Russell County Hospital
1610 Dowell Road
Russell Springs, KY 42642
270-866-4141
DEPARTMENT:
Laboratory
POLICY/PROCEDURE TITLE
Microstrep Plus Panel
POLICY/PROCEDURE NUMBER:
400-PE-MIC-164
Issued: 5/10/06
Revised:
Supercedes:
Prepared by: R. Johnson
Approved:
Reviewed:
Medical Director
Approved:
Reviewed:
Laboratory Director
_________________________________________________________________________________________________
I.
PRINCIPLE. The Microstrep plus panels are designed for use in determining quantitative and/or qualitative
antimicrobial agent susceptibility of colonies grown on solid media of aerobic streptococci, including S.
pneumoniae. The antimicrobial susceptibility tests are miniaturizations of the broth dilution susceptibility test
that have been diluted in water and dehydrated. Various antimicrobial agents are diluted in water, buffer, or
minute concentrations of broth to concentrations bridging the range of clinical interest. Panels are
rehydrated with 115 ul Mueller-Hinton broth supplemented with 2.5% lysed horse blood after inoculation of
the broth with a standardized suspension of the organism. After incubation in a non-CO2 incubator for 20-24
hours, the MIC for the test organism is manually read by observing the lowest antimicrobial concentration
showing inhibition of growth.
The antimicrobial agents are:
Antimicrobial agent
Ampicillin
Amoxicillin/K Clavulanate
Azithromycin
Cefactor
Ceftepime
Cefotaxime
Ceftriaxone
Cefuroxime
Chloramphenicol
Clindamycin
Erythromycin
Gatifloxacin
Levofloxacin
Meropenem
Penicillin
Tetracycline
Trimethoprim/Sulfamethoxazole
Vancomycin
II.
Abbreviation
Am
Aug
Azi
Cfr
Cpe
Cft
Cax
Crm
C
Cd
E
Gat
Lvx
Mer
P
Te
T/S
Va
Dilutions, ug/ml
0.06-4
0.5/0.25-4/2
0.25-2
0.5-4
0.25-2
0.25-2
0.25-2
0.25-2
1-16
0.06-0.5
0.06-0.5
0.12-2
0.25-4
0.06-0.5
0.03-4
0.5-4
0.25/4.75-2/38
0.12-1
SPECIMEN.
A.
Use pure isolates freshly grown (18-36 hr incubation) on appropriate non-inhibitory culture media, such
as BAP. Test morphologically similar, well-isolated colonies.
B.
HANDLE ALL BACTERIAL SPECIMENS AS POTENTIALLY BIOHAZARDOUS AND USE STANDARD
PRECAUTIONS IN ACCORDANCE WITH HOSPITAL AND LABORATORY SAFETY AND INFECTION
CONTROL POLICIES.
Page 1 of 4
Russell County Hospital
Policy/procedure name: Microstrept Plus Panel
Policy/procedure number: 400-PE-MIC-164
Issued: 5/10/06
Revised:
_________________________________________________________________________________________________________
III.
REAGENTS/SUPPLIES/EQUIPMENT.
A.
Protective gloves.
B.
Microstrep plus panel type 1.
1.
Store panels at 2-25 C. All wells on the panel, except the locator well, should appear clear prior to
inoculation.
2.
Do not use after printed expiration date.
3.
HANDLE ALL BACTERIOLOCIAL ORGANISMS AND CONTAMINATED MATERIALS AS
POTENTIALLY BIOHAZARDOUS AND USE STANDARD PRECAUTIONS IN ACCORDANCE
WITH HOSPITAL AND LABORATORY SAFETY AND INFECTION CONTROL POLICIES
C.
0.5 McFarland turbidity standard.
D.
Sterile swabs or micro loops.
E.
100 ul pipettor with disposable sterile tips.
F.
Cover trays.
G. Inoculator-D set.
IV.
V.
H.
3 ml inoculum saline.
I.
Mueller-Hinton broth w/ 3% lysed horse blood.
J.
Microdilution viewer.
K.
QC organism – Streptococcus pneumoniae, ATCC 49619.
QUALITY CONTROL.
A.
Quality control must be performed at least once each week of patient testing, and when a new lot
number or shipment is put into use. Refer to the Microstrep plus panel procedural and QC manual for a
description and expected results for the QC organism.
B.
Use Streptococcus pneumoniae, ATCC 49619.
1.
A control well is provided on each panel for reading of MIC’s.
2.
A growth well is provided on each panel to demonstrate how the organism will grow in the test
medium without antimicrobial inhibition.
3.
Acceptable endpoint ranges for the recommended control organism are listed in the Microstrep plus
panel procedural and QC manual. Deterioration of an antimicrobic agent or other circumstances
would result in MIC’s higher than the acceptable ranges.
C.
QC organisms must be tested in the same manner as unknown test organisms. Do not accept test
results without valid QC results. Should the QC organisms fail to react appropriately, discard and
repeat. Always check culture purity by subculturing from the inoculum onto suitable agar media.
Document all QC results in the QC logbook.
D.
HANDLE ALL CONTROL ORGANISMS AND CONTAMINATED MATERIALS AS POTENTIALLY
BIOHAZARDOUS AND USE STANDARD PRECAUTIONS IN ACCORDANCE WITH HOSPITAL AND
LABORATORY SAFETY AND INFECTION CONTROL POLICIES.
PROCEDURE.
Page 2 of 4
Russell County Hospital
Policy/procedure name: Microstrept Plus Panel
Policy/procedure number: 400-PE-MIC-164
Issued: 5/10/06
Revised:
_________________________________________________________________________________________________________
A.
Broth preparation.
1.
Allow the M-H broth to come to room temperature before use.
2.
Examine broth before use:
a.
b.
B.
C.
D.
E.
F.
Broth should be clear and red/dark red in color.
Do not use broth that is turbid and discolored.
Panel preparation.
1.
Obtain a panel from storage, cut open the pouch, and remove tray.
2.
Allow panel to come to room temperature before rehydration.
3.
Do not use if desiccant is not present or is broken, or if panel wells are discolored.
Inoculum preparation.
1.
Using either a sterile swab or micro loop, touch the surface of 4-5 large morphologically similar,
well-isolated, presumptive Streptococcus colonies from a 16-20 hours non-inhibitory agar place.
2.
Emulsify in 3 ml of inoculum saline to obtain a turbidity of a 0.5 McFarland standard.
3.
Cap tightly and vortex the suspension for 2-3 seconds.
4.
Pipet 0.1 ml (100 ul) of the suspension into 25 ml of broth. Cap tightly and invert 8-10 times to mix.
5.
Inoculate the panel within 15 minutes of adjusting the inoculum.
Panel rehydration/inoculation is performed using the RENOK system with inoculator-D sets.
1.
Remove the transfer lid from the inoculator set and pour the inoculated broth into the seed trough.
2.
Replace the lid and gently tap the lid in all four corners to ensure absence of bubbles. Allow to
stand at least 20 seconds to equibrilate.
3.
Attach the RENOK rehydrator/inoculator to the transfer lid and draw up the inoculum (to prime) and
then press the center release button to dispense the inoculum back into the seed trough. Draw up
the inoculum a second time into the transfer lid.
4.
Place the transfer lid on the panel and release the inoculum into the panel.
Incubation.
1.
Panels may be incubated in stacks of 3-5 trays.
2.
Place a clean cover tray over the top of each group of panels to prevent evaporation.
3.
Incubate for 20-24 hours at 34-36C in a non-CO2 incubator.
Read the panels.
1.
Following incubation, remove the panels from the incubator.
2.
Wipe the bottom of the panel with a lint-free tissue to remove condensation or debris.
3.
Read the panel only if the growth well is turbid. Inadequate growth is defined as a slight haze in
the well or if the broth is clear.
Page 3 of 4
Russell County Hospital
Policy/procedure name: Microstrept Plus Panel
Policy/procedure number: 400-PE-MIC-164
Issued: 5/10/06
Revised:
_________________________________________________________________________________________________________
VI.
4.
If the growth well is turbid, read the panel using indirect light. Growth in a well appears as turbidity:
haze throughout well, a button in the center of the well, or a fine granular growth throughout the
well.
5.
Record results on the panel worksheet as follows:
a.
Record the MIC as the last well showing inhibition of growth starting at the highest
concentration.
b.
When growth occurs in all concentrations, record the MIC as greater than the highest
concentration.
c.
When no growth occurs in any of the concentrations of antibiotics, record the MIC as less than
or equal to the lowest concentration.
d.
A clear well in a series of growth wells is called a skipped well and should be ignored.
e.
Spot growth in isolated wells indicates contamination and the test should be repeated.
REPORTING RESULTS.
A.
See the streptococci interpretive criteria chart on the table in the Microstrep plus panel procedural and
QC manual.
B.
Record the MIC’s. Record QC results.
VII.
LIMITATIONS. For purposes of diagnoses and treatment, results of this test should always be interpreted in
conjunction with the patient’s medical history, clinical presentation, and other findings.
VIII.
REFERENCE. MicroScan MicroSTREP plus Panel Procedural and QC Manual, Dade Behring, rev. April
2004.
Page 4 of 4