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Microbiology Unknown Report

Microbiology Unknown Report

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Published by acj56

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Published by: acj56 on Nov 12, 2012
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07/28/2014

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UNKNOWN REPORT
Alyssa BredaAfternoon: TWDr. Debra Costa-NinoDue: 1 August 2012
 
Breda 2
NAME: Alyssa BredaBACTERIA:
Klebsiella pneumoniae
and
Staphylococcus aureus
SECTION ONE: My Steps.
One
: Inoculate a back-up slant of nutrient agar from unknown broth.
Purpose
: Create a back-up inoculation in case of contamination or loss of media.
Observation
: Gas production, opaque/cream colored growth.
Two
: Make a streak isolation from the unknown broth onto NA
Purpose:
To isolate separate and distinct colonies in hopes of isolating my different organisms
Observations:
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.
Conclusion/Thoughts:
Since growth appeared on streaks, both organisms appear non-motile atthis point. All colonies have round edges and a convex elevation.
Three:
Gram stain from unknown broth
Purpose:
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.
Observation:
There is a Gram negative bacillus organism as well as a Gram-positive coccusorganism that was arranged in long strands.
Conclusion/Thoughts:
I have a Gram-negative rod and a Gram-positive cocci.
Four (G-)
: Make a Gram stain from one of the distinct colonies present on the streak isolation plate.
Purpose
: 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
rd
quadrantshow more of a yellow tinge thanthe opaque colonies toward theedges.
 
Breda 3
Observations:
The stain is a pure Gram-negative stain. The organism is a bacillus and arrange inno specific pattern.
Conclusion/Thoughts:
I have isolated a Gram-negative bacillus present in the larger opaquecolonies of my streak isolation plate
Five (G-
): Make a streak isolation plate on Eosin Methylene Blue Agar from the Gram-negative colonyon the NA streak isolation plate.
Purpose:
To determine/verify if the organism is a coliform and whether it is a lactose fermenter,and if so, to what degree. Also to ensure that the organism is not inhibited by eosin and methyleneblue.
How the test works:
Eosin Methylene Blue is a complex, selective, and differential medium. Itinhibits Gram-positive growth with eosin and methylene blue. It also reacts with lactosefermenters by turning the growth either pink, dark purple, or black. Green metallic growth alsooccurs with
 Escherichia coli
specifically.
Observations:
Good growth,
 
growth started as pink to dark purple at 24 hours, but after 48 hourswas a dark purple color. Isolation achieved by third quadrant.
Conclusion/Thoughts:
The dark purple growth indicates a possible coliform and the Gram-negative rod may be of the
 Enterobacter 
or
Klebsiella
species. The organism also ferments lactosewith acid production.
Six (G-
): Make an Agar Deep Stab from the EMB plate
Purpose
: To determine the aerotolerance of the organism and to determine if it was the organismthat produced gas in the original back-up slant.
How the test works
: Agar deep stabs are made with Tryptic Soy Agar. This is made with yeastextract to accommodate growth from a broad range of organisms. The oxygen is removed from the

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