clostridium) AND Corynebacterium species

General rule for gram negative (-):

ALL BACILLI ARE GRAM NEGATIVE(-) EXCEPT THE

crystal violet

primary stain

grams iodine

mordant

95% ethanol or acetone

decolorizer

safranin

secondary stain

gram (+) cells

Magnesium ribonucleate is found only in

Zeihl-Neelsen

modified the stain by adding phenol or carbolic acid and basic fuchsin

Cold Kinyoun

does not require heating so the concentration of carbol fuchsin is increased

CARBOL FUCHSIN

primary stain (acid fast)

ACID ALCOHOL

decolorizer (acid fast)

METHYLENE BLUE

counter stain (acid fast)

acridine orange

○ Fluorochrome dye that stains to gram (+) and gram (-) bacteria, living or dead.

○ It binds to the nucleic acid to the cell and fluorescents a bright orange when fluorescent microscope is used

○ It is used to locate bacteria in blood cultures and other specimens where discerning bacteria is otherwise difficult

calcoflour white

○ White fluorochrome that binds to chitin in fungal cell walls, it fluoresces a bright apple green or blue while allowing visualization of fungal structures with a fluorescent microscope

○ Was the original bluing used in high volume laundry to whiten yellow-appearing white cotton and other fabrics

methylene blue

○ Traditionally been used to stain Corynebacterium diphtheriae for the observation of metachromatic granules.

○ Sometimes used to as a simple stain to detect WBC such as in stool samples

lactophenol cotton blue

Used to stain the cell walls of medically important fungi grown in slide culture.

india ink (nigrosin)

○ A negative stain used to visualize capsules surrounding certain yeasts (such as Cryptococcus specie)

○ This fine ink particles are excluded in the capsule leaving a dark background and a clear capsule surrounding the yeast