QC OF MEDIA FOR SUSCEPTIBILITY TESTING

The procedures used to test media for disk diffusion are listed below. These types of media are inoculated according to "Method F" described in "Inoculation Procedures for Media QC". See "Troubleshooting Guide for Disk Diffusion Susceptibility Testing" for disk diffusion problem solutions.

MUELLER HINTON AGAR

For performing susceptibility tests on non-fastidious rapidly growing bacteria using the disk diffusion (Kirby-Bauer) method, CLSI (formerly NCCLS) recommends the use of Mueller Hinton Agar.

Mueller Hinton Agar is monitored for excessive levels of thymine and thymidine, using Enterococcus faecalis (ATCC® 29212 or 33186), which can yield small or non-existent zones for sulfonamides and trimethoprim. See "Acceptable QC Limits of Zone Diameters for Nonfastidious Organisms" for the recommended method for testing thymine and thymidine levels in Mueller Hinton Agar.

Mueller Hinton Agar is also monitored for the proper levels of the divalent cations, magnesium and calcium. Variation in these concentrations will effect the zone sizes of tetracycline, polymyxin, and aminoglycosides when tested with Pseudomonas aeruginosa (ATCC® 27853).

Each batch of Mueller Hinton Agar is tested with at least twelve antibiotic disks that are representative of the major classes of antibiotics. The plates are inoculated according to "Method F" described in "Inoculation Procedures for Media QC". The zone size control limits are listed in "Acceptable QC Limits of Zone Diameters for Nonfastidious Organisms" as recommended by CLSI (formerly NCCLS).(1)

Each batch of Mueller Hinton Agar is also tested for proper and consistent depth, the proper fill, and a pH value within the control limits. The plates should have a consistent depth of approximately 4-5mm. The pH should be between 7.2 and 7.4.

MUELLER HINTON WITH SHEEP BLOOD

Mueller Hinton with 5% Sheep Blood is recommended by the CLSI for disk diffusion testing of Streptococcus spp. Zone diameter limits are listed in the CLSI (formerly NCCLS) M100 publication for quality control testing of S. pneumoniae (ATCC® 49619). The sheep blood in these plates may also cause a film of fine growth within the zones of inhibition around sulfonamide and trimethoprim disks. See "Acceptable QC Limits of Zone Diameters for Fastidious Organisms" for zone size limits of disks routinely tested by Hardy Diagnostics.

MUELLER HINTON WITH CHOCOLATE

CLSI (formerly NCCLS) M2 publication no longer recommends the use of this media for Haemophilus influenzae or Neisseria gonorrhoeae. There are no official guidelines for the quality control of this medium. The medium of choice for H. influenzae is "HTM" (Cat. no. E20) as listed below. The medium of choice for N. gonorrhoeae is "GC Agar with Supplements" also listed below.

HAEMOPHILUS TEST MEDIUM (HTM)

This medium (Cat. no. H07) is recommended for testing Haemophilus spp. It consists of a Mueller Hinton Agar Base with NAD, bovine hematin, and yeast extract.

The procedure for inoculation is the same as Mueller Hinton Agar, except that Mueller Hinton Broth is recommended for preparing the suspension (see inoculation "Method F" "Inoculation Procedures for Media QC"). It is important that only the control organisms Haemophilus influenzae, ATCC® 49247 and 49766 (for certain cephalosporins) be used for quality control testing of zone diameters. The accurate adjustment of this suspension to a 0.5 McFarland Standard (Cat. no. ML05), or equivalent, is especially critical for this medium. The use of a photometric device is recommended. An inoculum concentration that is too high may lead to zone sizes that are too small.

These plates are to be incubated in an atmosphere of 5 to 7% CO2 for 16 to 18 hours before reading the results. Incubation for periods over 18 hours may lead to falsely resistant results, i.e. zones that are too small. See "Acceptable QC Limits of Zone Diameters for Fastidious Organisms" for the zone size control limits.

GC BASE WITH SUPPLEMENTS

This medium is recommended for the susceptibility testing of Neisseria gonorrhoeae, when using the disk diffusion method. The medium consists of GC Agar Base with Supplements. This medium is clear and contains no hemoglobin. In addition to the prepared GC Agar (Cat. no. E14) we offer GC Agar Base as Dehydrated Culture Media (Cat. no. C5790).  

The medium is inoculated with growth from an overnight culture on a chocolate plate. The inoculation procedure is the same as for Mueller Hinton Agar except that Mueller Hinton Broth is recommended for the suspension (see "Method F" in "Inoculation Procedures for Media QC").

These plates are incubated at 35°C. in an atmosphere of 5 to 7% CO2 for 20 to 24 hours before reading the zone sizes. See "Acceptable QC Limits of Zone Diameters for Fastidious Organisms" for the zone diameter control limits.

MRSA SCREEN PLATE

The MRSA Screen Plate (Cat. no. G47) is used to test for methicillin or oxacillin resistance of Staphylococcus aureus. This medium contains Mueller Hinton Agar with 4% sodium chloride and oxacillin (6ug/ml).

The inoculating procedure consists of using a 1ul loop that has been dipped into a suspension of the organism that is equivalent to a 0.5 McFarland Standard (Cat. no. ML05), then spot an area 10 to15mm in diameter. Alternatively, using a swab dipped in the suspension and excess fluid is expressed from the swab, it is then streaked onto the plate with a single line or spot. The plate is then incubated at no more than 35°C. and observed at 24 hours for colonies of Staphylococcus aureus.

BRAIN HEART INFUSION AGAR WITH VANCOMYCIN

Brain Heart Infusion Agar (BHIA) with Vancomycin, 6ug/ml, (Cat. no. G14) is an agar screen for detection of vancomycin-resistant enterococci from pure cultures.

BHIA with Vancomycin is recommended by the Clinical Laboratory Standards Institute (CLSI - formerly NCCLS) for the detection of vancomycin-resistant enterococci. If the organism does not grow on this medium, it is considered sensitive; if the organism does grow, it is considered resistant.

The inoculating procedure consists of using a 1ml or 10ml loop to apply a spot inoculum of a bacterial suspension equivalent to a 0.5 McFarland Standard (Cat. no. ML05). The plate is then incubated at 35 °C. for a full 24 hours at which time it is observed for growth or no growth.

Growth of more than one colony indicates a vancomycin-resistant organism; no growth indicates that the organism is susceptible.

Screening tests for vancomycin-resistant enterococci have been shown to be comparable in reliability to standard methods in detecting clinically significant resistance.

REFERENCES

1. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, M100. 2009. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.


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