Unknown strategies -general flowchart.ppt

Begin a lab journal (diary type) and document EVERYTHING.
This may be in spiral form or computer printed (preferred)
Record DAY/TIME – often this is critical info
Record each procedure/test done
media used (do NOT double test!!!)
Results of test (visual descriptions or
digital photos are helpful)
• Stain procedures performed & visual
description of stain results
(step-by-step staining description is not necessary)
Day 1: Receive Mixed Culture
Prepare Gram stain of mixture and
note appearance of each organism
Prepare streak plate for isolation
Day 2: Check streak plates for colonies
(Note all activities in your lab journal!)
Transfer a portion of a single isolated colony for each type observed to broth
(TSB) labeled with your name, unknown # and “Gram-negative” or “Grampositive” if known. Otherwise simply label as Unknown# and “A” or “B” until you
are able to determine Gram reaction. It is more important to get each colony
type growing well in broth than to make an initial confirmation of Gram stain
If possible, prepare Gram stain from THE EXACT SAME colony
transferred to the broth and compare appearance to those seen in
original mixture.
Day 3: Gram stain broth cultures. If only one cell type appears
To be present, proceed to next step. If not, repeat previous steps as needed
Determine from previous observations of your unknown mixture if cell shape &
Gram reaction matches.
If yes, begin inoculations of
appropriate biochemical test media.
If no, return to previous isolation
plates and repeat colony selection
IMPORTANT: At this stage you should
establish stock supply on a general
nutrient slant such as TSA or Nutrient
Day 4 through end of semester: Read biochemical test results and perform
additional tests as necessary to identify or eliminate. Continue until both
unknowns are identified

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