INOCULATION PROCEDURES FOR MEDIA QC
The following inoculation procedures are used by Hardy Diagnostics for performance testing. Refer to the Quality Control section of each Technical Data Sheet in the Hardy Diagnostics software program HUGO™, for the specific Method Code used. An explanation of each method is listed below.
To test the Nutritive Capacity of plated or tubed media.
1. Resuspend a lyophilized pellet of the desired organism in an appropriate broth (see manufacturer's product insert). 2. Transfer several drops to an appropriate plate and streak for isolation. Incubate the plate for the appropriate time (24 to 72 hours) in the correct temperature and atmosphere.
3. Alternatively, a stock culture can be taken from a frozen culture, or from lyophilized strains available in "KWIK-STIK™" or "LYFO DISK®" configurations.
4. Suspend three to five isolated colonies in a small volume of Tryptic Soy Broth (TSB; Cat. no. R30) and incubate for 4 to 5 hours. Adjust the turbidity to match that of a 0.5 McFarland standard (Cat. no. MS05). This basic suspension should contain approximately 107 to 108 CFU/ml. Alternately, a direct suspension can be made if the culture is 18 to 24 hours old (or depending on isolate).
5. For testing the nutritive capacity of a nutrient medium, dilute the cell suspension to 1:100 in TSB or normal saline. 6. Inoculate the test plate or tube with a 10uL calibrated loop of the diluted suspension. This will provide
approximately 103 to 104 CFU per plate or tube. Plates are streaked in four quadrants for isolation. If this does not provide isolated colonies for the media being tested, use a tenfold lighter inoculum.
To test the inhibitory capacity of plated and tubed media.
1. Use the same cell suspension (equivalent to a 0.5 McFarland standard) described in "Method A" and dilute to 1:10 in Tryptic Soy Broth (TSB).
2. Inoculate the inhibitory medium, as described in "Method A", with a 10uL calibrated loop onto the plate or tube. This should result in 104 to 105 CFU per plate or tube. A tenfold lighter inoculum may be required to avoid
overwhelming some selective media. A non-inhibitory plate is also inoculated at the same time, to serve as a positive control.
1. Using a needle, pick a colony from a culture plate that has been prepared as in "Method A".
2. Stab the butt of the medium with the needle to a depth of about ! inch from the bottom of the tube (LIA is stabbed twice).
3. As the needle is being withdrawn from the butt, streak the slant by fishtailing the needle up the slant. Leave caps loose during incubation.
To test Agar Deeps in tubes by stabbing with a needle.
1. Using a needle, pick a colony from a culture plate that has been prepared as in "Method A".
2. Stab the test medium to within ! inch of the bottom of the tube in a straight line. (Motility Test Medium is stabbed only to a depth of " inch. For the two tubes of the OF Dextrose Medium, both tubes are stabbed four times and one is overlaid with sterile mineral oil.)
To test Certain Types of Media for Biochemical Reactions (e.g. Urea, CTA, Simmons Citrate, Acetate, DNase, etc.).
1. Using a loop, pick a colony from a culture plate that has been prepared as in "Method A".
2. Inoculate the slant or plate surface with the needle by fishtailing it over the surface. Inoculate a broth by dipping the loop into the bottom of the tube. All tube caps should be kept loose during incubation (except anaerobic media). Simmons Citrate and Acetate Differential media are to be inoculated with a very light inoculum. Urea slants are streaked with a heavy inoculum. DNase plates are given a single heavy streak (inoculum must be very heavy, especially when testing M. catarrhalis). CTA tubes are given a heavy inoculum by stabbing a few times to a depth of ! inch. Moellers Decarboxylase tubes are given a sterile mineral oil overlay (1ml) after inoculation.
To test Antimicrobial Susceptibility Testing Media according to CLSI (formerly NCCLS) guidelines.
1. Select at least 4 to 5 well isolated colonies from a stock culture from a non-selective plate. Touch the top of each colony with a wire loop and transfer the growth to a tube containing 5ml of TSB (Soybean-Casein Digest). Mueller Hinton Broth is recommended for N. gonorrhoeae, Haemophilus spp., and S. pneumoniae.
2. Allow the broth culture to incubate at 35 degrees C. until it achieves or exceeds the turbidity of the 0.5 McFarland standard (usually 2 to 8 hours). If the stock cultures are 18 to 24 hours old, and are growing on a non-selective medium, a suspension may be prepared directly, as in step 3 below, without incubation. (H. influenzae, S. pneumoniae, and N. gonorrhoeae must be tested in this way, since they do not grow in a broth medium.)
3. Adjust the turbidity for the active growing broth culture with sterile saline to obtain a turbidity visually comparable to that of the 0.5 McFarland standard, vortex the turbidity standard before each use. To perform this test properly, use adequate light, and to aid in the visual comparison, read the tube against a white background with contrasting black lines. The correct density of the turbidity standard can be verified by using a spectrophotometer with a 1cm light path and matched cuvettes to determine the absorbance. The absorbance at 625nm should be 0.08 to 0.10 for the 0.5 McFarland standard.
4. Within 15 minutes after adjusting the turbidity of the inoculumsuspension, dip a sterile non-toxic swab on an applicator into the adjusted suspension. Rotate the swab several times, pressing firmly on the inside wall of the tube above the fluid level. This will remove excess inoculum from the swab. If the surface of the media shows excess moisture (droplets on the surface of the media or on the petri plate lid), then incubate the plates for 10 to 30 minutes with the lids ajar.
5. Inoculate the dried surface of the test plate by streaking the swab over the entire sterile agar surface (make sure to inoculate all the way to the edges of the plate). Repeat this streaking procedure two more times, rotating the plate approximately 60 degrees each time to ensure an even distribution of inoculum. If the plate is satisfactorily streaked and the inoculum is correct, the zones of inhibition will be uniformly circular and there will be a confluent or an almost confluent lawn of growth. If only isolated colonies grow, the inoculum was too light and the test should be repeated. Replace the plate top and allow 3 to 5 minutes, (but no longer than 15 minutes) for any excess surface moisture to be absorbed before applying the drug-impregnated disks.
6. Place the disks on the agar surface, distributing them no closer than 24mm apart. Ordinarily no more than 12 disks should be placed on one 150mm plate or more than 5 disks on a 100mm plate. (When testing fastidious strains such as Haemophilus spp., no more than nine disks should be placed on one 150mm plate and no more than four disks on one 100mm plate.) With sterile forceps, gently tap each disk to ensure complete contact with the surface of the agar. Do not move a disk once it has contacted the surface.
7. Invert the plates and place them in a 35 degrees C. incubator within 15 minutes after the disks are applied. Do not use increased CO2 atmosphere, except for Haemophilus spp. or N. gonorrhoeae which require 5 to 10% CO2. 8. After 16 to 18 hours of incubation, examine each plate and measure the diameters of the zones of complete inhibition. Measure the zones to the nearest whole millimeter, using caliper held on the back of the plate illuminated with reflected light against a black non-reflecting background. If blood is added to the agar base, measure the zones from the surface with reflected light, with the cover removed.
9. The endpoint should be taken as the area showing no obvious growth that can be detected with the unaided eye (not to include faint growth of tiny colonies which can be detected only with difficulty at the edge of the zone of inhibited growth). The zones of inhibition are usually clearly outlined and the veil of swarming growth of Proteus spp. is ignored.
10. The zone sizes should fall within the limits listed on Table 3 of the CLSI (formerly NCCLS) standards. Hardy Diagnostics routinely tests each lot of disk diffusion susceptibility media with representative antibiotics. Refer to the "QC Zone Diameters for Disk Diffusion Testing" for CLSI (formerly NCCLS) recommended acceptable quality control zone ranges.
11. The Mueller Hinton Agar is tested to see that the depth of the media is 4mm. It is measured in several places to make sure that the medium is level. The conveyer belts on which the plates are poured are checked periodically with a level to ensure a horizontal surface on which the media cools.
12. The amount of media for a large Mueller Hinton Agar plate (150mm diameter) is 60 to 70ml; for the small plate (100mm diameter) the fill is approximately 18ml.
13. The pH range is 7.2 to 7.4 at 25 degrees C.
14. Each lot of Mueller Hinton Agar is evaluated for the absence of thymine and thymidine, by testing Enterococcus faecalis (ATCC® 29212) with Trimethoprim/Sulfamethoxazole disks. Satisfactory media will provide essentially clear, distinct zones of inhibition 20mm or more in diameter.
To test media for filamentous fungi:
1. Inoculate one or two tubes of Potato Dextrose Agar or Sabouraud Dextrose (SabDex) Agar, depending on the organism. For heavily sporulating cultures such as Aspergillus and Penicillium, only one tube is necessary. 2. Incubate the slants at room temperature for 5 to 10 days.
3. Add approximately 5ml of sterile distilled water to the slant. Using a sterile stick or sterile needle, gently scrape all surface growth from the slant into the water, taking care not to remove any agar. The suspension should be equivalent to a 4.0 McFarland standard (Cat. no. MS4).
4. Aseptically pipet the suspension into empty cryogenic vials.
5. Label the vials, tightly seal the caps, and store at room temperature. Organisms will remain viable for four to five years.
Using the suspension:
6. To use the suspension, select the appropriate vial and to prepare a dilution in sterile distilled water, equivalent to a 0.5 McFarland standard.
7. Prepare a 1:10 dilution of the 0.5 McFarland.
8. Pipet 0.1ml of the diluted suspension to the media to be tested. Streak the agar surface and then score the media with a needle. Discard the vial after two week of use.
To test Mycobacteria:
9. Prepare a suspension equivalent to a 0.5 to 1.0 McFarland standard using an actively growing culture of the Mycobacterium spp. in 7H9 Broth (Cat. no. C32). Saline or broth may be used to adjust the suspension. 10. Vortex to mix. (concentration 1.5 to 3.0 x 108) Dilute to 1:100. Inoculate 100uL to test media.
To test the Ability of Sheep Blood plates to Produce the CAMP Phenomenon. 1. Staphylococcus aureus (ATCC® 33862) is streaked on the plate in a straight line.
2. Streptococcus agalactiae and Streptococcus pyogenes, taken from a plate, are streaked in lines perpendicular to, but not quite touching the S. aureus streak.
3. Plates are read no longer than 24 hours later and examined for the "bow-tie" or "arrowhead" shaped area of enhanced hemolysis at the junction of the S. agalactiae and S. aureus streak. S. pyogenes will not show the enhanced hemolysis.
To test GN, Selenite Cystine, Tetrathionate and Zephiran TSP Broths.
1. A loopful (0.01ml) from the dilution as described in "Method B" is transferred to the broth to be tested. (For Tetrathionate Broth, 0.2ml of Iodine Solution is added immediately before inoculation.) Also, inoculate a MacConkey Agar control plate.
2. GN Broth is incubated for 6 to 8 hours. Selenite Cystine Broth is incubated 18 to 24 hours. Zephiran TSP Broth is incubated " hour. Tetrathionate is incubated for 12 to 24 hours. Incubate all tubes at 35 degrees C.
3. One loopful (0.01ml) is transferred to a MacConkey Agar plate and streaked for isolation. After incubation for 18 to 24 hours, the resulting number of colonies on each plate is compared with the MacConkey Agar control plates.
To test the Nutritive Capacity of Tubed and Plated Media as Required by United States Pharmacopeia (USP) Standards.
Dilute each organism, as shown below to yield the stated inoculum concentration.
1. Prepare the organism to be tested according to steps 1 and 2 in "Method A" using 5ml of TSB (Cat. no. R30). It is recommended that a spectrophotometer be used to adjust the density of the inoculum. A 0.5 McFarland standard is equivalent to an ABS of 0.08 to 0.1 at 625nm. A blank 5ml of TSB can be used to "zero" the ABS before measuring the
ABS of the suspension. 2. Mix suspension well.
3. Aseptically remove 0.5ml of the suspension and transfer to a sterile 5ml of TSB (The new suspension created is 1:10 or 10-1). Mix well.
4. Aseptically remove 0.5ml of the suspension in step 3 above and transfer to a sterile 5ml of TSB (The new suspension created is 1:100 or 10-2). Mix well.
5. Aseptically remove 0.5ml of the suspension in step 4 above and transfer to a sterile 5ml TSB (The new suspension created is 1:1000 or 10-3). Mix well.
Repeat as necessary to attain the desired concentration.
Organism* InoculumDesired Concentration Spread Plate-Dilution Needed (0.1ml inoculation volume) Pour Plate-Dilution Needed (1.0ml inoculation volume)
Escherichia coli 1-100 CFU 10-6 10-5
Bacillus subtilis 1-100 CFU 10-6 10-5
aeruginosa 1-100 CFU 10-7 10-6 Salmonella enterica 1-100 CFU 10-7 10-6
Staphylococcus aureus 1-100 CFU 10-7 10-6
Candida albicans 1-100 CFU 10-6 10-5
Clostridium sporogenes 1-100 CFU 10-6 10-5
formerly A. niger 1-100 CFU 10-6 10-5
* Refer to Quality Control Report form (QCR) for atmosphere, time and temperature of incubation. Some organisms may be tested at more than one incubation temperature as required by various industry standards such as USP. Note: All testing using this method should be conducted in duplicate. Colony counts for the inoculum should be verified by plating on a non-selective media such as Standard Methods, TSA or SabDex Agar (filamentous fungi). Note: Growth promotion of other organisms on culture media may be evaluated using this method, however inoculum dilutions may require adjusting to yield 10-100 CFUs.
To test the Nutritive Ability of Media Intended for Mycoplasma and Ureaplasma Propagation. 1. Prepare the stock solution and working solutions.
2. Inoculate the entire content of the working solution from the cryogenic vial into the media to be tested.
From the 1:100 dilution of the control organism prepare serial dilutions using 5ml of TSB for each dilution. 1. Mix the suspension well.
2. Aseptically remove 0.5ml of the suspension and transfer it to a sterile 5ml of TSB. (10-3) Mix well. 3. Aseptically remove 0.5ml of the suspension and transfer it to a sterile 5ml of TSB. (10-4) Mix well. 4. Aseptically remove 0.5ml of the suspension and transfer it to a sterile 5ml of TSB. (10-5) Mix well. 5. Prepare plates and label with the dilutions to be plated.
6. Select appropriate dilution to plate. Salmonella enterica and E. faecalis should be used at the 10-4 dilution. E. coli and the rest of the organisms can generally be used at the 10-3 dilution. (When using older suspensions, use a higher dilution.)
7. Mix each dilution well and transfer one loopful (0.01ml) to the appropriately labeled plate.
8. For 60mm plates, inoculate the surface of the test plate by streaking the loop over the entire agar surface. (Make sure to inoculate all the way to the edges of the plate.) Repeat this streaking procedure 2 more times, rotating the plate 90 degrees from the first streaking and then rotate again at 45 degrees from the 2nd streaking.
9. For 100mm plates, inoculate with the appropriate dilution, then streak in a normal manner. 10. Incubation is recommended.
11. If multiple dilutions are plated, record QC results from the 60mm plate with 20 to 80 colonies present. ATCC is a registered trademark of the American Type Culture Collection.
LYFO DISK is a registered trademark of MicroBioLogics, Inc., St. Cloud, MN. KWIK-STIK is a trademark of MicroBioLogics, Inc., St. Cloud, MN.
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