The Kwikculture IDSX Kit for presumptive Identification and direct (without prior isolation) Antimicrobial Susceptibility Testing of urinary tract pathogens
Rev. 09232011
CCNAB
Columbia CNA agar with 5% blood
MAC
MacConkey agar
MH
Mueller-Hinton agar
MH
Mueller-Hinton agar
Sterile Water(2)
Rayon tipped swab(1) Color coded Antibiotic applicators(2) with antibiotic papers(up to 14) attached under protective covers. Loop/Needle(1) (ends bent) Transparent measuring films (2) (for post-incubation placement)
IDSX kit tools used for sample application and colony isolation
The rayon tipped sterile swab is used for applying sample to the chambers of the IDSX plate by: (1)uniformly spreading the sample over the surface of the agars in the two MH chambers [ where susceptibility testing is done] and (2) applying the sample as a streak in the CCNAB and MAC chambers [where isolation, quantitation and eventual identification are performed]. The loop/needle dilutes out the sample from the streaks in the CCNAB and MAC chambers to allow for isolated colony growth (isolation).
sterile swab
Loop/needle
LEUKOCYTES
10
CCNAB
MAC
MH
MH
Apply sample to one MH chamber by first dipping rayon swab in specimen to just cover the tip for 5 seconds. Without touching the edge of the sample tube, transfer this to chamber and uniformly spread over the surface. This will apply approximately 0.05ml of the 0.14ml picked up by the swab to the surface of the agar. Repeat for the other MH chamber. Antimicrobial agents will be added at a later step to these two chambers. Do the following if a 1:10 dilution is necessary (see urine dip stick slide): Since the swab picks up 0.14ml under the above conditions, add the sample saturated swab to 1.26ml sterile water supplied with the kit and mix by swirling prior to applying as described above.
Sample application to MAC and CCNAB chambers to provide for colony isolation
Following the addition of organisms to the MH chambers, as described in the previous slide, sample is now added to the MAC and CCNAB chambers. The swab is dipped in sample and a single streak is made across the MAC chamber (see 1 at left) at a distance of approximately 5mm from the edge by holding the swab at a 45 degree angle from the plane of the chamber. Re-dip swab and repeat for the CCNAB chamber. (The CCNAB step locations are a mirror image of the MAC isolation steps.) Allow streaks to dry one to two minutes and then effect an isolation using the loop/needle: Using the loop end, spread as shown at 2 using 8 to 10 passes within the shaded area boundary. Repeat for the CCNAB using the same loop end. Now use the bent needle end to complete the process for the MAC and CCNAB as shown at 3 also using 8 to 10 passes per chamber. This should result in isolated colonies.
8
CCNAB
MAC
MH
MH
MAC
MH
MH
bottom
bottom
The antimicrobial papers come loaded on two applicators. Pictured at left is an example of applicators with antibiotic papers, attached only at corners, before and after removal of white protective covers. The colored applicator is inserted in the left MH chamber (directly below the red CCNAB chamber). The clear applicator is inserted in the right MH chamber. The release rod shown in right picture is used to apply the papers to the two chambers. The rod is poked into each of the holes down to the yellow stop and then applicators are removed from the chambers. Check to make sure all papers are seated on the agar.
9
CPD = Cefpodoxime CF = Cephalothin CC = Clindamycin AMC = Amoxicillin/ Clavulanate FOX = Cefoxitin AM = Ampicillin
CCNAB
MAC
left
right
SXT = Trimethoprim/ Sulfamethoxizole F/M = Nitrofurantoin TZP = Piperacillin/ Tazobactam AN = Amikacin GM = Gentamicin CIP = Ciprofloxicin D = Doxycycline
Incubation
1. Cover plate with lid, return to pouch and tape closed. 2. Place plate in incubator upside down. 3. incubate at 35C for 12 to 24 hours.
10
(3) Display of Isolated colonies for identification and ability to repeat antimicrobial susceptibility testing when inoculums are too light(<105 cfu/ml) or too heavy(>108 cfu/ml).
(4) Colony forming units (cfu) per ml is determined. See slide #15 (5) Following the placement of transparencies, a result of resistant, intermediate or susceptible is visually determined for each antibiotic tested without the need to measure zones and consult Standard Interpretive Charts. This allows for targeted prescribing with the most prudent choice of antimicrobial agent.
11
(1) Is there a pure culture on the plate or are there several different species present?
Each colony originates from a single bacterium. Each species of bacterial or fungal organism exhibits a fairly unique colony. See table to right for a listing of various characteristics that brings about uniqueness. For example, in regards to the effect of the colony on culture medium with respect to hemolysis, observe the hemolytic reactions on the blood containing CCNAB medium . Beta hemolysis is a clear zone around the colony, that results from the complete hemolysis of red blood cells (rbcs). Alpha hemolysis is a greening of the agar surrounding a colony due to partial hemolysis of rbcs, and gamma hemolysis is the term for non-hemolytic colonies.
12
(2) Identification to gram (+) and gram (-) levels: MAC supports the growth of gram
negative bacteria and gram positive bacteria are inhibited. CCNA supports the growth of gram positive bacteria and gram negative organisms are inhibited.
(3) The isolated colonies on the MAC and/or CCNA allow for further identification. The
pictures below show two simple spot tests of the many that are available: A sample of a colony placed in 3% hydrogen peroxide forms bubbles of oxygen when the enzyme catalase is present.(If possible, sample a colony from the MH chamber since red blood cells also contain catalase) The cytochrome oxidase test produces a purple color when a gram negative colony contains the oxidase enzyme. If a microscope is available, cellular morphology may be observed (see next slide).
A presumptive identification can be made to the genus level for three of the most common gram positive bacteria:
and Enterococcus
Staphylococcus: catalase positive, non-hemolytic, alpha hemolytic or beta-hemolytic (see slide 13). Colonies are generally larger than other types of gram positive species. Staph. aureus has colonies that are golden in color (generally following a 24hr.incubation), beta-hemolytic and are catalase positive. Streptococcus: catalase negative, and generally alpha or beta hemolytic colonies. Enterococcus: catalase negative and non-hemolytic colonies. 13
14
104
105
106
108 107
An approximation can be made as to the concentration of organisms present in the final sample. Compare your result to these pictures. This example shows various concentrations of E.coli. Plates were incubated at 35C for 15 hours. Multiply the matched approximate concentration by 10 if a 1:10 dilution was made from patients specimen. 15
Transparencies placed
Transparencies placed
The films labeled left are dropped into the left MH chambers, and right films are dropped into the right MH chambers. A determination is made as to resistant, intermediate or susceptible for the antibiotics tested by observing the bacterial growth margins relative to the resistant (R) or susceptible (S) arcs. If the margin is less than or equal to the R arc, the species is resistant to the antimicrobial agent. If the margin is greater or equal to the S arc, the species is susceptible to that agent. When the margin falls between the R and S arc, the species has intermediate susceptibility(I).
16
17
Autoclave kit components, at 121C (250F) for 15 minutes. Cool to room temperature and dispose in regular trash.
Treat kit components with a fresh 50%(v/v) solution of household bleach* in water. Wear gloves. Treat plate by adding approximately 10ml of the 50% Clorox solution to each plate chamber. Allow solution to remain in chambers for 10 minutes and then carefully pour into a flushable toilet. The solid media is then easily removed: Tip the plate upside down at a 45 angle over toilet; and run the loop from the kit around the inside edge of each chamber to dislodge the media to the toilet bowl. Flush several times to complete the disposal. The empty plates and other treated components may then be placed in the regular trash. Household bleach (such as Clorox) has an available chlorine content of 5.25%. Hazard: Do not mix with other chemicals such as cleaners, acid or ammonia-containing products. To do so may release hazardous gases. Note: Not harmful to septic systems.
18
Following is an example of Kwikculture's IDSX kit, in this case, used to analyze a urine specimen.
20
Kwikcultures IDSX kit comes complete with everything needed to perform an antibiotic susceptibility test.
The pouch holding the IDSX dish is opened. Pouch is retained for the upcoming incubation step.
21
The swab is transferred to the first Mueller Hinton chamber where the sample is uniformly spread over the surface of the agar to form an eventual lawn of growth.
The above process is then repeated for the other Mueller Hinton chamber.
22
The sample is now added as a single streak to the Columbia CNA agar.
23
Dilution of the streaks in the MAC and CCNAB chambers for colony isolation The streaks are allowed to dry for one to two minutes and then an isolation is performed using the loop/needle tool.
Lightly run the loop end of the tool back and forth across the initial streak in the MacConkey chamber.
Repeat this spreading technique now for the Columbia CNA chamber.
Rotate the tool now to the bent needle end. Further dilute to isolate by passing the tool back and forth perpendicularly across the 2nd spreadings in each of the two chambers.
24
The pair of applicators are removed from the inner envelope and un-taped.
Applicators are now rejoined together and inserted into the twin Mueller Hinton chambers.
25
Empty applicators are now removed from the chambers. Papers are now gently seated onto the agar surfaces using the release rod. The IDSX dish with antibiotic papers in place is nearly ready for incubation.
The dish is now returned to its pouch, and incubated at 35*C overnight.
26
Measuring transparencies provide a direct susceptibility reading right from the chambers.
Transparencies are removed from package and oriented for placement in the chambers. Low-tack paper strips are used to pick-up and place the transparencies into the chambers.
Add measuring transparencies to incubated dish and read results directly from dish
Qualitative and quantitative analysis, concurrent with susceptibility is now possible within 12 to 20 hours.
The right labeled transparency is now placed into the right Mueller Hinton chamber.
The IDSX kit allows a direct readout of antibiotic susceptibility instead of having to manually measure zones and consult interpretive tables to obtain a result.
28
104 cfu/ml
105 cfu/ml
106 cfu/ml
107 cfu/ml
108 cfu/ml
Quantitative
Using the panel of standard concentrations above, one can approximate the cfu/ml of the sample added to the dish. The example to the left is approximately 106 cfu/ml. Knowing the concentration of bacteria allows the user to repeat the assay when inoculums are too light (<105 cfu/ml) or too heavy (>108 cfu/ml). See inoculum effect discussed on slides 6 and 17.
Qualitative
The example shows a pure culture of a lactose fermenting gram negative organism ( based on colony morphology and growth on MAC and inhibited growth on CCNA).
Susceptibility
Susceptible to SXT, F/M,TZP, AN, GM, CIP, D, FOX, AMC, CPD, AM; Intermediate to CF; Resistant to CC.
29