Chapter 2: Tools of the Laboratory Flashcards
The Five I’s
Inoculation Incubation Isolation Inspection Identification
Culture
the propagation of microorganisms with various media
Medium
a nutrient used to grow microorganisms outside their natural habitat
Inoculation
the implantation of microorganisms into or onto culture media
Usual incubation temperatures
between 20°C and 40°C
Pure Culture
container of medium that grows only a single known species
Mixed Culture
Contains two or more identifiable species
Contaminated Culture
Was once pure or mixed, but has since been contaminated; can’t identify all the organisms
Physical States of Media
liquid semisolid solid (can be converted to liquid) solid (cannot be liquefied)
Agar
complex polysaccharide from the alga Gellidium; liquefies at 100°C and solidifies at 42°C and can be poured in liquid form that will not harm the microbe or the handler
General Purpose Media
Grows a broad spectrum of microbes; complex; Nutrient agar, broth, brain-heart infusion, & TSA
Selective Media
Contains one or more agents that inhibit growth of certain microbes but not others; for samples containing dozens of different species (saliva, skin, water, soil)
Enriched Media
Contains complex organic substances; for growing certain species (fastidious microbes); Blood agar, Chocolate agar
Differential Media
Allows multiple types of microorganisms to grow; causes different reactions (dyes make microorganisms easily identifiable)
Transport Media
Used to maintain and preserve specimens that have to be held for a period of time before clinical analysis; also used to sustain species that die quickly
Reducing Media
Contains sodium thioglycollate or cystine that absorbs oxygen or slows the penetration of oxygen; grows anaerobic bacteria or determines oxygen requirements of isolates
Carbohydrate Fermentation Media
Contains sugars that can be fermented (converted to acids) and a pH indicator to show this reaction; identifies bacteria and fungi
Streak Plate
Easy and effective; method of choice
Loop Dilution
Allows colonies to grow deep in the solution, not just on the surface
Viruses measurements
between 20 – 800 nm
Smallest bacteria measurements
200 nm
Protozoa and Algae measurements
3-4 mm
Objective Lens
closest to the specimen, forms the initial image called the real image
Ocular Lens
forms the second image called the virtual image that will be received by the eye and converted to the retinal and visual image
Resolution
the capacity of an optical system to distinguish or separate two adjacent objects or points from one another.
Human eye can resolve objects that are no closer than___________apart
0.2 mm
Oil Immersion Lens
uses oil to capture light that would otherwise be lost to scatter which in turn increases resolution; oil immersion lens can resolve images that are at least 0.2 μm in diameter and at least 0.2 μm apart
Bright-field Microscope
Forms its image when light is transmitted through the specimen; produces image that is darker than the surrounding illuminated field.
Dark-field Microscope
Puts a “block” on a bright-field microscope that blocks all light from entering the objective lens except peripheral light; creates image of brightly illuminated specimens surrounded by a dark field
Phase-Contrast Microscope
Transforms subtle changes in light waves passing through specimen into differences in light intensity (different parts of specimen have different densities); best for internal detail
Interference Microscope
Manipulates light like phase-contrast but adds contrasting colors to the image and two beams of light rather than a single one; produces extremely well-defined images that are vividly colored and appear 3-D
Fluorescent Microscope
Modified compound microscope with an Ultraviolet radiation source and a filter that protects the viewer’s eye; specimen must be coated with a source of fluorescence
Confocal Microscope
Overcomes problem of cells or structures being too thick causing microscopes to have trouble focusing; Uses a laser beam of light to scan various depths in the specimen; captures highly focused view at any level
Transmission Electron Microscope
Produces image by transmitting electrons through the specimen; specimens must be sectioned in extremely thin slices and stained or coated with metals
Scanning Electron Microscope
Bombards surface of a whole metal-coated specimen with electrons while scanning back and forth over it; electron pattern is displayed on a TV screen; color is added afterwards
Wet Mount
consists of a drop or two of culture placed on a slide and overlaid with a cover slip
Hanging Drop
a drop of culture is placed in a concave (depression) slide, Vaseline adhesive or sealant, and cover slip are used to suspend the sample
Purpose of short term mounts
provide a true assessment of size, shape, arrangement, color, and motility
Steps in Smear Technique
(1) spread a thin film made from a liquid suspension of cells on a slide (2) air dry (3) heat fix: heat gently to kill the specimen and attach to the slide
Basic dyes have a ________ charge
positive
Acidic dyes have a ________ chage
negative
Positive Stain
dye sticks to the specimen and gives it color
Negative Stain
does not stick to the specimen but settles some distance from its outer boundary, forming a silhouette
Simple Stain
only require a single dye and an uncomplicated procedure; causes all the cells in the smear to appear more or less the same color, regardless of type; reveal shape, size, and arrangement
Differential Stain
use two differently colored dyes: the primary dye and the counterstain
Crystal Violet
Primary Stain in Gram Staining
Iodine
The mordant in Gram Staining
Alcohol Rinse
the decolorizer in Gram staining
Safranin
the counterstain in Gram staining
Gram-Positive Color
Blue/purple
Gram-Negative Color
Pink/red
Acid Fast Stain
differentiates acid-fast bacteria (pink) from non-acid-fast bacteria (blue)
Endospore Stain
Forced by heat into resistant bodies called spores; distinguishes between spores and vegetative cells
Capsule Stain
used to observe the microbial capsule, an unstructured protective layer surrounding the cells of some bacteria and fungi
Flagellar Stain
flagella are enlarged by depositing a coating on the outside of the filament and then staining it
