SELECTION OF SUSCEPTIBILITY TEST BROTH Use Sensititre CAMHBT for rapid growing mycobacteria, Nocardia and other aerobic Actinomycetes or Mueller Hinton broth with OADC for slow growing mycobacteria.
Sensititre broths are performance tested for use with
SENSITITRE® Broth Microdilution (MIC) Method: INOCULATION AND INCUBATION For Rapidly Growing Mycobacteria (RGM), Slowly Rapid growing mycobacteria, Nocardia spp and other Growing Nontuberculosis Mycobacteria, Nocardia and aerobic actinomycetes other Aerobic Actinomycetes 1.Sweep the confluent portion of growth from growth on an
agar plate with a swab. Emulsify in sterile water and adjust to
a 0.5 McFarland Standard visually or using the Sensititre
nephelometer. If particles are visible, vortex well.
Actinomycetes typically have very hard crusty colonies. It may
be necessary to vortex with glass beads to make a
homogeneous suspension. If large clumps remain after
INTENDED USE
vortexing, they should be allowed to settle and the
Susceptibility testing of rapidly growing mycobacteria including
supernatant used for the inoculum suspension.
Mycobacterium fortuitum group (M. fortuitum, M. peregrinum, M. fortuitum third biovariant complex), M. chelonae, M.
Moistening the swab with sterile water may facilitate a more
abscessus, M. mucogenicum) and M. smegmatis group (M. smegmatis, M. goodii, M. wolinskyi). Nocardia spp and other
aerobic actinomycetes. Slowly growing nontuberculosis
Warning- the use of water supplemented with Tween may
mycobacteria (NTM), i.e. Mycobacterium avium complex,
affect MICs Mycobacterium kansasii and Mycobacterium marinum.2.Transfer 50µl of the suspension into a tube of cation
Please refer to CLSI (NCCLS) (1) for details of testing M.
adjusted Mueller-Hinton broth with TES buffer to give an
inoculum of 5x105 cfu/mL (range 1x105 to 1x106 cfu/mL). . Mix
PRINCIPALS OF USE
Each plate is dosed with antimicrobial agents at appropriate
Steps 1 and 2 must be completed within 30 minutes.
dilutions. Results can be read manually by visual reading of
3. Plates containing ≥ 32µg/mL doxycycline or minocycline
may show a precipitate after incubation. This can be
PRECAUTIONS
prevented by reconstituting these well with 5µL sterile distilled
Only personnel trained and qualified in susceptibility testing
techniques should use the system. The laboratory should have established biosafety guidelines for handling 4. Transfer 100µL to each well by either: mycobacteria.
a. SensititreAutoInoculator. Replace the tube cap with a
Sensititre single-use dosehead and inoculate the plate
STORAGE AND SHELF LIFE
according to the AutoInoculator instructions.
The plates should be stored at room temperature (15-25°C)
b. Manual pipette. Pour the broth into a sterile seed trough
away from direct sunlight and direct heat. Each plate is
and inoculate the plate using an appropriate pipette. Dose the
packaged in foil with a silica gel desiccant. Do not use the
panel with the Sensititre label facing the user.
plate if past its expiration date, or the desiccant colour is not
Pipettes should be periodically serviced and checked for
Inoculate plate within 5 hours of removal from pouch.
Inoculate broth into a plate within 30 minutes.
PROCEDURE 5. A periodic check of the colony count of the positive control
well should be done. (See Appendix 1). Isolates should have
an inoculum of 5x105 CFU/mL, (range 1x105 – 1x106)
Materials required and not provided [TREK Product Code]
6. Cover all wells with the adhesive seal. Press all wells firmly
to assure adequate sealing. Avoid creases as these can lead
Sensititre cation adjusted Mueller-Hinton broth with TES buffer
7. Incubate rapid growing mycobacteria aerobically at 300C in
a non-CO2 incubator for 72 hours. Check for growth. If poor,
Sensititre cation adjusted Mueller-Hinton broth with OADC
reincubate for up to a further 48 hours. Incubate Nocardia and
other aerobic Actinomycetes at 350C in a non-CO2 incubator
Sensititre doseheads (for use with AutoInoculator) [E3010]
Plates can be stacked up to three high. Incubation of 4-5 days
may be required for isolates of M. chelonae and M. abscessus
0.5 McFarland turbidity polymer standard [E1041]
50µL or 100µL pipettor and disposable tips
Slow Growing Nontuberculous Mycobacteria (NTM including MAC)
Same procedure as above except transfer 50µL of the
organism suspension into 10mL of Sensititre Mueller-Hinton
broth with 5% v/v OADC growth supplement. Invert the tube 8-
Incubate at 350C in a non-CO2 incubator and read after 7
days. If growth is good in the positive control , read results.
SPECIMEN COLLECTION AND PREPARATION
Otherwise re-incubate for up to 14 days. Prolonged incubation
Specimens should be collected, transported, stored and then
may require taking steps to prevent loss of well contents
plated on to primary isolation medium to give isolated colonies
through evaporation. Place the plates in a plastic container
with the top ajar to facilitate gas exchange.
READING TEST RESULTS
Results can be read using the Sensititre manual viewer or the
Tentative Quality Control Ranges for mycobacteria QC
SensiTouch. See Sensitouch User Manual. It is not necessary
to remove the adhesive seal. Place the plate with the label
facing the user. Growth appears as turbidity or as a deposit of
cells at the bottom of a well. The MIC is recorded as the
lowest concentration of antimicrobic that inhibits visible
growth. Please refer to CLSI (NCCLS) M24-A (1) for guidance
on reading endpoints. Reading faint growth on SensiTouch
can be improved by use of bright indirect lighting against a
The positive growth control wells should be read first. If any
Mycobacteria end points can be difficult to interpret. CLSI
(NCCLS) M24-A (1) provides reading guidelines and
illustrations of various growth patterns. Negative wells can
show a slight precipitate related to the inoculum. Reading QC
strains with known MICs should be used for training
Growth can range from a few colonies with no turbidity to
heavy growth comparable to positive growth control. The MIC
is the lowest concentration that completely inhibits growth
except for sulphonamides, where the MIC is read as the
lowest concentration that inhibits 80% growth compared to the
a. Contamination
1 Ranges based on Sensititre in-house testing
Contamination may result in growth in a well bordered by
2 The MIC range listed is for sulfizoxazole for
wells showing no growth. Such a single well contamination
can be ignored, but if multiple well contaminants are
*** Range based on data from Reference 4
Occasionally a “skip” may be seen - a well showing no growth
**** Incubation time dependent. Range based on 72
bordered by wells showing growth. There are variety of
explanations including contamination, mutation, creased seal
and misaligned dosing. A single skip can be ignored.
Contact Sensititre Distributor or TREK Diagnostic Systems in
However, in order to ensure effective antimicrobic therapy
the event that quality control discrepancies cannot be
NEVER read the skipped well as the MIC; always read the
lowest well concentration above which there is consistently no
PERFORMANCE c. Mixed Cultures
Panels are designed to give comparable performance to CLSI
Except as referred to in (a) above, if two end points are seen
(NCCLS) reference micro-broth procedures (1). Performance
as a distinct “button” of cells followed by several wells of
has been independently investigated (references 5-7)
diffuse growth with the “button” no longer visible (or seen as
For further information contact TREK Diagnostic systems or
smaller buttons), there may be a mixed bacterial population.
Purity should be checked by sub-culturing growth onto
suitable agar. Test results are invalid if a mixed culture is
LIMITATIONS 1. Imipenem results for M.chelonae and M.abscessus should INTERPRETATIVE GUIDELINES 2. Tobramycin is the aminoglycoside of choice for M. chelonae
Refer to the MIC Interpretive guidelines as provided by the
and should only be reported for this organism (1)
CLSI (NCCLS) (1), EUCAST or your national reference group.
APPENDIX 1: Colony Count Procedure QUALITY CONTROL 1.Immediately following inoculation plate, using a 1µl loop,
Frequency of quality control testing should be according to
sample from the positive growth control well and inoculate
local guidelines. Inocula should be cultured on a suitable
medium to check for purity. Test results are invalid if a mixed
2.Take another loop (1µl) and sample from the same growth
well and mix with 50µl sterile deionised water. Inoculate 1µl of
All Sensititre plates include positive control wells. Tests are
this dilution onto an appropriate agar plate to obtain countable
invalid unless there is distinct growth in all positive control
3.Incubate both plates at 30 or 35 0C (depending on type of
A number of factors influence MIC’s including organism state,
inoculum density, temperature and broth. In practice, replicate
4.Read as follows:
MIC’s form a normal distribution with the majority of results
lying within one dilution of the modal value. The test
procedure can be considered satisfactory if control organism
MIC’s are within range. Results should not be reported if QC Colony Count 0.001 plate 0.001of 1/50 dilution
Table 1 lists tentative QC ranges using mycobacteria strains.
Until other data becomes available, S.aureus ATCC 29213
and other non-mycobacterial QC strains and ranges from
CLSI (NCCLS) document M100 (2) can be used for panel QC.
BIBLIOGRAPHY 1. NCCLS M24-A. Susceptibility Testing of Nocardia, and
CLSI (NCCLS) M100 QC strains should use the same
Other Aerobic Actinomycetes; Approved Standard- Second
inoculation method as for rapid growing mycobacteria except
Edition (2003). National Committee for Clinical Laboratory
that 50µL of inoculum should be added to Sensititre Mueller
Hinton broth with TES. Do not use broth supplemented with
OADC. Panels should be read after 18 to 24 hours incubation
2.NCCLS M100-S13 (M7). Performance Standards for
Antimicrobial Susceptibility Testing; Thirteenth Informational
Supplement. (2003). National Committee for Clinical Laboratory Standards, Wayne, PA 3.Brown-Elliott, B; Wallace, R; Crist, C; Mann, L; and Wilson, R. (2002). Comparison of In Vitro Activities of Galtifloxacin and Ciprofloxacin against Four Taxa of Rapidly Growing Mycobacteria. Antimicrobial Agents and Chemotherapy46: 3283-3285 4.Wallace. R; Brown-Elliott, B; Crist, C; Mann, L;and Wilson, R. (2002). Comparison of In Vitro Activities of Gatifloxacin and Ciprofloxacin against Four Taxa of Rapidly Growing Mycobacteria. Interscience Conference on Antimicrobial Agents and Chemotherapy Abstract E-541 5.Woods. G; et.al. (2003). Multisite Reproducibility of Results Obtained by Two Broth Dilution Methods for Susceptibility Testing of Mycobacterium avium Complex. Journal of Clinical Microbiology41: 627-631 6.Woods, G; et.al (1999). Multisite Reproducibility of results Obtained by Two Broth Dilution Methods for Susceptibility Testing of Mycobacterium abscessus, M. chelonae and Mycobacterium fortuitum. Journal of Clinical Microbiology37: 1676-1682 7.Brown, B; Wallace, R; and Onyi, G. (1996). Activities of the Glycylcyclines N,N-Dimethylglycylamido-Minocycline and N,N- Dimethylglycylamido-6-Demethyl-6-Deoxytetracycline against Nocardia spp. And Tetracycline- Resistant Isolates of Rapidly Growing Mycobacteria. Antimicrobialial Agents and Chemotherapy40: 874-878 8. Clinical Microbiology Procedures Handbook, 2nd Edition. 2004. Editor H.D. Isenberg. ASM Press. DISCLAIMER Read the instructions for use and references before using the product. Any change or modification of the instructions may affect results. TREK Diagnostic Systems will not be liable for any damages resulting from any changes in storage, precautions, handling or testing procedures of the current version of instructions. The information provided in this technical insert is current at the time of printing and may change without notice. Contact the local TREK distributor for latest information Manufacturer: TREK Diagnostic Systems Limited Imberhorne Lane, East Grinstead, West Sussex RH19 1QX UK Tel: +44 1342 318777 Distributed in USA by: TREK Diagnostic Systems, 982 Keynote Circle, Suite 6, Cleveland, Ohio, 44131 Sensititre® and SensiTouch® are registered trademarks of TREK Diagnostic Systems. RGMYCO_US_GB_V1.19
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SELECTION OF SUSCEPTIBILITY TEST BROTH