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MICROTEST LABORATORIES STANDARD OPERATING PROCEDURE #1015B, G, Y rev 006

Title: Identification of Bacteria (B: Genus & Species, G: Genus Only, Y: Yeast)

Written by: Date:_____________

1. SCOPE
1. This SOP outlines procedure utilized to determine the genus and species of bacteria isolates.
2. MATERIALS

2.1 Crystal Violet Stain
2.2 Sterile phosphate buffered water - appropriately labeled
2.3 Gram's Iodine
2.4 Gram's Decolorizer (95% ethanol or ethanol/acetone)
2.5 CounterStain (Safranin O)
2.6 Lab timer
2.7 Microscope slides (pre-cleaned with alcohol)
2.8 Wax marking pencil or equivalent
2.9 Tap water bottle
2.10 Blotting paper
2.11 Olympus Microscope
3.12 Immersion Oil
2.13 E.coli ATCC #8739
2.14 B.subtilis ATCC #6633
2.15 Bunsen burner
2.16 Inoculating loop
2.17 Sterile disposable glass pipets (1.0 mL or equivalent)
2.18 Forceps
2.19 Staining reservoirs with slide holders
2.20 Lens paper (Kimwipes or equivalent)

3. PROCEDURE
1. Streak organism for isolation, then place isolated organism on a slant. (To obtain an isolated working culture).
2. If culture is a strict anaerobe, it may be sent to vendor, or done in-house, for fatty acid hydrolysis (MIDI). Follow the procedure below for sub contracting a sample to a vendor.

3.2a Prepare a slant culture as in 3.2.

3.2b Take the next consecutive number from the Purchase Order Log Book located in front office.

3.2c Prepare a test request form for the appropriate vendor making sure to include the above purchase order number (test request forms are located in Sample Log-In).

3.2d The test request should read "Identification of Bacteria using MIDI".

3.2 e Make sure that the slant is sent via next day shipping in an insulated box with cooling packs.

3.3 A gram stain should be performed (preferably on a 24 hour culture). Proceed with the Identification of Bacteria or Yeast for both Genus Only and Genus and Species Identification.

4. BIOLOG IDENTIFICATION SYSTEM
1. MATERIALS
1. TSA Agar Plate (R2A, TSA with 5% Blood)
2. BUGM + B or TSA + B or Chocolate Agar
3. BUGM + M + T
4. BUY AGAR
5. Inoculating Fluid (GN/GP-IF GN/GP-IF + T or sterile water) in disposable tubes
6. 25° C, 30-32° C and 35-37° C incubators or appropriate conditions
7. Sterile cotton swabs
8. Biolog Turbidity Standards (GP-coc, GP-Rod, GN-FAS, GP-SB, GN-NENT, GN-ENT, YT)
9. Biolog MicroPlates (GN2, GP2 YT)
10. Eppendorf Repeat pipet
11. Biolog Turbidimeter
12. Gram stain and observe cell morphology. Follow attached Sample preparation Table to determine appropriate agar for unknown isolate.
13. Plate organism onto appropriate agar.
14. Incubate under appropriate conditions for approximately 4-24 hours.

BUGM + B or TSA + B: Gram negative enterics and nonenterics, Gram positive cocci and nonsporeforming rods

Chocolate: Gram negative fastidious organisms

BUGM + M + T: Gram positive rods with spores

BUY: Yeasts

2. INOCULUM PREPARATION
1. Place 20mL of appropriate inoculating fluid into disposable sterile tubes. Add Sodium thioglycolate (5mM) to the GN/GP-IF for tubes that will be used for GN-ENT, GN-FAS, GPC and FPR bacteria. This can be done by adding precisely three drops from an ampule containing a concentrated solution of sodium thioglycolate. A few species of GPC may also require the addition of 5mM sodium salicylate along with the thioglycolate, if the A-1 well turn purple.

Dip the swab into the inoculating fluid to moisten it. Pick cell onto the swab by rolling it over colonies rather than sliding across them. Twirl and press the swab against the inside surface of the tube above the water line to break up clumps and to release cells into the fluid.

2. Vortex tube for an approximately 45 seconds to create a homogenous mixture.
3. To blank the turbidimeter (transmittance = 100%), turn the turbidimeter switch on an insert a tube containing uninoculated GN/GP-IF or sterile water. Because the tubes used are not optically uniform, they should be blanked individually and not rotated in the light path of the turbidimeter.
4. Insert the sample tube and adjust the inoculum density. The acceptable range is defined by the turbidity standard plus or minus 3% transmittance. The density can be lowered by adding more inoculating fluid or raised by adding more cells.
3. INOCULATION OF THE MICROPLATE WELLS
1. Inoculate the cell suspension into the prewarmed MicroPlate promptly. Some strains lose metabolic activity if held too long (more than 20-30 minutes) in inoculating fluid without nutrients. Fill all wells with precisely 150ul for bacteria, 100m L for yeast. Take care not to carry over chemicals or splash from one well into another. The inoculating fluid fill form a soft gel within seconds after the inoculum is added to the wells.
2. Cover the microplate with its lid and incubate under conditions appropriate for the microorganism being tested.
4. INTERPRETATION OF RESULTS
1. It is good practice to take a 4-6 hour reading. Place Plate in Biolog Microlog plate reader. Follow MTL SOP EQ042 for instructions using the Biolog Identification System.
2. Enter data either manually or via plate reader into Biolog Computer System and review the percentage of identification.
3. For plates read after 4-6 hours of incubation, the similarity index must be at least 0.75 to be considered an acceptable species identification. After 16-24 hours of incubation, the similarity index must be at least 0.50 to be considered acceptable. If percentage of identification is SUFFICIENT, verify the species ID by comparing macroscopic colony characteristics (plate) to the description in Bergey’s Manual.
4. For any isolate that is identified as Salmonella, Shigella, E. Coli 0157:H7, Listeria sp., confirmation by an alternative method, is recommended.
5. RELATED SOP’S
1. MTL SOP #1017, Gram Stain
2. MTL SOP #EQ042, use of Biolog Microlog Microbial Identification System
6. REFERENCES
1. Biolog System Literature, Release 4.0
2. Bergey’s Manual of Systematic Bacteriology, Volumes 1, 2, 3, 4, Years 1984, 1986, 1989.
3. Bergey’s Manual of Determinate Bacteriology, Ninth Edition, 1994.
4. Kreger-Van Rij, NJW, The Yeasts: A Taxonomic Study