NAME: Alyssa BredaBACTERIA:
SECTION ONE: My Steps.
: Inoculate a back-up slant of nutrient agar from unknown broth.
: Create a back-up inoculation in case of contamination or loss of media.
: Gas production, opaque/cream colored growth.
: Make a streak isolation from the unknown broth onto NA
To isolate separate and distinct colonies in hopes of isolating my different organisms
Separate colonies achieved by third quadrant. One was larger and more creamcolored while the other was smaller and more circular and had a more cream color with a tint of yellow in it.
Since growth appeared on streaks, both organisms appear non-motile atthis point. All colonies have round edges and a convex elevation.
Gram stain from unknown broth
To see both organisms in one gram stain and to determine if I can observe two differentorganisms (Gram negative and Gram positive) on the slide.
There is a Gram negative bacillus organism as well as a Gram-positive coccusorganism that was arranged in long strands.
I have a Gram-negative rod and a Gram-positive cocci.
: Make a Gram stain from one of the distinct colonies present on the streak isolation plate.
: To determine if the colony is pure and if so, whether it is gram negative or grampositive.
How the test works:
Gram stains start with transparent cells that have been aseptically transferredand heat fixed to a slide. Staining with crystal violet (positively charged) stains the cells purplesince it is attracted to the negatively charged heads of the phospholipid bilayer. Next iodine createsa mordant by combining with the crystal violet, holding it stronger near the gram-positive cellswith the thicker cell wall. Next the decolorizer washes the stains from gram negative while leavingthe mordant on gram-positive. The counter stain of safarin then stains the gram-negative cells pink because it is positively charged and is attracted to the negative cell membrane. Thus, gram-negative cells appear pink and gram-positive cells appear purple.
Picture: streak isolation plate.The distinct colonies in towardthe middle of the 3
quadrantshow more of a yellow tinge thanthe opaque colonies toward theedges.