GRAM STAIN/CULTURE of CSF and OTHER BODY FLUIDS by HEMATOLOGY LAB (11:30pm 07:30am)
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1 Created Rebecca Riemann Clin. Lab 5/22/2012 Tech 1 Revised Eric Spitzer Chief of 5/22/2012 Micro/Assoc. Dir Labs Reviewed Jay Bock M.D. 5/22/2012 Approved Eric Spitzer Chief of Micro/Assoc. Dir Labs mic /22/2012 GRAM STAIN/CULTURE of CSF and OTHER BODY FLUIDS by HEMATOLOGY LAB (11:30pm 07:30am) I. PRINCIPLE To provide for appropriate turnaround time for processing of critical specimens, the Hematology Lab will perform Gram stain and culture on all CSF specimens received from 1130pm-0730am. In addition, the Hematology Lab will perform a Gram stain only on any body fluid received from 1130pm-0730am. (Refer to SPE 3.4) II. REAGENTS AND SUPPLIES A. Media and Reagents 1. BBL 5% sheep blood agar, chocolate agar and THIO Broth 2. BBL Gram stain reagents B. Supplies 1. Cytoslides 2. Methanol, absolute 3. Cytospin 4. Cetrifuge, vortex mixer 5. Sterile snap cap tubes (Falcon 2054), sterile Pasteur pipettes, inoculating loops III. QUALITY CONTROL (Gram Stain Reagents) 1. All new Lot number/shipments are quality controlled by the Microbiology Lab. 2. Weekly quality control will be performed by Hematology and recorded in the appropriate QC log. 3. Expected results: a. Staph aureus ATCC positive (deep violet) b. E. coli ATCC negative (pink or red) Corrective Action: If the above expected results are not obtained, request a new lot number of reagent from the Microbiology Lab. Gram stain for Hematology night shift Page 1 of 7
2 IV. PROCEDURE A. Perform all the initial processing of specimens in a biological safety cabinet (or, if not available, behind a shield). B. Gram Stain: 1. Prepare a smear using the cytospin. 2. Heat fix the smear on an electric slide warmer until dry. Add one drop of Methanol. Methanol fixation prevents lysis of RBCs, results in cleaner background and prevents wash-off of specimen from slide. Allow slide to dry and then cool before staining. 3. Flood the fixed smear with the crystal violet solution. Allow stain to remain for 30 s. 4. Decant crystal violet and rinse slide gently with running tap water (excessive rinsing can cause crystal violet to be washed from gram-positive cells). 5. Rinse off excess water with iodine solution, then flood the slide with fresh iodine solution. Allow iodine to remain for 30 s. 6. Rinse gently with flowing tap water. 7. Decolorize by letting acetone/alcohol reagent flow over smear while slide is held at an angle. Stop when runoff becomes clear. Adjust decolorization time to thickness of smear. 8. Remove excess decolorizer with gentle flow of tap water (excess washing can cause dye-iodine complex to be washed from gram-positive cells). 9. Flood slide with safranin counterstain, allow to remain for 30 s. 10. Remove excess counterstain with gentle flow of tap water. 11. Drain slide and air dry upright or use slide warmer. 12. Examine the smear microscopically using the oil immersion objective. 13. Turnaround Time: CSF = 0-60 minutes Non-CSF Body Fluids = minutes C. Culture: (Culture will only be set up on CSF) 1. If >1 ml is received, centrifuge for 20 min at x g at 20 C. Allow the centrifuge to stop. Do not brake. a. Aspirate the supernatant with a sterile pipette, leaving approximately 0.5 ml of fluid in the specimen tube. Reserve the supernatant in a sterile snap cap tube for antigen studies. b. Vortex the sediment vigorously for at least 30 s to resuspend the pellet. Do not use a pipette to mix the sediment, because the bacteria and cells may adhere to the sides of the tube and cause false-negative findings. c. Use a sterile pipette to inoculate the media by placing 1 or 2 drops of the vortexed sediment onto each plate and into the broth. 2. If ml is received, vortex, then inoculate. 3. Place media in 37 C incubator and Microbiology Lab will pick up first thing in the a.m. Gram stain for Hematology night shift Page 2 of 7
3 V. INTERPRETATION AND REPORTING OF RESULTS 1. Gram reactions Gram positive: deep violet Gram negative: pink or red 2. Evaluate smear Background should be clear or gram-negative. WBCs should be completely Gram negative. If excess precipitated stain is observed, prepare another smear and stain it with freshly filtered crystal violet or decolorize and repeat the stain. 3. Reporting results Examine several areas of smear under oil immersion for the presence of WBCs and microorganisms. a. If no organisms/wbcs are detected report No organisms seen and/or No WBCs as appropriate. b. If organisms/wbcs are observed, report relative numbers Rare (<1 per oil immersion field) Few (1 to 5 per oil immersion field) Moderate (6 to 10 per oil immersion field) Many (>10 per oil immersion field) (Refer to the Entering Gram Stain Results in Misys GUI section of this procedure.) VI. ENTERING GRAM STAIN RESULTS IN MISYS (SUNQUEST) GUI 1) Click on the Laboratory folder in Citrix. 2) Click on the Sunquest Laboratory Icon. 3) Click OK for the work area: Prod-Lab 4) Sunquest Login Screen-Enter your LIS tech code as the User, followed by your password and then change Location from R to HE. Gram stain for Hematology night shift Page 3 of 7
4 5) Double click on the Microbiology Result Entry folder. 6) The Data entry mode will default to Specimen Mode and the Data search will default to Lookup by: Accession Number. Type the accession number of the CSFC or FLDC in the Value Box and click on Search. 7) Double click on the test you want. 8) You will automatically be brought to the Direct Exam Tab and be at the Observations. Gram stain for Hematology night shift Page 4 of 7
5 9) Hit the F8 key to get the Micro Direct Exam Keyboard. 10) Click on the appropriate key on the displayed keyboard for the result you wish to enter or just hit the corresponding key for the result you want to enter. Enter the quantity followed by the result as shown in the example below. (Hitting F8 again will remove the displayed keyboard.) KEY RESULT CODE TRANSLATION 1 RARE RARE 2 FEW FEW 3 MOD MOD 4 MANY MANY Q WBCS WBCS SEEN Y RBS RBCS SEEN A GPC GRAM POSITIVE COCCI Z GNC GRAM NEGATIVE COCCI S GPR GRAM POSITIVE RODS N GNR GRAM NEGATIVE RODS P YEAS YEAST Codes not on keyboard. Use one semicolon (;) to enter the GPCP GRAM POSITIVE COCCI IN PAIRS code. GPCS GRAM POSITIVE COCCI IN CHAINS GPCC GRAM POSITIVE COCCI IN CLUSTERS Gram stain for Hematology night shift Page 5 of 7
6 11) Each element of the result (ie. WBCS, RBCS, GPC) should be entered as a separate observation, along with the quantity, as seen above. If you are entering codes you will have to TAB to get another line within the observation. If you are selecting from the displayed keyboard or just hitting the key you will automatically get another line within the cell. When you are done entering results in the observation, arrow down to get out of the observation. Click on the SAVE button at the bottom of the screen to file the results. 12) If you need to do a look up for a code, enter one semicolon (;) and then click on this box. 13) Enter a key word(s) in the Search value box, then just select the code you want and it will drop it into the observation. Gram stain for Hematology night shift Page 6 of 7
7 Errors in Misys GUI 1) The most frequently encountered errors are moving too quickly from one folder to the next. If you throw an error, the Messages folder will be flashing. Click on the folder and the error message will display at the bottom of the screen. Click CLOSE to remove the message from the Misys screen. Exit out of the culture and also exit out of GUI. You will also have to clear the error in LIS ALAB. 2) To clear the message in LIS ALAB, sign onto ALAB. The error will be displayed with your tech code. The errors from Misys GUI will usually have MyMRE listed as the application. 3) Hit Enter till you get to the Function prompt and enter ER. You will then get the message CONFIRM ERROR REPORT CANCELLATION. Type Y and the message will be cleared. Battery Locked in Misys GUI 1) If after you go back into GUI you find that the battery that you want to enter results on is locked, you will have to exit out of GUI again and go back onto ALAB. 2) Enter LOCK at the FUNCTION prompt. Then select Unlock an Entity. Enter the accession number that you want to unlock. You will then get the message Attempt to stop the process? (Y/N) Type Y. It will take several seconds or longer to clear. 3) Go back into GUI and enter your results. Gram stain for Hematology night shift Page 7 of 7
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