Exam 2 Lab Flashcards
Simple stains
composed of a single basic dye, such as crystal violet, safranin, methane blue, malachite green, carbolfuchsin, and brilliant green
Basic dyes- simple stains
they work best under basic (higher pH) conditions and are more commonly used than acidic dyes as most cells are negatively charged
Negative (acid) stains
stain the background and leave cells colorless with the color’s eosin-y, nigrosine, cobra red, and picric acid; stains the background by having a negative charge toward acidic dyes and cells
Endospore Stain
cannot be stained normally because the walls are impermeable to room temperature. By staining toluidine blue and waiting 15 minutes, the endospores are ready to be examined
Capsule Stain
First the bacteria need to be stained with crystal violet, then the whole slide needs to be dipped into 20% copper sulfate to stain the background
Flagella Stains
They cannot be seen normally because they are extremely thin and invisible. Therefore, by using mordant tannic acid, we would be able to increase the diameter and stain the bacteria properly.
Gram stain
Hans Christian Gram produced the most frequently used differential stain, that differentiates between two large groups Gram postive and Gram negative; used first to identify bacterial pathogens
Acid - Fast Stain
Using acid alcohol as decolorizer and mycolic acid being the mordant.
Mannitol Salt Agar (MSA)
Active ingredients: 7.5% salt (NaCl), mannitol (sugar alcohol), phenol red (pH indicator)
Selective: Staphylococcus (salt inhibits most other bacteria)
Differential for: Pathogenic Staphylococcus (S. aureus)
(fermentation of mannitol = yellow colonies)
MacConkey Agar (Mac)
Active ingredients: crystal violet (dye) & bile salts (G.I.)
lactose (sugar) & neutral red (pH indicator)
Selective for: Gram negative bacteria (crystal violet and bile salts inhibits G+)
Differential for: coliform bacteria-ferment lactose
“colorless” colonies = non-coliforms
red colonies = coliforms
Eosin Methylene Blue Agar (EMB)
Active ingredients: eosin & methylene blue (dyes)
lactose & sucrose (sugars)
Selective for: Gram negative bacteria (eosin and methylene blue inhibits G+)
Differential for: coliform bacteria
“colorless” colonies = non-coliform bacteria
red colonies = likely fermenting only one sugar either lactose or sucrose
purple/black or metallic green =
coliform bacteria (strong fermentation of both sugars)
Salmonella and Shigella Agar
Active ingredients: bile salts, brilliant green, & sodium citrate
ferric citrate & sodium thiosulfate
lactose & neutral red
Selective for: Gram negative bacteria
Differential for: Salmonella sp. = black pigment-combination of ferric citrate and sodium thiosulfate
coliform bacteria = red colonies fermenting lactose
“colorless” colonies = possible Shigella sp.
Staining
most microorganisms are difficult to view by bright field microscopy, coloring specimens with stain increases contrast and resolution, kills microbes, and must be prepared for staining
Wet mounts
a drop of medium containing organisms is placed on a slide and used to view living microorganisms
Smears
Microorganisms are spread onto the surface of a glass slide and used to view destroyed organisms
Heat fixation
kills the organisms, causes organism to adhere (stick) to slide, and alters organism to accept stains (dyes)
Preparing a Specimen for Staining
Spread culture in thin film over slide in combination with water, air dry, and pass slide through heat to fix it quickly
Chromophore
colored portion of the dye
Differential stain
makes use of two or more dyes and distinguishes between organisms based on structural differences; they are gram stain, ziehl-neelson stain, Kinyoun stain, and Schaeffer Fulton stain
Gram Stain
Add crystal violet and wait for 1 minute, drain and rinse.
Add mordant which is iodine and wait for 1 minute, drain and rinse.
Add decolorizer with alcohol and run until comes clear, rinse with water afterwards. The gram negatives should be clear and gram positives purple.
Add safranin and wait for 1 minute, the gram negatives should be pink and gram positives should be purple. Drain, rinse with water, and blot.
