Simple Stains Flashcards
Chromophore
the portion of the stain that gives it a color
Heat fixing
process performed on a DRY bacterial smear
bunsen burner or hot plate is used as a heat source & makes the bacterial smear adhere to the slide
Bacterial surface
negatively charged
Primary Stain
Positively charged
First stain applied in many differential staining techniques & is usually subjected to a decolorizing step that forms the basis for the differential stain
Secondary Stain (counterstain)
Negatively charged
stain applied after decolorizing to provide contrast between cells that were decolorized and those that weren’t
Bacteria with capsules
Bacillus anthracis, Klebsiella pneumoniae, and Streptococcus pneumoniae
Basic (cationic) stain examples
Bacillus anthracis, Klebsiella pneumoniae, and Streptococcus pneumoniae
Endospore outer coating
Keratin
Stain used to pick up keratin
Malachite green
Mordant
a stabilizer that causes the dye to form large crystals that get trapped by the peptidoglycan meshwork of cell wall
Decolorizer
removes stain from vegetative cell
used in the gram stain, acid-fast stain, and endospore stain
Mycolic acid
waxy substance in cell wall of acid-fast organisms
gives acid-fast cells (1) a higher affinity for the primary stain and (2) resistance to declaration by an acid alcohol solution
Endospore
a dormant, highly resistant form of the bacterium that allows it to survive poor environmental conditions
Glycocalyx
a polysaccharide coating surrounding some bacteria
capsule - tightly attached
slime layer - loosely attached (advantage of resistance to phagocytosis, increased adherence to surfaces, & decreased dessication)
Basic (cationic) stains
use a dye solution in which the chromogen is basic and carries a positive charge (cationic)
attracted to negative charges on the surface of most bacterial cells, as well as nucleic acids, and proteins
applied to bacterial smears that have been heat-fixed
Basic (cationic) dyes
crystal violet, methylene blue, carbolfuchsin, safranin, and malachite green
Acidic (anionic) stains
use a dye solution in which the chromogen is acidic and carries a negative charge
repelled by negative charge on the surface of most bacterial cells and thus stains the background, not the bacterial cell itself
Acidic (anionic) dyes
nigrosin & india ink
Bacterial morphologies
Spheres, rods, spirals, and slightly-curved rods
Sphere morphology
cocci, singular coccus
Rods morphology
bacilli, singular bacillus
Spirals morphology
spirilla, singular spirillum
Slightly-curved rods
vibrios
Bacterial arrangements
diplo, strepto, tetrad, sarcinae, staphylo
Diplo arrangement
pairs of cells
Strepto arrangement
chains of cells
Tetrad arrangement
group of four cells
Sarcinae arrangement
group of eight cells
Staphylo arrangement
irregular cluster of cells (grape like clusters)
Acid fast stain
used to identify bacteria with a waxy cell wall content (mycolic acid)
differential stain - differentiates between Mycobacteria and other types of bacteria
Steps of acid fast stain
Primary stain - carbolfuchsin (red)
Decolorizer - acid alcohol
Counterstain - methylene blue
Acid fast organisms
pink/red
e.g. Mycobacterium spp. (M. leprae & M. tuberculosis)
Non-acid fast organisms
blue
Enterococcus faecium
Capsule stain
differential stain - identifies bacteria with a polysaccharide coating known as a glycocalyx, or capsule
negative stains background while basic stains colorize the cell, leaving the capsule unstained
Capsule stain steps
Primary stain - nigrosin or India ink
Counterstain - basic stain (stains cell)
Organisms with a capsule…
appears the color of basic stain, surrounded by a clear halo against a background color of the acidic stain
Endospore stain
differential stain - identifies presence and location of spores in bacterial cells
Endospores are…
highly resistant to heat, chemicals, and staining b/c of tough outer covering made of the protein keratin
metabolically inert or dormant structures produced by the cell when it is stressed (poor environmental conditions or depleted nutrients)
can survive dehydration and then begin to grow when conditions are more favorable
Endospore stain steps
Primary stain - malachite green (stains spore bc keratin)
Decolorizer - water; removes stain from vegetative & spore mother cell
Counterstain - Safranin (red) ; stains the vegetative & spore mother cell
Organisms with endospores….
the vegetative and spore mother cell take on the color of the basic stain (pink/red), the endospore is green
Bacteria with endospores
Bacillus anthracis, Clostridium tetani, C. botulinum, C. perfringens (gas gangrene), C. difficile (pseudo-membranous colitis)
Identify endospores
Top: central spore
Middle: terminal spore
Bottom: subterminal spores
Identify blue and red stains
Blue - Enterococcus faecium; non acid fast (-)
Red - Mycobacterium smegmatis; acid fast (+)
Identify stain
Klebsiella pneumoniae capsule stain
Identify stain
Bacillus anthracis endospore stain w/ 27 hours incubation period
Identify stain
Bacillus anthracis endospore stain w/ 48 hour incubation period (more spores)
Identify stain
Clostridium botulinum endospore stain
sub-terminal, unstained spores
Identify species
White: Staphylococcus epidermidis
Green: Pseudomonas aeruginosa
Violet: Chromobacterium violaceum
Red/orange: Serratia marcescens
Rose: Kocuria rosea
Yellow: Micrococcus luteus
Identify species
1. flat/dry
2. spreading edge
3. crusty/friable
4. transparent
Flat/dry: Bacillus spp.
Spreading edge: Alcaligenes faecalis
Crusty/friable: Mycobacterium phlei
Transparent: Lactobacillus plantarum
Identify species
Enterobacter aerogenes & Citrobacter diversus (motile)
Enterococcus faecalis & Staphylococcus (non-motile, gram positive cocci)
Mycobacterium phlei & Mycobacterium smegmatis (non-motile w/ a waxy cell wall)
Identify species
Streptococcus species from a throat culture demonstrating flocculence in todd-hewitt broth
