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SHEEP/HORSE BLOOD STERILITY TESTING



Initial Testing (performed at the Blood Culture bench)
On receipt in the laboratory, each bottle is assigned a letter (A, B, C, etc.). The type of blood, lot #, expiry date, and hematocrit (found on label of bottle) is entered into the LIS and BacT/Alert in the following manner:

i. Enter the vial location.

ii. Enter the accession # as bottle letter (space) hematocrit.

e.g. A 39, B 40, C 39, etc.

iii. Enter the name as follows: type of blood, lot #, expiry date.

e.g. sheep 130820 112387, horse 130825 112387, etc

As for routine patient specimens, will bring up the information entered for the previous specimen.
With a needle and syringe 2.5 mL of blood is aseptically removed from each bottle of blood and inoculated into separate BacT/Alert FAN aerobic bottles. The original bottles of blood are immediately refrigerated and the BacT/Alert bottles are incubated in the BacT/Alert incubator and processed as routine specimens.
Print-out of LIS results will be filed with the invoice on QA bench. If any bottle gives a positive reading, the QA technologist must be informed as soon as possible and the original bottle of blood is removed from use. The BacT/Alert bottle is Gram stained and subcultured to BA (AnO2) and CHOC (CO2). Identification to the species level (e.g. Staphylococcus, diphtheroid, etc) will be sufficient.
After Use (performed by media preparation and the QA technologist)
As each bottle of blood is used, the last few drops of blood are inoculated onto a BA plate which is labelled with the lot # and letter. This plate is incubated at 35oC for 48 hours, then at RT for 48 hours. The results are recorded as a QC item in the LIS for the medium that the blood has been added to.


KIRBY BAUER QUALITY CONTROL

A. CONTROL STRAINS


1. To control the precision and accuracy of the test procedure, the following organisms are to be maintained:
Staphylococcus aureus ATCC 25923

Escherichia coli ATCC 25922

Pseudomonas aeruginosa ATCC 27853

Haemophilus influenzae ATCC 49247

Enterococcus faecalis ATCC 29212
2. Working cultures are stored on TSB agar slants at 4o to 8oC (Chocolate agar for H. influenzae). Stock cultures are stored in trisodium citrate glycerol at -70oC.
3. Before testing cultures are subcultured from the working culture slants (Haemophilus is subcultured on alternate days).
4. Replace working cultures monthly from frozen stock cultures.
5. For testing, inoculate the culture onto Blood agar or Chocolate agar to obtain single colonies.
6. Choose colonies for testing according to the recommended procedures.
7. Continue to use these cultures as long as there is no significant change in the mean zone diameter that cannot be attributed to methodology. Obtain fresh cultures from the ATCC or any reliable commercial source.

B. ANTIBIOTICS TO BE TESTED




  1. Test the control organisms using the antimicrobial discs which are used to test clinical isolates. The discs currently in use and the appropriate organisms for testing are listed in Table 1 (see Antimicrobial Abbreviations for codes). The stock supply of discs is found in freezer MIFA. The working racks of discs are placed in the freezer each night. Note: There is also a rack labelled “Rarely Used Antibiotics” in MIFA. When replacing a vial from the stock supply, write the date in use on the vial.




  1. The disks on the working discs rack are tested for Quality Control weekly and the “Rarely Used Antibiotics” are tested for Quality Control whenever patient test is done.




  1. Each new batch of Mueller Hinton agar must be tested for unsatisfactory levels of inhibitors. This is done by performing the tests with E. faecalis (ATCC 29212) and sulfonamide and trimethoprim/sulfamethoxazole (co-trimoxazole) discs.

C. ZONE SIZE LIMITS


Table 2 lists the maximum and minimum zone diameters (the accuracy) that should be observed with a single control test CLSI M100-S22 . Enter zone diameters into the LIS KB-QC charts.
(a) No more than one out-of-control result in 20 consecutive control tests is allowed. Any more than this requires corrective action.
(b) Anytime corrective action is taken the count of 20 begins again.
D. FREQUENCY OF TESTING
1. Each new lot of Mueller Hinton agar must be tested with the control strains when the medium is prepared. In addition media depth, pH and sterility must be tested and documented.
2. Each new lot of antimicrobial discs must be tested with appropriate control strains before being introduced into routine use. Preferably this will be done when the discs arrive in the laboratory.
3. The overall performance of the procedure should be monitored daily. Weekly monitoring will be done in this laboratory provided that the following conditions exist:
(a) Documentation that the control strains were tested for 30 consecutive test days
(b) No more than 3 of the 30 zone diameters were outside the accuracy control limits stated in Table 2.
When these requirements are fulfilled, each control strain must be tested:

(i) Once a week

(ii) Whenever any reagent component is changed.
4. If any zone diameter is outside the control limit when tested weekly, you must return to daily testing until the problem is resolved. If resolution of the problem cannot be documented, you must continue daily control tests. To return to weekly testing, documentation of satisfactory performance for another 30 consecutive days must be done.
E. RESOLUTION OF THE PROBLEM


  1. Resolution of any problem must be documented in the LIS as a "Procedure Comment" or "Result Comment".




  1. See CLSI M100-S22 Table 3D- Disc Diffusion QC Troubleshooting Guide for corrective action suggestions.




  1. Corrective Action during Daily Testing.

(a) One out-of-control measurement is not cause for immediate attention.


(b) Corrective action must be taken if any of the following circumstances arise:


  1. 2 consecutive measurements of any drug-microorganism combination fall outside the range

(ii) 3 or more in 20 consecutive test results fall outside the range


  1. Corrective Action during Weekly Testing.

If a zone falls outside the accuracy control/limits, the following are required:


(a) Appropriate control strain(s) must be tested for 5 consecutive test days.
(b) For each drug-microorganism combination, all 5 zones must be within the accuracy control limits.
(c) If any result is outside the accuracy or precision control limits, daily control testing must be resumed for a minimum of 30 consecutive test days.
TABLE 1: ANTIBIOTICS TO BE TESTED FOR KIRBY BAUER QC

S. aureus E. coli Ps. aeruginosa H. influenzae*

ATCC 25923 ATCC 25922 ATCC 27853 ATCC 49427

E X


OX X

DA X


VA X

CN X


KZ X

P X


SXT X

TE X


RD X

MUP X


AM X X


CN X X

KZ X


CRO/CAX X X

SXT X


TE X

F X


C X

TOB X X


CAZ X X

CP X


MEM X

AMC X


ATM X

CPD X


CXM X

FOX X


FEP X

ETP X


PRL X

AMK X




*Haemophilus (to be tested on Haemophilus Test Medium)



TABLE 2: ACCEPTABLE ZONE DIAMETERS FOR KIRBY BAUER QC (mm)

S. aureus E. coli Ps. aeruginosa H. Influenzae*

ATCC 25923 ATCC 25922 ATCC 27853 ATCC 49427



E 22 - 30

OX 18 - 24

DA 24 - 30

VA 15 - 19

CN 19 - 27

KZ 29 - 35

P 26 - 37

SXT 24 – 32

TE 24 - 30

RD 26 - 34

MUP 22 - 27






AM 16 – 22 13 - 21

CN 19 - 26 16 - 21

KZ 23 - 29

CRO/CAX 29 – 35 31 - 39

SXT 24 - 32

TE 18 - 25

F 20 - 25

C 21 - 27

TOB 18 - 26 19 - 25

CAZ 25 - 32 22 - 29

CP 30 - 40

MEM 28 - 34

AMC 19 -25

ATM 28 - 36

CPD 23 - 28

CXM 20 - 26

FOX 23 - 29

FEP 31 - 37

ETP 29 - 36

PRL 25 - 33

AMK 18 - 26






Reference: CLSI M100-S22 document



Performance Standards for Antimicrobial Susceptibility Testing


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