COMMON UNKNOWN ISOLATION PROCEDURES 8/22/22

UNKNOWN ISOLATION Procedures:

ATLAS page 9

Your unknown is given to you containing two (2) microbes.  A known, Staphylococcus aureus which is a Gram positive cocci in grapelike clusters and some "UNKNOWN."  You must "isolate" the UNKNOWN from the known.  To do this you perform an isolation to a slant by: (1) pour plate (2) spread plate and the (3) streak plate.

BEGINNING the UNKNOWN ISOLATION:

A) Isolation (The Streak Plate Lab) - separating the 2 microbes using a streak plate

B) "Dots-R-Us" Lab - numbering the individual "dots" and doing gram stains on each one to determine the first one that is NOT the known.  Then taking the other 1/2 of the colony to a slant by zig-zag and overnight incubation

C) The Purity Lab - Testing the Dots Slant in at least 2 regions of the zig-zag by gram stain to make sure everything is the same size, shape, & gram stain

D) The Work/Stock Subculture Slant Refrigeration Procedure - Making another slant by taking a small amount from the pure culture slant every 10 days (subculturing) and incubating it overnight, testing it for purity and then moving it to the refrigerator to keep as a backup or STOCK...

POUR PLATE ISOLATION:

The POUR PLATE ISOLATION PROCEDURE involves doing SERIAL DILUTIONS of your Unknown Broth in Saline or adding 0.1 ml of the mixed unknown broth to the center of a sterile plate.  Pour 10 ml of melted nutrient agar into the liquid broth in the plate. Swirl the plate and leave the lid "cracked" until 3/4ths of the condensation has left the lid.  Close and tape the plate shut.  Incubate the plate upside-down in the incubator for 12 hours.  Remove the plate from the incubator and observe for separate individual perfectly round colonies.  Check each colony by Gram staining 1/2 the colony.  If the observed microbe is NOT Gram positive cocci in grapelike clusters, take the other 1/2 of the colony with your sterile loop and "zig-zag" it onto a nutrient agar slant.  Incubate the slant 12 hours. Check the slant in 3 regions checking for Gram positive cocci in grapelike clusters.  If you do NOT find Staph then make a second slant and check it in the same manner. If both slants do not have Staph, label one as STOCK and place it in the refrigerator as a backup.  Label the other slant as "WORKING" and begin your Unknown Tests.  The pour plate has the disadvantage of having colonies trapped within the agar, thus confusing the isolation process...

SERIAL DILUTIONS PROCEDURE: 
Atlas page 83-4


SPREAD PLATE ISOLATION:

The SPREAD PLATE ISOLATION PROCEDURE involves doing SERIAL DILUTIONS of your UNKNOWN BROTH or adding 0.1 ml of the mixed unknown broth to the top of a cooled sterile nutrient agar plate... Use a sterile glass spreading rod which has been flamed to spread the broth all over the agar surface.  Close and tape the plate shut.  Incubate the plate upside-down in the incubator for 12 hours.  Remove the plate from the incubator and observe for separate individual perfectly round colonies.  Check each colony by Gram staining 1/2 the colony.  If the observed microbe is NOT Gram positive cocci in grapelike clusters, take the other 1/2 of the colony with your sterile loop and "zig-zag" it onto a nutrient agar slant.  Incubate the slant 12 hours. Check the slant in 3 regions checking for Gram positive cocci in grapelike clusters.  If you do NOT find Staph then make a second slant and check it in the same manner. If both slants do not have Staph, label one as STOCK and place it in the refrigerator as a backup.  Label the other slant as "WORKING" and begin your Unknown Tests.  The spread plate has the disadvantage of needing a sterile glass spreader which involves "flaming" a glass rod using ETOH which is a safety problem with students - FLAMING STUDENTS! An additional concern is the extra time needed with the plate open to the air while spreading.  This allows for contamination of the plate with airborne contaminants.

STREAK PLATE ISOLATION:

(Photo of Continuous Quadrant Streak Isolation Method)Remember to use 5  PULLING STRIPES crossing only the last stripe after flaming INSTEAD of Continuous Streaks (demo to left) as that will NOT tear the agar plate

STREAK PLATE ISOLATION:

The STREAK PLATE ISOLATION PROCEDURE & T-stripe (ask me about the T-stripe in class) involves dipping a sterile loop into the mixed unknown broth one (1) time and dragging it across a poured sterile plate agar in a pattern determined by the user.  The pattern of "dragging and flaming" which works best  for students is some variation of the QUADRANT streak.  After streaking quickly (a total of 10 seconds with an open plate is considered enough time).  Close and tape the plate shut with 2 short pieces of tape.  Incubate the plate upside-down in the incubator for 12 hours.  The streak plate disadvantage is that sometimes students re-dip into their mixed unknown sample and multiply the number of bacteria instead of the purpose of getting 1 bacteria, also sometimes they multiply by crossing too many lines and many times, or they use too much time with the plate open "painting", or lay the top down on the table-top causing contamination,  or put too much pressure on the loop and "cut" into the agar leaving all the bacteria at the cut instead of drawn across the plate.  The steak plate is the preferred way of isolation for our classes.

ISOLATION steps/Dots-R-Us Lab: Remove the plate from the incubator and observe for separate individual perfectly round colonies.  Check each colony by Gram staining 1/2 the colony.  If the observed microbe is NOT Gram positive cocci in grapelike clusters, take the other 1/2 of the colony with your sterile loop and "zig-zag" it onto a nutrient agar slant.  Incubate the slant 12 hours. Check the slant in 3 regions checking for Gram positive cocci in grapelike clusters.  If you do NOT find Staph then make a second slant and check it in the same manner. If both slants do not have Staph, label one as STOCK and place it in the refrigerator as a backup.  Label the other slant as "WORKING" and begin your Unknown Tests.  
A photo of the streak plate and a resulting plate is located below...