Gram stain Flashcards
Clinical significance of gram stain
-results can have a dramatic effect on patient care
-can give a great deal of information quickly and is inexpensive
-can be done directly from patient specimens and facilitate early treatment
-the initial choice of antibiotic therapy is guided by the gram stain results
-the first step to identifying bacteria (gram pos or neg)
What four reagents are used in gram stain?
- Crystal violet
- gram’s iodine
- acetone-alcohol
- safranin
Direct smear preparation
- if its a fluid, place a drop on a clean glass slide and allow it to air dry
- if the specimen is received on a swab, gently roll the swab on a clean glass slide to avoid rupturing the host cell
- in both cases the specimen is fixed to the glass slide by passing it a few times over a flame
Staining procedure steps
step 1: flood the heat-fixed slide with crystal violet for 30sec and rinse with water
step 2: flood the slide with Gram’s stain for 1-minute rinse with water (does not cause a color change)
step 3: decolorize the slide by gently rinsing it with an acetone-alcohol solution for 10 seconds
step 4: flood the slide with safranin for 30 seconds and rinse with water and dry (this counterstains gram-negative bacteria)
* gram-positive bacteria will retain a crystal violet stain
* gram negative do not retain it
Gram stain theory
-gram-positive and gram-negative bacteria stain differently because of the structure of their cell walls
-Gram-positive cell wall has a much thicker peptidoglycan layer with many teichoic acid cross-links which helps it to resist decolorization
* is not possible to determine the species of the bacteria from gram stain results alone needs culture and biochemical testing
What stain is host cell morphology best viewed from?
wright’s stain
squamous epithelial cells
-commonly found on direct gram stains from a variety of sites
-they are distinguished by their large size, small nucleus, and abundant cytoplasm
-their diagnostic relevance is dependent on the site
tetrads
-groups of four cocci
-referred to as small clusters
gram-positive coccus
-the gram-positive coccus is a spherical bacterium
-when in pairs they are called diplococci and may be elongated in shape
-may also form chains of variable length
-groups of exactly four cocci are called tetrads (can be referred to as small clusters)
-groups of cocci are referred to as clusters
Gram-positive rod (bacillus)
-bacillus is a rectangular-shaped bacterium
-rods are variable in length, width, and staining characteristics
-Long, wide rods usually have blunt ends
-Long narrow rods have blunt ends and may form chains of variable length
-coccobacillus share characteristics of rods and cocci, they are very short, coccoid rods
-branches rods are long and narrow and may have a beaded staining pattern
Gram-negative cocci
-may appear in two clinically significant groupings
-it is spherical
-when they appear in pairs they are called diplococci and may also be kidney shaped
gram-negative rod (bacillus)
-is a rectangular-shaped bacterium, rods are variable in length, width, and staining characteristics
-Long narrow rods typically show uniform staining they may appear in short chains
-coccobacilli share characteristics of rods and cocci, they are short and narrow rods
-curved rods are short, narrow, and curved, they show minimal staining and may form short chains
- fusiform rods are long and narrow with pointed ends
-spiral rods are long, narrow, with multiple curves
Antibiotics effects
-the presence of antibiotics may alter the size, shape, and arrangement of some species of bacteria
-ex: they may appear swollen or elongated
Key to a good gram stain
-do not make the smear too thick
-make sure the slide is dry and heat fix
-rinse carefully
-do not over or under decolorize
-be sure to leave the iodine on 2X as long as crystal violet
-do not leave the safranin on too long
-host cells like red and white cells should appear pink on properly stained smears
