What Factors Influence The Size Of The Zone Of Inhibition For An Antibiotic

Introduction

At a meeting organized by WHO in Geneva in 1977,¹ business organization was expressed about the worldwide increase in antibiotic resistance associated with the growing, and frequently indiscriminate, use of antibiotics in both man and animals. In recent years, drug-resistant bacteria have given rise to several serious outbreaks of infection, with many deaths. This has led to a need for national and international surveillance programmes to monitor antibody resistance in bacteria by susceptibility testing using reliable methods that generate comparable data. The availability of microbiological and epidemiological data would help clinicians in selecting the most advisable antimicrobial agent for the handling of a microbial infection.

If predictions are to be valid, the susceptibility exam must be performed by an accurate and reproducible method, the results of which should be direct applicable to the clinical situation. The ultimate criterion of the reliability of any susceptibility testing method is its correlation with the response of the patient to antimicrobial therapy.

The WHO meeting considered that the modified disc technique of Kirby-Bauer, for which requirements had been established by WHO in I976,² could be recommended for clinical and surveillance purposes in view of its technical simplicity and reproducibility. The method is particularly suitable for use with bacteria belonging to the family Enterobacteriaceae, but it can also be recommended as a general purpose method for all chop-chop growing pathogens, except strict anaerobes. It was therefore recommended that the details of this examination exist made available for laboratory workers.ⁱⁱⁱ

^one WHO Technical Report Series, No. 624, 1978 (Surveillance for the prevention and control of wellness hazards due to antibiotic-resistant enterobacteria: report of a WHO Meeting).
ⁱⁱ WHO Technical Report Series, No. 610, 1977 (Twenty-eighth report of the WHO Proficient Committee on Biological Standardization), Addendum 5.

ⁱⁱⁱ A comparable method, based on the same principles and quality control requirements as the Kirby-Bauer method, is the NEO-SENSITABS method, produced by ROSCO Diagnostica, Taastrup, Denmark. This method uses 9-mm colour-coded, antibody tablets, instead of paper discs. The tablet form results in an boggling stability with a shelf-life of iv years, fifty-fifty at room temperature. This increased stability is very important for laboratories in tropical countries.

Full general principles of antimicrobial susceptibility testing

Antimicrobial susceptibility tests measure the ability of an antibiotic or other antimicrobial amanuensis to inhibit bacterial growth in vitro. This ability may be estimated by either the dilution method or the diffusion method.

The dilution test

For quantitative estimates of antibody action, dilutions of the antibiotic may exist incorporated into broth or agar medium, which is and so inoculated with the test organism. The everyman concentration that prevents growth after overnight incubation is known every bit the minimum inhibitory concentration (MIC) of the agent. This MIC value is so compared with known concentrations of the drug obtainable in the serum and in other body fluids to assess the likely clinical response.

The diffusion test

Paper discs, impregnated with the antibody, are placed on agar medium uniformly seeded with the test organism. A concentration gradient of the antibiotic forms by diffusion from the disc and the growth of the exam organism is inhibited at a altitude from the disc that is related, among other factors, to the susceptibility of the organism.

There is an approximately linear relation betwixt log MIC, as measured by a dilution test, and the inhibition zone diameter in the diffusion examination. A regression line expressing this relation tin be obtained by testing a big number of strains past the 2 methods in parallel (encounter Fig. 7 and eight).

Fig. 7. Graphic representation of the relationship between log₂ MIC and the inhibition zone diameter obtained past the improvidence test using discs containing a single concentration of antibiotic

Fig. 8. Interpretation of zone sizes as susceptible, intermediate, and resistant past their relationship to the MIC

Clinical definition of terms "resistant" and "susceptible": the three-category system

The result of the susceptibility test, as reported to the clinician, is the classification of the microorganism in 1 of two or more categories of susceptibility. The simplest organization comprises only 2 categories: susceptible and resistant. This classification, although offer many advantages for statistical and epidemiological purposes, is too inflexible for the clinician to utilise. Therefore, a 3-category classification is often adopted. The Kirby-Bauer method and its modifications recognize 3 categories of susceptibility and it is important that both the clinician and the laboratory worker understand the exact definitions and the clinical significance of these categories.

· Susceptible. An organism is called "susceptible" to a drug when the infection caused by information technology is likely to reply to treatment with this drug, at the recommended dosage.
· Intermediate susceptibility covers two situations. It is applicative to strains that are "moderately susceptible" to an antibiotic that can be used for handling at a higher dosage considering of its depression toxicity or because the antibiotic is concentrated in the focus of infection (e.grand., urine).

The nomenclature also applies to strains that show "intermediate susceptibility" to a more toxic antibiotic that cannot be used at a higher dosage. In this situation, the intermediate category serves every bit a buffer zone between susceptible and resistant.

As most clinicians are not familiar with the subtle, although clinically important, distinction between intermediate and moderate susceptibility, many laboratories use the designation "intermediate" for reporting purposes.

· Resistant. This term implies that the organism is expected not to respond to a given drug, irrespective of the dosage and of the location of the infection.

For testing the response of staphylococci to benzylpenicillin, only the categories "susceptible" and "resistant" (corresponding to the production of b-lactamase) are recognized.

The ultimate decision to apply a item antibiotic, and the dosage to be given, volition depend not only on the results of the susceptibility tests, but also on their interpretation by the physician. Other factors, such as pathogenic significance of the microorganism, side-furnishings and pharmacokinetic properties of the drug, its diffusion in different torso sites, and the immune status of the host, will besides have to be considered.

Indications for routine susceptibility tests

A susceptibility test may be performed in the clinical laboratory for ii main purposes:

· to guide the clinician in selecting the best antimicrobial agent for an private patient;
· to accumulate epidemiological information on the resistance of microorganisms of public health importance within the community.

Susceptibility tests as a guide for handling

Susceptibility tests should never exist performed on contaminants or commensals belonging to the normal flora, or on other organisms that have no causal human relationship to the infectious process. For case, the presence of Escherichia coli in the urine in less than meaning numbers is non to be regarded as causing infection, and it would be useless and even misleading to perform an antibiogram.

Susceptibility tests should exist carried out merely on pure cultures of organisms considered to be causing the infectious process. The organisms should likewise exist identified, equally not every microorganism isolated from a patient with an infection requires an antibiogram.

Routine susceptibility tests are not indicated in the following situations:
· When the causative organism belongs to a species with predictable susceptibility to specific drugs. This is the case for Streptococcus pyogenes and Neisseria meningitidis, which are still generally susceptible to penicillin. (However, there have recently been a few reports of desultory occurrences of penicillin-resistant meningococci.) It is as well the example for faecal streptococci (enterococci), which, with few exceptions, are susceptible to ampicillin. If resistance of these microorganisms is suspected on clinical grounds, representative strains should be submitted to a competent reference laboratory.

· If the causative organism is boring-growing or captious and requires enriched media, e.g., Haemophilus influenzae and Neisseria gonorrhoeae, disc-diffusion susceptibility tests may give unreliable results.

The emergence of b-lactamase-producing variants of these species has led to the introduction of special tests, such as the in vitro exam for b-lactamase production. It will be the responsibleness of the central and regional laboratories to monitor the susceptibility of pneumococci, gonococci, and Haemophilus. If issues arise with resistant strains, the peripheral laboratories should be alerted and instructions should be given on advisable testing methods and on alternative treatment schemes.

· In uncomplicated intestinal infections acquired by salmonellae (other than Due south. typhi or Due south. paratyphi), susceptibility tests are not routinely needed. Antibiotic treatment of such infections is not justified, even with drugs showing in vitro activeness. At that place is at present ample prove that antimicrobial treatment of common salmonella gastroenteritis (and indeed of virtually types of diarrhoeal illness of unknown etiology) is of no clinical do good to the patient. Paradoxically, antibiotics prolong the excretion and dissemination of salmonellae and may lead to the selection of resistant variants.

Susceptibility tests as an epidemiological tool

Routine susceptibility tests on major pathogens (S. typhi, shigellae) are useful as function of a comprehensive programme of surveillance of enteric infections. They are essential for informing the physician of the emergence of resistant strains (chloramphenicol-resistant S. typhi, co-trimoxazole-resistant and ampicillin-resistant shigellae) and of the demand to modify standard handling schemes. Although susceptibility testing of non-typhoid salmonellae serotypes causing abdominal infection is not relevant for treating the patient, the appearance of multiresistant strains is a warning to the physician of the overuse and misuse of antimicrobial drugs.

Continued surveillance of the results of routine susceptibility tests is an fantabulous source of information on the prevalence of resistant staphylococci and Gram-negative bacilli that may exist responsible for cantankerous-infections in the hospital. Periodic reporting of the susceptibility design of the prevalent strains is an invaluable aid to forming a sound policy on antibiotic usage in the hospital by restriction and/or rotation of life-saving drugs, such as the aminoglycosides and cephalosporins.

Option of drugs for routine susceptibility tests in the clinical laboratory

The choice of drugs used in a routine antibiogram is governed past considerations of the antibacterial spectrum of the drugs, their pharmacokinetic properties, toxicity, efficacy, and availability, equally well as their cost to both the patient and the community. Among the many antibacterial agents that could be used to treat a patient infected with a given organism, only a limited number of advisedly selected drugs should be included in the susceptibility test.

Table 12 indicates the drugs to be tested in diverse situations. The drugs in the table are divided into two sets. Set 1 includes the drugs that are available in most hospitals and for which routine testing should exist carried out for every strain. Tests for drugs in set 2 are to be performed only at the special asking of the physician, or when the causative organism is resistant to the first-choice drugs, or when other reasons (allergy to a drug, or its unavailability) make farther testing justified. Many antibiotics with expert clinical activity have been omitted from the table, but it must exist emphasized that they are rarely needed in the direction of the infected patient. In very rare cases, one or more additional drugs should exist included when there is a special reason known to the physician, or when new and improve drugs become available. Periodic revision of this table is therefore desirable, and this should be done later appropriate discussions with clinical staff. Many problems arise in practise, because clinicians are not e'er enlightened that only one representative of each group of antimicrobials is included in routine tests. The result obtained for this particular drug may then be extrapolated to all, or most, of the other members of the group. Difficulties ascend in some countries when the physician is familiar only with the commercial brand proper name of the drug and non with its generic nonproprietary proper noun. A serious effort should be fabricated to inform medical personnel nearly the international nonproprietary names of pharmaceutical substances, and to encourage their employ.¹

¹ International Nonproprietary Names for Pharmaceutical Substances, Cumulative List No. 7. Geneva, World Health System, 1988.

Tabular array 12. Basic sets of drugs for routine susceptibility tests^a

	*Staphylococcus*	Enterobacteriaceae			*Pseudomonas aeruginosa*
		Intestinal	Urinary	Blood and tissues
Set 1 First choice	benzylpenicillin oxacillin erythromycin tetracycline chloramphenicol	ampicillin chloramphenicol co-trimoxazole nalidixic acrid tetracycline	sulfonamide trimethoprim co-trimoxazole ampicillin nitrofurantoin nalidixic acid tetracycline	ampicillin chloramphenicol co-trimoxazole tetracycline cefalotin gentamicin	piperacillin gentamicin tobramycin
Fix 2 Additional drugs	gentamicin amikacin co-trimoxazole clindamycin	norfloxacin	norfloxacin chloramphenicol gentamicin	cefuroxime ceftriaxone ciprofloxacin piperacillin amikacin	amikacin

^a Notes on the individual antibacterial agents are given in the text.

one. The benzylpenicillin disc is used to examination susceptibility to all b-lactamase-sensitive penicillins (such as oral phenoxymethylpenicillin and pheneticillin). Isolates of staphylococci that fall into the resistant category produce b-lactamase and should be treated with a b-lactamase-resistant penicillin or with another antibiotic, such as erythromycin.

2. Oxacillin. The oxacillin disc is representative or the whole group of b-lactamase-resistant penicillins (including meticillin, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin). Moreover, at that place is proficient clinical show that cross-resistance exists betwixt the meticillin and the cephalosporin groups. Therefore, it is useless and misleading to include cefalotin in the antibiogram for staphylococci. Resistance to meticillin and related drugs is ofttimes of the heterogeneous blazon, i.e., the majority of cells may be fully susceptible and produce a wide inhibition zone, while the resistant office of the population appears in the form of infinitesimal discrete colonies growing within the inhibition zone. This type of resistance is more apparent when the temperature of the incubator is prepare at 35 °C¹ or when the incubation time is prolonged.

A serious disadvantage of meticillin, as a representative disc for the b-lactamase-resistant penicillins, is its swell lability even under conventional storage atmospheric condition. The oxacillin disc is much more resistant to deterioration and is therefore preferred for the standardized diffusion test. The cloxacillin and dicloxacillin discs are not used as they may not indicate the presence of a heteroresistant strain.

¹ SAHM, D. F. et al. Current concepts and approaches to antimicrobial agent susceptibility testing. In: Cumitech 25, Washington, DC, American Order for Microbiology, 1988.

3. The results for the tetracycline disc may be applied to chlortetracycline, oxytetracycline, and other members of this grouping. Notwithstanding, nigh tetracycline-resistant staphylococci remain normally sensitive to minocycline. A disc of minocycline may thus exist useful to test multiresistant strains of staphylococci.

iv. The result with the chloramphenicol disc may be extrapolated to thiamphenicol, a related drug with a comparable antibacterial spectrum, but without known gamble of aplastic anaemia.

5. Only 1 representative sulfonamide (sulfafurazole) is required in the exam.

6. The co-trimoxazole disc contains a combination of trimethoprim and a sulfonamide (sulfamethoxazole). Although the use of combinations of drugs in discs has been condemned in previous WHO reports,^two co-trimoxazole is an exception because the ii components of this synergistic combination have comparable pharmacokinetic backdrop and generally act "as a single drug".

² WHO Technical Written report Serial, No. 796, 1990 (The use of essential drugs: fourth study of the WHO Proficient Committee).

seven. Ampicillin is the epitome of a group of broad-spectrum penicillins with action against many Gram-negative bacteria. As it is susceptible to b-lactamase, information technology should not exist used for testing staphylococci. By and large, the susceptibility to ampicillin is likewise valid for other members of this group: amoxycillin, pivampicillin, talampicillin, etc. (though amoxycillin is twice equally active confronting salmonellae and only half every bit active against shigellae and H. influenzae).

8. Cefalotin. Only cefalotin needs to be tested routinely, equally its spectrum is representative of all other first-generation cephalosporins (cefalexin, cefradine, cefaloridine, cefazolin, cefapirin). Where second- and third-generation cephalosporins and related compounds (cefamycins) with an expanded spectrum are available, a separate disc for some of these new drugs may be justified in selected cases (cefoxitin, cefamandole, cefuroxime, cefotaxime, ceftriaxone). Although some cephalosporins can be used to treat severe staphylococcal infections, the susceptibility of the infecting strain can be derived from the result with oxacillin as already mentioned nether two above.

9. Erythromycin is used to exam the susceptibility to some other members of the macrolide grouping (oleandomycin, spiramycin).

10. Aminoglycosides. This group of chemically related drugs includes streptomycin, gentamicin, kanamycin, netilmicin and tobramycin. Their antimicrobial spectra are not always closely enough related to allow assumption of cross-resistance, but against susceptible pathogens these agents have been shown to be equally effective. Numerous studies have compared the nephrotoxicity and ototoxicity of gentamicin, netilmicin and tobramycin, only there is no conclusive testify that any 1 of the drugs is less toxic than the others. It is strongly recommended that each laboratory select a single agent for primary susceptibility testing. The other agents should be held in reserve for treatment of patients with infections caused by resistant organisms.

xi. Nitrofurantoin is limited to utilise only in the handling of urinary tract infections, and should not be tested against microorganisms recovered from material other than urine.

The modified Kirby-Bauer method

The disc diffusion method, originally described in 1966,¹ is well standardized and has been widely evaluated. Official agencies take recommended it, with modest modifications, as a reference method which could be used as a routine technique in the clinical laboratory.

¹ BAUER, A. Due west. et al. Antibody susceptibility testing by a standardized unmarried disc method. American journal of clinical pathology, 44: 493-496 (1966).

Reagents

Mueller-Hinton agar

one. Mueller-Hinton agar should be prepared from a dehydrated base according to the manufacturer'south recommendations. The medium should be such that command zone sizes within the published limits are produced (see Table xiii). It is of import not to overheat the medium.

2. Cool the medium to 45-50 °C and cascade into the plates. Permit to gear up on a level surface, to a depth of approximately 4 mm. A 9-cm plate requires approximately 25 ml of medium.

3. When the agar has solidified, dry the plates for firsthand use for 10 - 30 minutes at 35 °C, by placing them in the upright position in the incubator with the lids tilted.

four. Whatever unused plates may be stored in a plastic bag, which should be sealed and placed in the fridge. Plates stored in this way will keep for ii weeks.

To ensure that the zone diameters are sufficiently reliable for testing susceptibility to sulfonamides and co-trimoxazole, the Mueller-Hinton agar must have low concentrations of the inhibitors thymidine and thymine. Each new lot of Mueller-Hinton agar should therefore be tested with a control strain of Enterococcus faecalis (ATCC 29212 or 33186) and a disc of co-trimoxazole. A satisfactory lot of medium volition give a singled-out inhibition zone of 20 mm or more than that is substantially free of hazy growth or fine colonies.

Antibody discs

Any commercially available discs with the proper bore and potency tin can be used. Stocks of antibiotic discs should preferably be kept at - twenty°C; the freezer compartment of a dwelling house refrigerator is user-friendly. A small-scale working supply of discs can be kept in the fridge for upwards to 1 month. On removal from the refrigerator, the containers should be left at room temperature for about one hour to allow the temperature to equilibrate. This process reduces the amount of condensation that occurs when warm air reaches the cold container. If a disc-dispensing apparatus is used, it should have a tight-fitting encompass and be stored in the refrigerator. Information technology should also exist immune to warm to room temperature before beingness opened.

Tabular array xiii. Zone diameter limits for control strains^a

		Diameter of zone of inhibition (mm)
Antibiotic	Disc potency	S. aureus (ATCC 25923)	*E. coli* (ATCC 25922)	*P. aeruginosa* (ATCC 27853)
amikacin	xxx m1000	xx-26	19-26	xviii-26
ampicillin	10 mg	27-35	xvi-22	-
benzylpenicillin	x IU	26-37	-	-
cefalotin	thirty mgrand	29-37	17-22	-
ceftriaxone	30 mone thousand	22-28	29-35	17-23
cefuroxime	xxx mchiliad	27-35	20-26	-
chloramphenicol	30 myard	19-26	21-27	-
ciprofloxacin	100 mone thousand	22-30	30-xl	25-33
clindamycin	2 mg	24-30	-	-
co-trimoxazole	25 kg	24-32	24-32	-
erythromycin	fifteen chiliadg	22-30	8-xiv	-
gentamicin	10 grandg	19-27	nineteen-26	16-21
nalidixic acid	30 thousandk	-	22-28	-
nitrofurantoin	300 mg	18-22	20-25	-
norfloxacin	10 mchiliad	17-28	28-35	22-29
oxacillin	1 mg	18-24	-	-
piperacillin	100 myard	-	24-30	25-33
sulfonamide^b	300 yardg	24-34	eighteen-26	-
tetracycline	thirty mthousand	nineteen-28	xviii-25	-
tobramycin	ten mg	xix-29	18-26	nineteen-25
trimethoprim	5 kk	19-26	21-28	-

^a National Commission for Clinical Laboratory Standards. Performance standards for antimicrobial disc susceptibility tests. Tentative standard. 4th ed. Villanova, PA, U.s.a., NCCLS, 1988.
^bSulfafurazole.

Turbidity standard

Prepare the turbidity standard by pouring 0.6 ml of a 1% (10 1000/litre) solution of barium chloride dihydrate into a 100-ml graduated cylinder, and filling to 100 ml with 1% (10 ml/litre) sulfuric acid. The turbidity standard solution should be placed in a tube identical to the one used for the goop sample. It can be stored in the night at room temperature for six months, provided information technology is sealed to prevent evaporation.

Swabs

A supply of cotton fiber wool swabs on wooden applicator sticks should exist prepared. They can exist sterilized in tins, culture tubes, or on paper, either in the autoclave or by dry out heat.

Procedure

To prepare the inoculum from the primary culture plate, touch with a loop the tops of each of iii - 5 colonies, of similar appearance, of the organism to be tested.

Figure

Transfer this growth to a tube of saline.

Figure

When the inoculum has to be made from a pure culture, a loopful of the confluent growth is similarly suspended in saline.

Compare the tube with the turbidity standard and adjust the density of the test suspension to that of the standard by adding more bacteria or more than sterile saline.

Effigy

Proper aligning of the turbidity or the inoculum is essential to ensure that the resulting lawn of growth is confluent or almost confluent.

Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess inoculum by pressing and rotating the swab firmly against the side of the tube above the level of the liquid.

Effigy

Streak the swab all over the surface of the medium 3 times, rotating the plate through an bending of sixty ° later on each awarding. Finally, pass the swab round the border of the agar surface. Leave the inoculum to dry for a few minutes at room temperature with the hat closed.

Figure

The antibiotic discs may be placed on the inoculated plates using a pair of sterile forceps. It is convenient to use a template (see Fig. ten) to place the discs uniformly.

Figure

A sterile needle tip may too be used to place the antibiotic discs on the plate.

Figure

Alternatively, an antibiotic disc dispenser can be used to apply the discs to the inoculated plate.

Effigy

A maximum of seven discs tin be placed on a ix-ten cm plate. Six discs may be spaced evenly, approximately 15 mm from the edge of the plate, and 1 disc placed in the center of the plate. Each disc should be gently pressed downwards to ensure even contact with the medium.

The plates should be placed in an incubator at 35 °C within thirty minutes of grooming. Temperatures above 35 °C invalidate results for oxacillin/meticillin.

Do not incubate in an atmosphere of carbon dioxide.

Subsequently overnight incubation, the bore of each zone (including the diameter of the disc) should be measured and recorded in mm. The results should then be interpreted according to the disquisitional diameters shown in Table 14.

The measurements can exist fabricated with a ruler on the under-surface of the plate without opening the lid.

Effigy

If the medium is opaque, the zone tin be measured by ways of a pair of calipers.

Effigy

A template (see Fig. 9) may be used to assess the final result of the susceptibility tests.

Effigy

The endpoint of inhibition is judged by the naked eye at the border where the growth starts, simply there are 3 exceptions:

· With sulfonamides and co-trimoxazole, slight growth occurs inside the inhibition zone; such growth should exist ignored.
· When b-lactamase-producing staphylococci are tested against penicillin, zones of inhibition are produced with a heaped-up, clearly defined edge; these are readily recognizable when compared with the sensitive control, and regardless of size of zone of inhibition, they should exist reported as resistant.

· Certain Proteus species may swarm into the expanse of inhibition around some antibiotics, but the zone of inhibition is unremarkably clearly outlined and the thin layer of swarming growth should be ignored.

Interpretation of the zone sizes

· Using a template. When the zone sizes are compared with the template (see Fig. 9) the consequence - susceptible, resistant, or intermediate - tin can be read at one time: "susceptible", when the zone edge is outside the black circle; "resistant", when there is no zone, or when it lies within the white circle; and "intermediate", when the border of the zone of inhibition lies on the black circle.
· Using a ruler. When the zone sizes are measured in mm, the results should be interpreted co-ordinate to the critical diameters given in Table 14.

Straight versus indirect susceptibility tests

In the standardized method outlined to a higher place, the inoculum is prepared from colonies on a primary culture plate or from a pure civilisation. This is called an "indirect sensitivity test". In certain cases, where a rapid respond is important, the standardized inoculum may be replaced past the pathological specimen itself, e.chiliad., urine, a positive claret culture, or a swab of pus. For specimens of urine, a microscopical test of the sediment should first exist made in order to see if there is evidence of infection, i.e., the presence of pus cells and/or organisms. The urine may so be used as the inoculum in the standard examination. Too, susceptibility tests may exist performed on incubated blood cultures showing evidence of bacterial growth, or a swab of pus may exist used as a direct inoculum, when a Gram-stained smear shows the presence of large numbers of a single type of organism. This is chosen a "directly susceptibility test"; its reward over the indirect test is that a outcome is obtained 24 hours earlier. The primary disadvantage is that the inoculum cannot be properly controlled. When the susceptibility plate shows also light or likewise heavy growth, or when the civilisation is a mixture, the results should be interpreted with circumspection, and the test repeated on pure cultures.

Technical factors influencing the size of the zone in the disc diffusion method

Inoculum density

If the inoculum is too light, the inhibition zones will exist larger although the sensitivity of the organism is unchanged. Relatively resistant strains may and so be reported as susceptible. Conversely, if the inoculum is too heavy, the zone size will be reduced and susceptible strains may be reported as resistant. Commonly optimal results are obtained with an inoculum size that produces about confluent growth.

Fig. 9. Zone diameters for the determination of susceptibility with the standard disc improvidence method

Table fourteen. Interpretative chart of zone sizes^a

	Diameter of zone of inhibition (mm)
Antibiotic or chemotherapeutic amanuensis	Disc potency	Resistant	Intermediate/ moderately susceptible	Susceptible
amikacin	thirty mg	£ 14	15-16	³ 17
ampicillin when testing:
- Enterobacteriaceae	10 one thousandgrand	£ xiii	fourteen-16	³ 17
- Enterococcus faecalis	ten mg	£ sixteen	-	³ 17
benzylpenicillin when testing staphylococci	10 IU	£ 28	-	³ 29
ceftriaxone	30 mg	£ 13	14-20	³ 21
cefuroxime sodium	xxx one thousandgrand	£ 14	15-17	³ 18
cefalotin	thirty mone thousand	£ 14	15-17	³ 18
chloramphenicol	xxx thousandg	£ 12	xiii-17	³ 18
clindamycin	two m1000	£ 14	15-20	³ 21
co-trimoxazole	25 mm	£ 10	11-15	³ 16
erythromycin	15 thouchiliad	£ xiii	14-22	³ 23
gentamicin	x kg	£ 12	13-fourteen	³ 15
nalidixic acid	30 yardg	£ thirteen	14-xviii	³ 19
nitrofurantoin	300 mg	£ xiv	15-16	³ 17
oxacillin when testing:
- staphylococci	one thoug	£ x	11-12	³ xiii
- pneumococci	1 mg	£ nineteen	-	³ 20
piperacillin when testing:
- Enterobacteriaceae	100 thoug	£ 17	eighteen-20	³ 21
- Pseudomonas	100 g1000	£ xiv	15-17	³ 18
sulfonamides	300 mg	£ 12	13-sixteen	³ 17
tetracycline	thirty kg	£ 14	15-18	³ 19
tobramycin	10 yardthou	£ 12	thirteen-14	³ 15
trimethoprim	5 grandm	£ 10	11-xv	³ 16

^a National Committee for Clinical Laboratory Standards. Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

Timing of disc application

If the plates, afterwards being seeded with the examination strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take identify before the discs are applied. This causes a reduction in the zone bore and may result in a susceptible strain beingness reported as resistant.

Temperature of incubation

Susceptibility tests are normally incubated at 35 °C for optimal growth. If the temperature is lowered, the fourth dimension required for constructive growth is extended and larger zones upshot. When a heterogeneous resistant strain of Staphylococcus aureus is being tested confronting meticillin (oxacillin), the resistant portion of the population can be detected at 35 °C. At higher temperatures the entire civilization appears to exist susceptible. At 35 °C or lower temperatures, resistant colonies develop within the zone of inhibition. These resistant colonies tin can be seen more than easily if the plate is left for several hours at room temperature before the consequence is read. Such colonies should always exist identified to cheque whether they are contaminants.

Fig. 10. Template for uniform placement of susceptibility discs on plates of 90 mm diameter

Incubation time

Most techniques adopt an incubation period of betwixt 16 and 18 hours. In emergencies, however, a provisional report may exist made after 6 hours. This is not recommended as a routine and the result should always be confirmed after the conventional incubation time.

Size of plate, depth of agar medium, and spacing of the antibiotic discs

Susceptibility tests are usually carried out with nine - 10 cm plates and no more than 6 or 7 antibody discs on each plate. If larger numbers of antibiotics have to be tested, two plates, or one xiv-cm diameter plate, is to be preferred. Excessively large inhibition zones may be formed on very thin media; the converse is true for thick media. Pocket-size changes in the depth of the agar layer have negligible effect. Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or deformation virtually the edge of the plates (see Fig. ten).

Potency of the antibiotic discs

The diameter of the inhibition zone is related to the amount of drug in the disc. If the authority of the drug is reduced owing to deterioration during storage, the inhibition zone volition prove a corresponding reduction in size.

Composition of the medium

The medium influences the size of the zone by its result on the rate of growth of the organism, the rate of diffusion of the antibody, and the action of the amanuensis. It is essential to use the medium advisable to the particular method.

The many factors influencing the zone diameters that may exist obtained for the same test organism clearly demonstrate the need for standardization of disc diffusion methods. Just if the weather condition laid down in a particular method are closely followed can valid results be obtained. Alteration of any of the factors affecting the exam can effect in grossly misleading reports for the clinician.

The precision and accuracy of the method should exist monitored by establishing the quality control program described below. Variations tin can then be immediately investigated and corrective action taken to eliminate them.

Quality control

The need for quality control in the susceptibility exam

The final result of a disc diffusion test is influenced by a big number of variables. Some of the factors, such equally the inoculum density and the incubation temperature, are easy to control, but a laboratory rarely knows the verbal composition of a commercial medium or the batch-to-batch variations in its quality, and it cannot take for granted the antimicrobial content of the discs. The results of the test must therefore be monitored constantly by a quality control plan, which should be considered part of the procedure itself.

The precision and accuracy of the test are controlled past the parallel use of a prepare of command strains, with known susceptibility to the antimicrobial agents. These quality control strains are tested using exactly the same procedure every bit for the examination organisms. The zone sizes shown by the command organisms should fall within the range of diameters given in Table 13. When results regularly fall outside this range, they should be regarded as evidence that a technical error has been introduced into the test, or that the reagents are at error. Each reagent and each step in the test should and so be investigated until the cause of the mistake has been institute and eliminated.

Standard process for quality control

The quality control programme should use standard reference strains of bacteria that are tested in parallel with the clinical cultures. They should preferably exist run every week, or with every fifth batch of tests, and, in addition, every time that a new batch of Mueller-Hinton agar or a new batch of discs is used.

Standard strains for quality control

Staphylococcus aureus (ATCC 25923)
Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC 27853)

These cultures can be obtained from national civilisation collections. They are commercially available in the form of pellets of desiccated pure cultures.

Cultures for day-to-24-hour interval utilise should exist grown on slants of nutrient agar (tryptic soy agar is user-friendly) and stored in the refrigerator. They should be subcultured on to fresh slants every 2 weeks.

Preparing the inoculum

The cultures may be inoculated into any type of broth, and incubated until the broth is turbid. Each broth should be streaked on to an agar plate and incubated overnight. Single colonies should then exist picked off and submitted to susceptibility tests as described on "Procedure".

Placing antimicrobial discs

After the inoculum has been streaked on to the plates, equally described on "Procedure", the appropriate discs should be applied. The discs to exist selected for each control strain are listed in Table 13.

Reading the plates

Later 16 to xviii hours' incubation, the diameters of the inhibition zones should exist measured with a ruler and recorded, together with the date of the examination, on a special quality control nautical chart. This chart should brandish data for each disc - strain combination. The chart is labelled in millimetres, with an indication of the range of adequate zone sizes. An example of such a chart is shown in Fig. xi. When the results consistently fall outside the acceptable limits, activity should be taken to better the quality of the test.

Grossly aberrant results, which cannot be explained past technical errors in the procedure, may betoken contamination or sudden changes in the susceptibility or growth characteristics of the control strain. If this occurs, a fresh stock-strain should be obtained from a reliable source.

Fig. eleven. Quality control chart for antimicrobial susceptibility testing

Control disc: Ampicillin disc 10 myard. Command strain: Due east. coli ATCC 25922