The following are the questions most often asked of our Technical Services Department. Please consult reference sources and request Instructions for Use (IFU) for complete information on the items mentioned in these sections.
Q. What kinds of clinical media require end user quality control?
A. Currently, exempt media listed in Table 1B of the current CLSI M22-A document, Quality Assurance for Commercially Prepared Microbiological Culture Media
, do not require the end user to repeat QC as long as the manufacturer provides the necessary certification and documentation of QC. Campylobacter Agar, Chocolate Agar, and media for the isolation of pathogenic Neisseria
spp. are listed as exempt, yet it is highly recommended these media be re-tested by the end user. At this time, Hardy Diagnostics provides a “QC Voucher” with each shipment certifying the media according to CLSI M22-A guidelines.
On January 1, 2016, the Centers for Medicare and Medicaid Services (CMS) will institute new guidelines for quality control testing. On this date, QC requirements will change to CMS/CLIA default requirements or labs will need to implement an Individual Quality Control Plan (IQCP) in accordance with applicable regulations and/or accreditation requirements. Due to this change, QC Vouchers will no longer accompany each shipment and end users should maintain Certificates of Analysis (CofA) for each lot as part of their records of manufacturer quality control testing. Hardy Diagnostics CofA can he obtained at the following location on our website indicating representative samples from each lot have been tested using appropriate QC organisms and specifications in accordance with Clinical Laboratory Standards Institute (CLSI) recommendations, where applicable, and meet the specifications published in the “Techncal Documents and Instructions for Use (IFU)” under the Technical Support menu at www.HardyDiagnostics.com. End users will need the catalog number and lot number in advance to retrieve a lot specific CofA.
Q. What organisms do I need to use to QC the media?
A. Most of our products have Instructions for Use (IFU) which can be accessed on the Hardy Diagnostics website. Each IFU lists the organisms used at Hardy Diagnostics to QC the medium.
Q. Do I have to use all of the organisms listed on the technical information sheet?
A. Currently, the end user is only required to check the performance of media using an organism that will produce a positive reaction and another organism that will produce a negative reaction for each reaction tested. Hardy Diagnostics may include organisms to monitor weak positive reactions or test the growth of multiple organisms, but this testing is not required of the end user. On January 1, 2016, end users will be required to follow CMS/CLIA default QC guidelines or implement an IQCP.
Q. What should the reactions look like?
A. Refer to the Instructions for Use (IFU) on our website for the product. Most of the IFU have color photographs of expected reactions.
Q. How can I keep track of lot numbers and expiration dates without doing a lot of writing?
A. Currently, a “QC Voucher” comes with each shipment and contains a listing of the media, lot numbers, and expiration dates for each media item that is shipped. Simply sign and date the voucher at the bottom and retain it with your QC records. On January 1, 2016, end users will be required to follow CMS/CLIA default QC guidelines or implement an IQCP.
Q. How often should I QC microbiology reagents like indole, oxidase, and beta-lactamase?
A. Current CMS/CLIA quality control standards for microbiology require testing of each lot or shipment. Follow appropriate regulatory and/or accreditation requirements to ensure compliance.
Media in General
Q. Where does your sheep blood come from?
A. Hardy Diagnostics obtains blood for its media from a ranch that maintains its animals for bleeding purposes only. These animals are maintained according to a veterinary supervised program to keep them healthy and disease free. The animal feed contains no antimicrobics, the addition of which is a common practice in feed lots. The blood containing media products are manufactured within five days after the sheep are bled. We never use less expensive slaughterhouse blood, which is commonly used by other media companies. These non-controlled sources of blood contain antimicrobics, are prone to spontaneous hemolysis, and tend to vary widely in hematocrits.
Q. Should I use a heavy or light inoculum when setting up biochemical tests?
A. For a more rapid result and clear reaction, inoculate your CTA and Urea Agar using a heavy inoculum. Simmons Citrate and Acetate Differential Slants should receive a light inoculum to avoid false-positives.
Q. Which tubes should be incubated with tight caps?
A. When incubating tubes for anaerobes (e.g., Thioglycollate or Cooked Meat), always tighten the caps. Caps on Strep B Carrot Broth™ should always be tightened to produce maximum color development by group B streptococci. Caps are also tightened on Transgrow bottles, which contain a CO 2 enriched atmosphere. All other tubes and bottles should be incubated with slightly loose caps. Fungi especially need an exchange of air for optimal growth. Biochemical reactions, such as with TSI and LIA slants, also require an exchange of air in order to produce a pH shift resulting in proper color development.
Q. Which kinds of media require an oil overlay?
A. OF Media and Moeller’s Decarboxylase broths require an overlay of sterile mineral oil. Always inoculate a “base” control (without carbohydrates or amino acids) along with your test, to compare the color.
Q. What should I do when my Fluid Thioglycollate Broths turn pink all over?
A. Fluid Thioglycollate contains the pH indicator resazurin, which turns pink in the presence of oxygen. It is normal to have a pink layer in the upper 30% of the broth. Often during shipment, the bottle or tubes become agitated and the entire medium turns pink due to dispersion of the small amount of oxygen present in the headspace of the tube. Sometimes just allowing the broth to stand undisturbed for a few hours will settle the pink color/oxygen to just the upper layer. If this is not successful and more than 30% of the media remains pink, then the tubes or bottles should be immersed in a boiling waterbath for about 10 minutes with the caps loosened to drive off the oxygen. The caps must be re-tightened while the medium is still hot to prevent re-oxygenation of the media.
Q. What should I do when my motility media has bubbles in it?
A. Sometimes motility media will become separated or have numerous bubbles due to rough treatment during shipping. If this happens, place the tubes briefly in a boiling waterbath, then allow them to cool in the upright position.
Q. Can I inoculate plates right after taking them out of the refrigerator?
A. Always warm your media to room temperature before inoculating. Some bacteria are sensitive to the cold (e.g. N. gonorrhoeae). Excess condensation on the surface of the plate can be evaporated by placing plates in the incubator for a brief period.
Q. Why does my Lauryl Tryptose Broth have a shiny precipitate?
A. A precipitate or cloudiness may form in refrigerated broth. Media will become clear when warmed to room temperature.
Q.When I received my media, the Durham tubes already contained bubbles.
A. Prior to inoculation of the medium, it may be necessary to invert the tube gently in order to release any bubbles that may be trapped in the Durham tube. Bubbles that are not removed before inoculation may lead to false-positive results.
Q.Why are some of my Dilu-Lok™ vials yellow?
A. The empty vials have been irradiated and, depending on their location in the run, some vials may have received more kGy than others in the same run. The color of the vial does not affect the performance of the product.
Stock Culture Maintenance and Preservation
Q. What is the best way to preserve cultures for long-term storage?
A. We recommend our CryoSavers™ for preserving cultures at ultra-low temperatures. Formulas available are Skim Milk (Cat. no. CSM100), Skim Milk with Glycerol (Cat. no. CSMG100) and Brucella with Glycerol (Cat. no. CS100BNB). Brucella with Glycerol is also available with beads (Cat. no. CS100B, CS100G, CS100N, CS100R and CS100Y). The vials are conveniently packaged in a plastic CryoSaver™ box that can be used to store your cultures in the freezer. Stock cultures will last indefinitely in an ultra low or -70 degrees C. freezer. If stored in a household refrigerator freezer (usually -20 degrees), results may be disappointing.
Q. Why does Campy fail to grow when I try to reconstitute the lyophilized pellets?
A. Campylobacter jejuni is one of the most difficult organisms to revive from a preserved state. Follow the manufacturer’s instructions carefully when reconstituting preserved microorganisms. Never attempt to grow any lyophilized organism in a broth. Always use an appropriate non-selective solid media for growing-up the reconstituted organism. The preferred medium on which to grow Campy is a Chocolate Agar plate. Once inoculated to the solid media, immediately transfer the culture to the appropriate atmosphere and incubation temperature. Campylobacter spp. must be placed immediately into a jar with a gas generator to create a microaerophilic atmosphere. The jar method is superior to the bag method for generating Campy from a pellet. Allow at least 48 hours at 35 degrees C. for growth; 42 degree C. incubation is not recommended when growing Campy from a preserved state.
Q. Once the Campy is growing, how do I keep it alive?
A. Transfer isolated colonies from the non-selective plate to a Thioglycollate with Supplements tube (Cat. no. K23). Keep the tube continuously in the 35 degree C. incubator. Oddly, this will maintain a viable culture of Campy for many months.
Q. Are there special requirements for reconstituting certain lyophilized microorganisms?
A. Many organisms require special media or incubation conditions upon rehydration. Consult Hardy Technical Services for questions or access the Document Library on the MicroBioLogics’ (MBL) website at www.microbiologics.com
. Click here
for the recommended growth requirements for LYFO DISK® and KWIK-STIK microorganisms.
Q. How can I reduce the frequency of skin contamination in my blood cultures?
A. Use an alcohol scrub applicator that contains iodine and produces a thorough friction scrub that penetrates the skin surface. Studies have shown contamination rates cut in half using this system, thus creating a monetary savings due to the reduced rate of false-positives (ref: Schifman, Pindur; Proc. of ASM Annual Meeting, 1991).
Q. What should I do with contaminated plates?
A. No media company guarantees 100% sterility of their products. The bags and the air surrounding the plates are not sterile. In addition, condensation may build up during brief temperature shifts in shipment and may drip onto media and cause contamination. The contamination rate with our plated media is usually no more than 1%. We recommend that our customers keep a tally of broken or contaminated plates, and report them to us once a month for replacement or credit. Any problem of excessive contamination (greater than 2%), discoloration, hemolysis, or breakage should be reported to us immediately.
Technical Data/Instructions for Use (IFU)
Q. How do I get a Technical Data Sheet/Instruction for Use (IFU) for your media?
A. Technical Data Sheets/Instructions for Use (IFU) are located on our website catalog. Call up the product and click on “More Info.” Then click on Technical Data “View File.” You may then print the Instructions for Use (IFU) for your records.
Safety Data Sheets (SDS)/Material Safety Data Sheets (MSDS)
Q. How do I get an SDS for your media?
A. SDS information is located on our website catalog. Call up the product and click on “More Info.” Then click on SDS “View File.” You may then print the SDS for your records.
Q. Why should media be stored in the dark?
A. Some types of media contain dyes and other light sensitive ingredients. Excessive exposure to light can produce the formation of toxic peroxides that can inhibit growth. Store your media away from the light, especially sunlight or UV light.
Q. Why are my plates sometimes too soft and have a rough surface?
A. Media exposed to freezing temperatures may become mushy, wrinkled on the surface, or, if containing blood, may become hemolyzed; thus rendering it useless. If storing media in a standard refrigerator, do not store it next to the freezer compartment. It is a good idea to take temperature readings at different locations in your refrigerator, in case there are excessively cold areas. Also, remember that a thermostat set at 2 degrees C. may, at times, permit the temperature to reach zero and destroy your media. It is safer to set it at a slightly higher temperature, such as 6 degrees C. (ref: Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.).
Q. How can I store my media so it will stay as fresh as possible?
A. Always store and incubate your plates in the inverted position (media on top, lid on bottom). This prevents moisture from reaching the surface of the agar, which can cause contamination. Always store media in the dark. Reseal the bag to prevent excessive moisture loss if less than the full pack is used. Never place plated media near the condenser fans in a large refrigerator. This will cause excessive drying of your media. Never leave plated media on the bench at room temperature for more than a few hours. Rotate your stock by using the oldest lots first.
Q. At what temperature should I adjust my incubator?
A. Set your incubator at 35 degrees C., instead of 37 degrees C., for the cultivation of most bacteria. An incubator set at 37 degrees C. may, at times, reach higher temperatures that can inhibit some types of bacteria. Methicillin-resistant S. aureus (MRSA) is inhibited by temperatures greater than 35 degrees C. It is recommended that the CO2 incubator for mycobacteria be set at 37 degrees C. (ref: Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.). If your incubator does not have a humidifier, place a beaker of water inside; add some detergent to the water to prevent bacterial growth.
Q. How should I clean my incubator?
A. Cleaning the inside of an incubator with disinfectant is known to result in many “no growth” cultures for several days, so allow it to “air out” thoroughly before using again. A 10% solution of household bleach is one of the best (and cheapest) disinfectants; be sure to make a fresh batch weekly, due to the rapid dissipation of chlorine ions. Periodic cleaning will prevent airborne fungal contamination of your cultures.
Q. What is the best method of streaking, and which kind of loops are the best?
A. Avoid streaking your plates close to the outer edge. If a contaminant is present, it is most likely to be at the edge of the plate. Older loops may have a rough surface that can gouge the media. Replace your loops occasionally. Nichrome loops are less expensive than platinum and last about 80% as long. The nichrome twisted wire loops are more flexible. Another alternative is disposable plastic loops which are available in soft (flexible) and more rigid shafts. These loops are calibrated and come with a certificate of calibration and sterility.
Q. Can I use a nichrome loop for the oxidase test?
A. Nichrome loops should not be used for the oxidase test, since surface oxidation formed during flaming may cause false-positives. Always use a platinum loop or disposable loop for testing. Oxidase testing should be performed on isolates taken from non-selective media to prevent erroneous results.
Q. What is the easiest way to check the accuracy of my calibrated loops?
A. Our CalCheck™ loop calibrators take only seconds to check your wire loops. These calibrators are designed to be used only with our 26-gauge wire. They consist of surgical steel posts sized to check the diameter of your loop. The product numbers for the 1ul and 10ul loop calibrators are Cat. no. 7020 and 7010, respectively. The operation is simple; the green post should go through the loop, and the red post should not.
Q. Why should I use an MRSA Screen plate in addition to a regular Mueller Hinton?
A. Methicillin-resistant S. aureus always exists as a mixed population of sensitive and resistant strains, hence the term “heteroresistant.” The sensitive strains may mask the resistant portion of the population on an ordinary Mueller Hinton plate. The MRSA Screen plate contains added NaCl that resistant strains prefer. It also contains oxacillin, so any growth in 24 hours is considered positive for MRSA. It is recommended to incubate no longer than 24 hours at no more than 35 degrees C. for S. aureus. Refer to the Instructions for Use (IFU) for MRSA Screen plate (Cat. no. G47) for more information.
Q. Can I use the MRSA Screen plate when screening for methicillin-resistant, coagulase-negative Staphylococcus spp.?
A. No, CLSI documents currently recommend the MRSA Screen plate (agar screen test) for use with coagulase-positive S. aureus only. Coagulase-negative staphylococci may be tested using disk diffusion, MIC, agar gradient methods.
Testing of oxacillin against S. saprophyticus is not recommended, because mecA-negative strains of S. saprophyticus often appear resistant by interpretive criteria used for coagulase-negative staphylococci.
Q. What should I do when my QC zone sizes are out of range for one or two antimicrobics when testing my Mueller Hinton Agar?
A. First, try a different lot number or a different manufacturer of the antibiotic disk. Make sure you are keeping your penicillin, ampicillin, and cephalosporins in the freezer, since they are particularly labile, even at refrigerated temperatures. Also, make sure your dispenser is kept in an airtight container with fresh desiccant and indicator (DesiView™, Cat. no. DV10) in order to keep out moisture.
Next, try reconstituting new pellets of stock organisms. CLSI recommends starting a new stock organism from a frozen or lyophilized culture every month, and subculturing to a new plate every week. Next, to make sure the inoculum is adjusted to the correct cell density for proper interpretation, see that your 0.5 McFarland Standard is in date and that you are vortexing the dilution tube before each use. We recommend the use of Latex McFarland Standards, rather than traditional barium sulfate standards, due to their increased stability and decreased sensitivity to light; the latex standard should not be vortexed. Carefully adjust the turbidity of the inoculum each time. When using the “direct suspension method,” suspensions should be made with overnight cultures. Zones that are too small can result if the inoculum is too turbid or the medium has been over inoculated. Read the plates no later than 24 hours (18 hours for HTM) or zone sizes may also be too small. Refer to the current CLSI M100 document for tips and tricks on troubleshooting issues with zone sizes.
Q. What is the most current source of information on zone size limits for disk diffusion tests?
A. CLSI updates the interpretive and QC charts for disk diffusion in their most current publications of the M2-A, Performance Standards for Antimicrobial Disk Susceptibility Tests
, M100-S, Performance Standards for Antimicrobial Susceptibility Testing
, (also see current publications of their Supplemental Tables) and M31-A, Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals
; Approved Standard. To obtain the most recent publication, check the following website: www.clsi.org
. CLSI documents are only available through CLSI. The source for all CLSI publications is:
Clinical and Laboratory Standards Institute (CLSI – formerly NCCLS)
940 West Valley Road, Suite 1400
Wayne, PA 19087-1898
Q. How do I measure a double zone?
A. If there are double zones, measure the inner-most zone. This may be caused by a disk sitting on top of the agar, not being pressed firmly enough to make good contact and not allowing sufficient diffusion of the antibiotic. This may also be a normal characteristic of the disk when testing heteroresistant strains (e.g. Ceftaroline for MRSA). Double check that the disk is pressed down firmly, even when using automatic dispensers. If the disk is fully moist after application, this means that the disk has good contact and the antibiotic should diffuse out as expected.
Q. When physicians request information on antimicrobials, they sometimes use their brand names rather than generic names; such as “Cefobid,” “Flagyl,” “Primaxin,” “Suprax,” “Timentin,” and “Unasyn.” Is there a good reference source on the newer antimicrobics?
A. The Guide to Antimicrobial Therapy by Sanford, MD (Cat. no. G15P) is an excellent source of current information on dosage, drug interactions, drugs of choice, side effects, etc. This pocket guide is updated annually. It has a complete chart of brand and generic names. The five brand names mentioned above are cefoperazone, metronidazole, imipenem + cilastatin, cefixime, ticarcillin/clavulanic acid, and ampicillin/sulbactam, respectively.
Q. What stock organism should I use to QC my beta-lactamase test?
A. H. influenzae Type B (ATCC ® 33533), Cat. no. 0338P, is beta-lactamase-positive. We also have a beta-lactamase-positiveNeisseria gonorrhoeae (ATCC ® 31426), Cat. no. 0375P.
Q. How do I perform QC on the HTM plates for Haemophilus influenzae susceptibility testing?
A. The current CLSI publication of the M100-S document contains revised tables of patient and QC zone sizes for disk diffusion testing of Haemophilus spp. on HTM plates. The QC organisms required by CLSI are H. influenzae , ATCC ® 49247 and 49766. Additionally, E. coli ATCC® 35218 is required to QC amoxicillin/clavulanic acid. It is imperative with this medium that the turbidity of the inoculum be carefully adjusted, and that the plates be observed no later than 18 hours after incubation. A suspension that is too heavy will produce false-resistance results.
Q. How do I perform a disk diffusion test on N. gonorrhoeae?
A. Neisseria gonorrhoeae should be tested on GC Agar Base with Supplements according to CLSI guidelines (ref: Performance Standards for Antimicrobial Disk Susceptibility Test, M2-A. Clinical and Laboratory Standards Institute (CLSI – formerly NCCLS), Wayne, PA). Use N. gonorrhoeae ATCC ® 49226 for QC. The use of Mueller Hinton with Chocolate plates for Haemophilus or GC is now discouraged in favor of these two new types of media. Refer to the most current CLSI publications: M2-A and M100-S.
Q. Which kind of beta-lactamase test should I be using?
A. An acidometric test such as the Beta-Lactamase Test (Cat. no. Z51) is acceptable for testing only Staphylococcus spp., N. gonorrhoeae , and Haemophilus spp. It produces a rapid and very distinct color change. Our “Nitrocef Matchbook™” (Cat. no. Z108) or Nitrocef HardyDisks™ (Cat. no. Z7301) are chromogenic tests acceptable for beta-lactamase testing of Branhamella ( Moraxella )catarrhalis , Enterococcus faecalis , and Bacteroides spp., in addition to the three organisms mentioned above.
Q. How should I screen for Extended-Spectrum Beta-Lactamases (ESBLs) and other mechanisms of resistance?
A. CLSI has revised their position on the detection of ESBLs: previous recommendations were to perform ESBL screen and confirmatory tests for E. coli
spp., and Proteus mirabilis
. However, new data suggests that ESBL phenotypic tests are not optimal due to the presence of multiple resistance mechanisms that may mask ESBLs in confirmatory tests; ESBLs are now known to exist in species of Enterobacteriaceae other than E. coli
spp., and P. mirabilis
where confirmatory tests are more problematic; and some MICs correlate better with patient outcomes rather than the knowledge of the resistance mechanism.
New CLSI recommendations for ESBL testing under the revised breakpoints in the current version of the M100-S, Performance Standards for Antimicrobial Susceptibility Testing , state that ESBL screen and confirmatory tests and editing of the “S” to “R” for cephalosporins, penicillins, and aztreonam are no longer necessary for patient management. However, for infection control purposes, ESBL screen and confirmatory tests, if requested, may still provide useful information on ESBL positive and negative strains, yet editing of the “S” to “R” for cephalosporins, penicillins, and aztreonam is still not required. Many ESBL positive organisms are commonly associated with nosocomial infections and, when under selective pressure in environments of heavy antibiotic usage, may adapt to confer resistance to all penicillins and all cephalosporins (1st, 2nd, and 3rd generation), with exception to cefepime (4th generation). It is highly suggestive of ESBL pro duction when resistance to 4th generation cephalosporins (cefepime and cefpirome) is observed; see the Supplemental Table 2A in the current CLSI M100-S document. Therefore, if and when ESBL screening is conducted, careful reading of susceptibility results, combined with judicious interpretation and enhanced communication with infectious disease physicians, can often lead to the correct diagnosis and therapeutic success.
Q. How do I recognize unusual susceptibility patterns?
A. Susceptibility results can provide valuable information when carefully read and interpreted. Some genera present unique susceptibility patterns that can be easily recognized. Appendix A, in the current CLSI document M100-S, Suggestions for Verification of Antimicrobial Susceptibility Test Results and Confirmation of Organism Identification , should be consulted for unusual or unexpected reactions.
Q. How do I screen for reduced susceptibility to vancomycin and vancomycin resistance in staphylococci?
A. Disk diffusion is not recommended for differentiation of reduced susceptibility to vancomycin (MICs 4 to 8ug/mL) from susceptible strains (0.5 to 2ug/mL). MIC determination should be performed. The vancomycin screen agar used for enterococci (BHIA with Vancomycin, Cat. no. G14) may be used, but results should be confirmed by MIC.
Q. How can I best store my antimicrobial disks?
A. Cartridges of commercially prepared paper disks used specifically for susceptibility testing are usually packaged to maintain anhydrous conditions.
Disks should be stored as follows:
Refrigerate cartridges at 8 degrees C. or below, or you can freeze at -14 degrees C. or below in a nonfrost-free freezer. Sealed packages of disks with beta-lactams should be stored frozen, except for a small working supply, which should be refrigerated for no longer than one week. Some of the more labile drugs (imipenem, meropenem, cefaclor, and clavulanic acid combinations) are more stable when stored frozen until the day of use.
Unopened disk containers should always be allowed to equilibrate to room temperature before opening. This will minimize excess condensation when warm air comes in contact with cold disks.
Once a sealed package of disks has been opened, it should be placed in a tightly sealed, desiccated container. Disk dispensers should always be stored refrigerated with a tight cover and desiccant (DesiView™, Cat. no. DV10). The disk dispenser should be allowed to come to room temperature before opening the cover. Replace desiccant when the indicator changes color.
Disks should always be discarded on the expiration date.
Q. How do I store my quality control strains for sensitivity testing?
A. For storage of less than one year, stock cultures may be maintained at -20 degrees C. or below with suitable stabilizer (e.g., Brucella Broth with Glycerol (Cat. no. D04), CryoSaver™ Skim Milk (Cat. no. CSM100), CryoSaver™ Skim Milk with Glycerol (Cat. no. CSMG100) or freeze-dried. Lower temperatures such as -70 degrees C. will preserve organisms indefinitely.
Working cultures should be stored on Tryptic Soy Agar (TSA) slants (Cat. no. L60) for the non-fastidious strains and Chocolate Agar slants (Cat. no. L37) for the fastidious strains at 2 to 8 degrees C. These should be subcultured each week for no more than 3 weeks. New working cultures should be prepared monthly from frozen, freeze dried or commercial cultures. Before using, these strains should be subcultured onto agar plates and streaked for isolation, since it is impossible to determine if slant cultures have become contaminated before use. Frozen or freeze dried preparations should be subcultured twice before testing.
For quality control strains of E. coli ATCC ® 35218 and K. pneumoniae ATCC ® 700603, it is best to store at -60 degrees C. or below and use a minimal number of subcultures, because spontaneous loss of the plasmid encoding beta-lactamase has been documented. This may lead to quality control results of increased zone diameters for; E. coli ATCC ® 35218 with ampicillin, piperacillin and ticarcillin, and increased zone diameters for K. pneumoniae ATCC ® 700603 with cephalosporins and aztreonam.
Q. When do I use transmitted light to read my sensitivity plates?
A. Use transmitted, rather than reflected, light when measuring staphylococci with oxacillin and vancomycin, and/or enterococci with vancomycin. If there are double zones, measure the inner zone. Look for hazy zones or satellite colonies which may denote a resistant sub-population.
Spot Tests and Rapid Tests
Q. Which indole reagent should I be using?
A. There are three indole reagents. Kovacs Indole (Cat. no. Z67) can be used with the “tube” test or with the “spot” test for aerobic bacteria. It is the least sensitive of the three. The Indole DMACA (Cat. no. Z65) is more sensitive and can only be used with the “spot” test. It is for aerobes and anaerobes. Ehrlichs Indole is for the tube test and requires a xylene extraction. It is the most sensitive of the three (ref: Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.).
Q. How can I do a simple confirmation test for Branhamella ( Moraxella ) catarrhalis?
A. Hardy Diagnostics rapid CatScreen™ (Cat. no. Z110) performs a butyrate reaction within minutes for this organism. The disk turns blue within five minutes when positive.
Q. Why are my CatScreen™ disks turning blue?
A. The disks are temperature sensitive. Take them out only when you need to use them and return them to the refrigerator as soon as possible.
Q. What types of oxidase reagents do you have?
A. Oxidase reagent is available in three different formats: OxiDrops™ (Cat. no. Z119), OxiStrips™ (Cat. no. Z93) and OxiSticks™ (Cat. no,. Z173). The OxiStrips*trade; are reagent impregnated paper strips, OxiSticks™ are reagent impregnated swabs, and the OxiDrops™ is a liquid reagent that can be used directly on growing colonies or on a piece of filter paper.
Q. What types of rapid test kits and reagents do you have?
A. Ask your sales representative for a copy of our most recent “Rapid Test Kits and Reagents” brochure. You can also access this information at the Hardy Diagnostics website by selecting a product like Indole (Cat. no. Z67) and choosing the link to this catalog under “Brochures and Studies.”
Q. Is there a single transport media I can use for Chlamydia, Herpes, and Mycoplasma?
A. Our CVM Transport (Cat. no. R96) can be used for all three organisms above. It inhibits most other bacteria and yeast. The transport is available with or without a collection swab. CVM Transport has been proven suitable for maintaining viability of most, chlamydia, viruses and mycoplasma when transported at ambient temperature (15 to 30 degrees C.) for up to 48 hours.
Q. The disposal of mercury-containing PVA is becoming more of a problem in our lab. What is a good substitute?
A. We now have Modified PVA which contains either copper sulfate or Zinc PVA: both of which do not contain mercury. Although most technologists feel these alternatives do not produce the same degree of clarity as traditional PVA, some studies show acceptable results.
Q. What is the best media for fungal cultures?
A. Many experts are now recommending Inhibitory Mold Agar (Cat. no. W25), which is an excellent medium for the primary set-up of fungal cultures. It is a highly enriched medium suitable for fungal pathogens. It contains chloramphenicol to inhibit bacterial contamination. BHI with or without blood may also be used as a non-inhibitory medium. Malt Extract Agar (Cat. no. W28) is another popular fungal medium. V9 Agar and Potato Flake Agar are especially good to enhance the conidia and spore formation of molds.
Q. How can I do a Germ Tube test for Candida without the hazards of using human serum?
A. Our Germ Tube Cryo™ (Cat. no. Z217) consists of newborn calf serum and Tryptic Soy Broth. It comes pre-measured and ready-to-use in small CryoSaver™ vials that are stored in the freezer. This eliminates the hazards of using human serum and saves the technologist time, since it is “ready-to-use.”
Q. How can I keep my mycology plates from drying out when I incubate them for long periods of time?
A. Our petri plate seals MycoSeals™ (Cat. no. SS9225) are designed to prevent media from drying out by forming an air permeable seal over the lid. MycoSeals™ are composed of a cellulose band that shrinks upon drying to keep the lid attached to the bottom dish. For greater safety, MycoSeals™ can also be used to prevent the dispersal of fungal spores and conidia when the lid is accidentally lifted outside of a safety cabinet.
Q. Do you have a chromogenic medium for Candida?
A. Yes, HardyCHROM™ Candida (Cat. no. G301). Colonies of Candida albicans , Candida krusei , Candida glabrata and Candida tropicalis produce distinctive colored colonies which aid in the identification of these species. Rapid Trehalose Broth (Cat. no. Z205) or GlabrataQuick™ (Cat. no. Z298) can be used for definitive identification of C. glabrata .
Q. What is the easiest way to perform a slide culture?
A. MycoVue™ (Cat. no. MV1) is available and contains Potato Flake Agar. It simplifies the slide culture technique by providing all the necessary components for this procedure in one ready-to-use, disposable unit. The device is designed to fit easily onto a microscope stage, thus allowing a direct view of the developing fungus through the device and eliminating disruption of the fungal colony. If desired, the lid of the device can be removed and the coverslip stained for further evaluation or preservation.
Q. Do you have a rapid test for the identification of Candida albicans?
A. AlbiQuick™ (Cat. no. Z121) can be used for the rapid identification of Candida albicans based on the detection of the enzymes beta-galactosaminidase and L-proline aminopeptidase in yeasts.
Q. Why do I occasionally get slight growth in the X quadrant of the Haemophilus ID Quad plate?
A. This “carryover” effect is often seen when testing H. influenzae on the Haemophilus Quad plate. Nutrients from the Chocolate Agar plate can be accidentally carried over with the inoculum to the Quad plate, resulting in slight growth in the X and V quadrants. Always compare the amount of growth in the X and V quadrants to the growth in the Chocolate (fourth) quadrant. Slight growth on the X or V quadrant, compared to luxuriant growth on the Chocolate quadrant, is interpreted as a negative. As a precaution against the “carryover” effect, always prepare a dilution of your inoculum and flame your loop between streaking each quadrant.
Q. How can I prevent an overgrowth of normal respiratory flora when culturing for Haemophilus?
A. Because Haemophilus influenzae colonies are slow growing and small, they are often missed on a Chocolate plate when there is heavy growth of normal flora. Our Chocolate Agar with Bacitracin plate (Cat. no. E11) will inhibit most normal flora ( S. viridans ,Neisseria , staph, and diphtheroids) and allow for the growth of Haemophilus without competition from other colonies for nutrients.
GC Media (Neisseria gonorrhoeae )
Q. Why should Transgrow Bottles not be tilted when inoculating?
A. Transgrow Bottles contain a CO 2 enriched atmosphere. CO 2 is heavier than air. Therefore, tilting the open bottle will cause the CO 2 to run out. Always hold the bottle upright when inoculating or whenever the cap is off. Excess moisture, if present, can be removed with a sterile swab; also while holding the bottle upright.
Q. After inoculating my Martin Lewis with Lincomycin Pill Pocket Plate, should I put a drop of water on the CO 2tablet?
A. After inoculating the plate, place the CO 2 generating tablet in the round well, then seal the zip-lock bag. Do not add any water to the well. There is enough moisture in the bag due to condensation to slowly release the CO 2 . If water is added, the pill will fizz up too rapidly and the CO 2 will be dissipated before the bag can be sealed.
Q. Why do bacteria other than GC grow on the Thayer Martin plates?
Q. How come N. gonorrhoeae can grow on a regular Blood Agar Plate?
A. Our TSA with 5% Sheep Blood plate is so enriched that some strains of N. gonorrhoeae will grow quite well. Never use “growth/no growth” on a blood plate as a criteria for identifying N. gonorrhoeae.
Q. What is a good way to confirm the identification of N. gonorrhoeae?
A. We recommend our “CarboFerm™ Neisseria Kit” (Cat. no. Z98), which is a reliable carbohydrate fermentation method. The carbohydrates are in a dried form, so the kit has a long shelf life. This method takes less than four hours to complete.
Q. What is the best media for isolating Shigella?
A. Selenite Cystine Broth and SS Agar are excellent for Salmonella , but poor for the isolation of Shigella . HE Agar and GN Broth are recommended for both Salmonella and Shigella.
Q. Why do the enteric plates sometimes look like they are contaminated with fungus?
A. During prolonged storage, SS and HE plates will occasionally exhibit “spider” or “snowflake” like formations in the media. These formations are caused by bile precipitates. Although it may look like fungal contamination, it is not. Both of these media contain a high concentration of bile which is used to inhibit gram-positive bacteria. Although a cosmetic defect, it does not influence the growth characteristics of the media and will often clear during incubation.
Q. Is there any advantage to using the new Blood-Free Campy media?
A. Some studies have shown an increased rate of recovery using the charcoal agar based Campy, Blood-Free (Cat. no. G06, Karmali formula). The charcoal helps to neutralize any toxins that might inhibit the growth of C. jejuni , C. coli , and C. laridis . The inhibitory characteristics of this medium are the same as our Campy CVA (Cat. no. A40). Information and samples are available upon request. (ref: J. Clin. Micro., Vol. 23:456-459, Mar. 1986.)
Q. Do you have a rapid test to help identify Campylobacter spp.?
A. Indoxyl Acetate Disks (Cat. no. Z111) help differentiate Campylobacter spp. on the basis of indoxyl acetate hydrolysis. Positives will show a blue-green color within 30 minutes.
Q. How can I isolate and identify E. coli O157:H7?
A. An initial screen can be performed by looking for clear colonies on MacConkey with Sorbitol (Cat. no. G36) or better yet, CT-SMAC (Cefixime Tellurite-Sorbitol MacConkey) Agar (Cat. no. G129). CT-SMAC is more selective for E. coli
O157 and reduces false-positives. CT-SMAC is currently being recommended by many Public Health Departments. These colonies can then be tested by latex agglutination with Hardy’s “E. coliPRO™ O157 Kit” (Cat. no. PL070HD). To test for the flagella H7 antigen use Denka Seiken’s “Antiserum Tube Test” (Cat. no. 295569). Please contact us for more information and product information sheets.
Our CIN / MacConkey with Sorbitol Agar biplate allows you to screen for Aeromonas and Yersinia on the CIN Agar side, as well as E. coli O157 on the other side.
Q. How do I interpret a yellow slant on an EnteroScreen 4™ tube?
A. The slant of an EnteroScreen 4™ tube (Cat. no. L225) determines the deaminase reaction. A negative reaction would be recorded for a purple or yellow slant. A red slant is indicative of a positive reaction for lysine deaminase. Though other Enterobacteriaceae will grow, EnteroScreen 4™ is best used for differentiating Salmonella and Shigella spp.
Q. Can I use the EnteroScreen 4™ to speciate enteric organisms?
A. This product was developed as a single tube screen for non-lactose-fermenting, oxidase-negative, enteric pathogens isolated from stool specimens. A positive lysine deaminase and/or strongly positive urea will immediately rule out Salmonella and Shigellaspecies. A weak positive urea with a negative lysine deamination, no H 2 S and no gas should be investigated further as a possibleYersinia enterocolitica.
Q. With my latex kit for Staphylococcus , I sometimes get rough negatives or weak positives.
A. Try the StaphTEX™ Latex Kit (Cat. no. ST50), which is noted for its easy to read reactions. The negatives are exceptionally smooth, and the positives are strong and easy to read, since the latex beads are blue on a white background.
Q. Can I do the latex test on colonies taken from Mannitol Salt Agar?
A. Do not use colonies grown on high salt containing media (such as Mannitol Salt Agar) to perform the latex agglutination test. Rough, stringy non-interpretable results can occur if isolates are tested from high salt containing media or colonies that are greater than 48 hours old.
Q. Do you have a test to help differentiate S. lugdunensis?
A. Our Rapid Ornithine tubes (Cat. no. K279) provide results in as little as two to four hours. The test can be used to determine ornithine decarboxylase activity in the family Enterobacteriaceae and can be used to differentiate Staphylococcus lugdunensis from other species of Staphylococcus.
Q. What additional tests can I do to confirm group A strep?
A. False-positives will often be encountered when using bacitracin disks to identify group A strep (up to 15%), because groups C and G will often be inhibited by bacitracin as well. Use the more specific PYR (Cat. no. Z75) method to confirm. Studies have shown PYR to have a specificity of 98% for S. pyogenes (ref: J. Clin. Micro., Vol. 15:987, 1982). It produces an instantaneous color change to red if positive. PYR is also a rapid way to assist in confirming Enterococcus faecalis , which will also be positive.
Q. What rapid tests do you have for the identification of gram-positive, catalase-negative cocci?
A. StrepQuick™ (Cat. no. Z122) is a card test containing PYR, LAP and esculin for rapid gram-positive, catalase-negative cocci based on pyroglutamate aminopeptidase (PYR), leucine aminopeptidase (LAP), and esculin hydrolysis (ESC) activity. This test kit simplifies identification of Enterococcus
spp., group A streptococci ( Streptococcus pyogenes
spp., and Pediococcus
spp. An interpretation can be made within 15 minutes.
We also have a Rapid Anginosus ID Kit (Cat. no. Z14), which can be used to detect arginine decarboxylase activity and perform the Voges-Proskauer test in as little as four hours. This kit can be used in the presumptive identification of streptococcal isolates suspected of belonging to the Streptococcus anginosus , formerly known as S. milleri, group ( S. anginosus, S. constellatus, S. intermedius ).
Q. Sometimes I have difficulty in getting reliable results using the CAMP test for group B strep.
A. Due to uncontrollable lot-to-lot variation and seasonal differences in the sheep blood, the CAMP test will sometimes produce weak or non-existent zones of enhanced hemolysis with group B strep. We suggest a more reliable means of identification; such as Hardy’s latex agglutination test, “StrepPRO™” Grouping Kit (Cat. no. PL030HD), or a rapid Hippurate Test (Cat. no. Z52). However, if you must do a CAMP test, always run a positive and negative control.
Q. How can I test for group B strep from a vaginal/rectal OB specimen?
A. Currently, the CDC recommends overnight incubation of the specimen in LIM Broth (Cat. no. L57) or other selective enrichment, and then subculture to a non-selective Sheep Blood Agar plate (Cat. no. A10). Catalase-negative, beta-hemolytic and non-beta-hemolytic colonies after 18-24 hours of incubation should be tested further using a slide agglutination test such as Hardy’s StrepPRO™ Grouping Kit (Cat. no. PL030HD).
An alternate method would be to plate the specimen directly to Granada Medium (Cat. no. G123) or use our Strep B Carrot Broth™ Kit (Cat. no. Z140). Our Strep B Carrot Broth™ Kit is a novel way of screening for positive group B strep cultures from pregnant women. It has been found to be more sensitive than LIM Broth and even PCR. The CDC found it to be 100% accurate when tested against 50 strains of Streptococcus and Enterococcus . A color change to bright orange in as little as six hours indicates a positive culture. Only beta-hemolytic group B streps will be positive, so the negatives need to be subcultured for the rare non-hemolytic strains (less than 4%). You can safely retain the tubes for up to two weeks at room temperature for future subculturing for susceptibility studies. See the Instructions for Use (IFU) or contact us for more information.
Subcultures from LIM Broth or Strep B Carrot Broth™ can be subcultured to GBS Detect (Cat. no. A300), a selective agar formulated to detect non-hemolytic GBS. This special formulation enables all GBS (even those that appear non-hemolytic on a regular blood agar plate) to demonstrate a strong hemolytic reaction in 24 hours.
Q. What is “Blood Agar EH”?
A. Blood Agar with Enhanced Hemolysis (Cat. no. A03) utilizes a new powder base that produces larger and clearer zones of hemolysis around the colonies of beta-hemolytic streptococci. These extremely distinct zones develop more rapidly than on regular Blood Agar. This media should not be used with the CAMP test, and may cause confusing results with bacitracin disks (due to the large hemolytic zones which may spread close to the disk).
Q. What exactly does your “Selective Strep Agar” select for?
A. This type of media (Cat. no. A70) allows for the growth of all
types of strep while inhibiting the growth of most other organisms. This media may also be used to selectively isolate groups A, B, C, F, and G strep as well as S. pneumoniae
We also have another media that selects for group A beta-hemolytic streptococci only . It will inhibit viridans strep and most other normal throat flora. It is called Group A Beta Strep Agar (Cat. no. A72).
Q. How can I differentiate between a possible pathogenic Enterococcus species and a non-pathogenic one?
A. Rapid MGP Medium (Cat. no. Z225) can be used to differentiate the non-pathogenic Enterococcus casseliflavus and Enterococcus gallinarum (MGP-positive) from Enterococcus faecalis and Enterococcus faecium (MGP-negative). Non-pathogens will turn yellow within 5 hours.
Q. How can I get the best recovery of anaerobes from my Thioglycollate Broth?
A. Inoculate Thioglycollate media all the way to the bottom of the tube where anaerobic conditions exist. Also, be careful not to agitate or invert the tube, which will cause oxygen to reach the lower area of the tube. Thioglycollate contains a small amount of agar which may appear as a white precipitate, not to be confused with growth. Consider using our Thio with Supplements (Cat. no. K23). It contains hemin and vitamin K which are required by some fastidious anaerobes. It also contains a calcium carbonate chip (CaCO 3 ), which serves as a buffer to neutralize the build-up of harmful acids as the organisms grow, thus prolonging their viability.
Q. What is the best media for preserving stock cultures of anaerobes?
A. Our Cooked Meat Medium (Cat. no. K19) contains chopped meat, hemin, vitamin K, iron filings, and yeast extract. It has been shown to be a good medium for long-term storage of anaerobic bacteria, when freezing is not convenient. If freezing of the cultures is desired, use our CryoSaver™ Skim Milk (Cat. no. CSM100). Each vial is pre-measured with skim milk, which is an excellent cryopreservative (ref: Summanen, P. et al. 1993. Wadsworth Anaerobic Bacteriology Manual, 5th ed. Star Publishing, Belmont, CA).
Q. How can I optimize my recovery of anaerobes?
A. Transport your specimens in a specialized anaerobic transport medium (such as AG25H, see the “AnaeroGRO™” section of our catalog), and plate them out immediately upon arrival. If using an anaerobe jar, avoid using a gas generating system that requires a catalyst. These types of systems are unreliable and produce explosive hydrogen gas. Instead, use an ascorbic acid oxygen scavenger system such as AnaeroGen™. See “AnaeroGen™” (Cat. no. AN25US or AN35US), Oxoid’s Gas Generator that does not require catalyst or the addition of water.
Q. Why does V Agar have such a short outdate?
A. V Agar which contains 5% human blood is very prone to spontaneous hemolysis. It is made with expired units of blood bank blood. The cells in each unit may vary in their ability to remain intact in the agar. This is why this product is given only a six week shelf-life. The units of blood have been thoroughly tested, and found to be negative for HIV antibody, RPR, and hepatitis B surface antigen. However, biohazard precautions should be exercised, as with all blood products and specimens. Autoclave all units before disposal. Remember that G. vaginalis will produce only a faint beta-hemolysis on this medium, which can take up to 48 hours of incubation. Incubation must be in a CO 2 atmosphere to stimulate growth and beta-hemolysis.
Q. What are some other tests to help confirm the identification of Gardnerella vaginalis?
A. G. vaginalis will be sensitive to a Metronidazole 50mcg disk (Cat. no. DD8) and resistant to a Sulfonamide 1mg disk (Cat. no. DD11). These disks can be placed directly on a V Agar or Chocolate Agar plate.
Q. My AFB cultures often turn out contaminated after doing the digestion procedure.
A. The stock bottle of the Phosphate Buffer and NALC Reagent can easily become contaminated if allowed to touch the rim of the centrifuge tube. Also, as the liquid is being poured out, air is being sucked back into the bottle. This can be another source of contamination. To eliminate this problem we offer Phosphate Buffer (Cat. no. X43) and TB Base Digestant (Cat. no. X45) in smaller, single use, sizes (50ml) that can be discarded after each use to reduce the chance of contamination.
Q. During digestion procedures, how do I know that the specimen has been adequately neutralized by the buffer?
A. Our TB Prep Kit Red™ (Cat. no. Z158) has a pH indicator. When the specimen is adequately neutralized the specimen will be colorless.
Q. How can I culture for the types of mycobacteria that will not grow on ordinary Middlebrook and Lowenstein Jensen?
A. Mycobacterium haemophilum
can be grown on a Middlebrook 7H11 plate that has an “X-Factor Strip” placed on the surface. This supplies hemin that this organism requires (ref: Clin. Micro. Newsletter, Vol. 14:11, June 1, 1992).
Mycobacterium genavense can be cultured on our Middlebrook 7H10 with 10% Human Blood (Cat. no. C42).
Q. What is the best reference source for clinical microbiology?
A. The most complete and easy to use reference text on the market, we believe, is the Color Atlas and Text of Diagnostic Microbiology , by Koneman (Cat. no. 730147). Another good one to have is Bailey and Scott’s Diagnostic Microbiology , by Finegold, Baron and Peterson (Cat. no. 3083300). It contains step-by-step procedures and a wealth of practical information on infectious diseases, with many color photographs. ASM’s Manual of Clinical Microbiology , 11th ed. (Cat. no. 5817374, hardcover) is another valuable source of information.
Q. What is the best source of help for writing a procedure manual for microbiology?
A. The CLSI publication GP2, Clinical Laboratory Technical Procedure Manuals , provides guidelines for writing procedures. The ASM’sClinical Microbiology Procedures Handbook (Cat. no. 5815271) provides hundreds of procedures, all written in the approved CLSI, and is available in a three ring binder. All of our Instructions for Use (IFU) on our online catalog at www.HardyDiagnostics.com are written in the recommended CLSI format so they can be used in your manual.
Inoculation Procedures for Media QC
Q. What are your inoculation procedures for media QC?
A. Refer to the inoculation procedures for media QC document available here
Limitations of Procedures and Warranty
Q. What are your limitations of procedures and warranty?
A. Refer to the limitation of procedures and warranty document available here
ATCC is a registered trademark of the American Type Culture Collection.
Tween is a registered trademark of ICI Americas, Inc.
AnaeroGen is a trademark of Oxoid, Ltd.