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Techniques in Microbiology: Antimicrobial Susceptibility Testing

Source: World Health Organization

Antibiotic susceptibility testing has become a very essential step for the proper treatment of infectious diseases. It is used

• To guide the clinician in selecting the best antimicrobial agent.
• To accumulate epidemiological information on the resistance of microorganisms of public health importance.

The choice of drugs used in a routine antibiogram is governed by various considerations since only a few antimicrobial agents can be tested. Table 1 suggests the drugs to be tested in various situations. The drugs in Table 1 are divided into two sets. Set 1 includes drugs that are available in most hospitals and for which routine testing should be carried out for every strain. Tests for drugs in set 2 are to be performed only at the special request 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 further testing justified.

 

	Set 1	Set 2
Staphylococcus	Benzyl penicillin Oxacillin Erythromycin Tetracycline Chloramphenicol	Gentamicin Amikacin Co-trimoxazole Clindamycin
Intestinal	Ampicillin Chloramphenicol Co-trimoxazole Nalidixic acid Tetracycline	Norfloxacin
Enterobacteriaceae Urinary	Sulfonamide Trimethoprim Co-trimoxazole Ampicillin Nitrofurantoin Nalidixic acid Tetracycline	Norfloxacin Chloramphenicol Gentamicin
Blood and tissues	Ampicillin Chloramphenicol Cotrimoxazole Tetracycline Gentamicin	Cefuroxime Ceftriaxone Ciprofloxacin Piperacillin Amikacin
Pseudomonas aeruginosa	Piperacillin Gentamicin Tobramycin	Amikacin

Table 1: Basic sets of drugs for routine susceptibility tests

Antimicrobial susceptibility tests measure the ability of an antibiotic or other antimicrobial agent to inhibit bacterial growth in vitro. This ability may be estimated by either the dilution method or the diffusion method. The recommended method for intermediate and peripheral laboratories is the modified Kirby-Bauer method, the methodology of which is given below:

Modified Kirby-Bauer method

Reagents

Mueller-Hinton agar

1. Mueller-Hinton agar should be prepared from a dehydrated base according to the manufacturerÍs recommendations. The medium should be such that with standard strains, zone sizes within the acceptable limits are produced. It is important not to overheat the medium.
2. Cool the medium to 45-50°C and pour into plates. Allow to set on a level surface, to a depth of approximately 4 mm. A 9 cm diameter plate requires approximately 25 ml of the medium.
3. When the agar has solidified, dry the plates for immediate use for 10-30 minutes at 36°C by placing them in an upright position in the incubator with the lids tilted.
4. Any unused plates may be stored in a plastic bag, which should be sealed and placed in a refrigerator. Plates stored in this way can be kept for two weeks.
5. In order 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 will give a distinct inhibition zone of 20 mm or more that is essentially free of hazy growth or fine colonies.
6. For testing the susceptibility of fastidious organisms, 5% blood should be added to the Mueller-Hinton agar base.

Antibiotic discs

Any commercially available discs with the proper diameter and potency can be used. Stocks of antibiotic discs should preferably be kept at -20°C, or the freezer compartment of a home refrigerator is convenient. A small working supply of discs can be kept in the refrigerator for up to one 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 procedure reduces the amount of condensation that occurs when warm air reaches the cold container.

Turbidity standard

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

Swabs

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

Procedure

• To prepare the inoculum from the primary culture plate, touch with a loop the tops of each of 3-5 colonies, of similar appearance, of the organism to be tested.
• When the inoculum has to be made from a pure culture, a loopful of confluent growth is similarly suspended in saline. Inoculum from colonies of streptococci cannot be made by emulsification. Hence, with streptococci, after inoculation the culture tubes are incubated for 4-6 hours to get uniform turbidity which should be matched with the turbidity standards.
• 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 sterile saline. Proper adjustment to the turbidity of 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 swabs firmly against the side of the tube above the level of the liquid.
• Streak the swab all over the surface of the medium three times, rotating the plate through an angle of 60o after each application. Finally, pass the swab round the edge of the agar surface. Leave the inoculum to dry for a few minutes at room temperature with the lid closed. The antibiotic discs may be placed on the inoculated plates using a pair of sterile forceps.
• A sterile needle tip may also be used to place the antibiotic discs on the plate. Alternatively, an antibiotic disc dispenser can be used to apply the discs to the inoculated plate.
• A maximum of seven discs can be placed on a 9-10 cm diameter plate. Six discs may be spaced evenly, approximately 15 mm from the edge of the plate, and one disc placed in the centre of the plate. Each disc should be pressed down gently to ensure even contact with the medium.
• The plates should be placed in an incubator at 35°C within 30 minutes of preparation. Temperatures above 35°C invalidate the results for oxacillin/ methicillin.
• Do not incubate in an atmosphere of carbon dioxide.
• After overnight incubation, the diameter 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 critical diameters by comparing with standard tables (Table 2).
• The measurements can be made with a ruler on the under surface of the plate without opening the lid.
• The end-point of inhibition is judged by the naked eye at the edge where the The end-point of inhibition is judged by the naked eye at the edge where the growth starts, but there are three exceptions:

  i. With sulfonamides and co-trimoxazole, slight growth occurs within the inhibition zone; such growth should be ignored.

  ii. 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 the size of the zone of inhibition, they should be reported as resistant.

  iii. Certain Proteus spp. may swarm into the area of inhibition around some antibiotics, but the zone of inhibition is usually clearly outlined and the thin layer of swarming growth should be ignored.

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