Kirby-Bauer Disk Diffusion Susceptibility Test Protocol

Created: Tuesday, 08 December 2009

Author • Jan Hudzicki

Information

History

The publication on penicillin by Alexander Fleming in 1928 is a milestone in the history of medicine. As more antimicrobial compounds were discovered, it was predicted that infectious diseases would be eliminated through the use of these antimicrobials (7). Unfortunately, the development of bacterial resistance to these antimicrobials quickly diminished this optimism and resulted in the need for physicians to request the microbiology lab to test a patient’s pathogen against various concentrations of a given antimicrobial to determine susceptibility or resistance to that drug. The original method of determining susceptibility to antimicrobials was based on broth dilution methods (5, 7), which although still the gold standard today, are time consuming to perform. This prompted the development of a disk diffusion procedure for the determination of susceptibility of bacteria to antimicrobials.

By the early 1950s, most clinical microbiology laboratories in the United States had adopted the disk diffusion method for determining susceptibility of bacteria to antimicrobials. Each lab modified the procedure to suit its own needs, which included using different types of media, inoculum concentration, incubation time, incubation temperature, and concentration of the antimicrobial compound. Interpretation of susceptibility and resistance was based only on the presence or absence of a zone of inhibition surrounding the disk, and two or three different concentrations of the same antimicrobial were routinely tested against the pathogen (1). Many researchers published variations for the procedure resulting in multiple protocols that resulted in widespread confusion (1). In 1956, W. M. M. Kirby and his colleagues at the University of Washington School of Medicine and the King County Hospital proposed a single disk method for antimicrobial susceptibility testing (6).

The lack of standardization for the determination of bacterial susceptibility continued to be a problem throughout the early 1960s. Kirby and his colleague, A. W. Bauer, extensively reviewed the susceptibility testing literature. They consolidated and updated all the previous descriptions of the disk diffusion method and published their findings (2). This publication led the World Health Organization to form a committee in 1961 to lay the groundwork for the development of a standardized procedure for single antimicrobial disk susceptibility testing (7). The result was a standardized procedure for the disk diffusion susceptibility test, henceforth called the Kirby-Bauer disk diffusion test (2).

Currently, the Clinical Laboratory Standards Institute (CLSI) is responsible for updating and modifying the original procedure of Kirby and Bauer through a global consensus process. This ensures uniformity of technique and reproducibility of results as pathogens develop new mechanisms of resistance and new antimicrobials are developed to fight these organisms. Interpretative guidelines for zone sizes are included in their publications (3). The CLSI publication, Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard 9th Edition, represents the standard for clinical laboratories performing susceptibility testing today.

Purpose

The purpose of the Kirby-Bauer disk diffusion susceptibility test is to determine the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds in order to assist a physician in selecting treatment options for his or her patients. The pathogenic organism is grown on Mueller-Hinton agar in the presence of various antimicrobial impregnated filter paper disks. The presence or absence of growth around the disks is an indirect measure of the ability of that compound to inhibit that organism.

Theory

Determination of bacterial resistance to antimicrobials is an important part of the management of infections in patients. The disk diffusion method of Kirby and Bauer has been standardized and is a viable alternative to broth dilution methods for laboratories without the resources to utilize the newer automated methods for broth microdilution testing.

When a 6-mm filter paper disk impregnated with a known concentration of an antimicrobial compound is placed on a Mueller-Hinton (MH) agar plate, immediately water is absorbed into the disk from the agar. The antimicrobial begins to diffuse into the surrounding agar. The rate of diffusion through the agar is not as rapid as the rate of extraction of the antimicrobial out of the disk, therefore the concentration of antimicrobial is highest closest to the disk and a logarithmic reduction in concentration occurs as the distance from the disk increases (7). The rate of diffusion of the antimicrobial through the agar is dependent on the diffusion and solubility properties of the drug in MH agar (2) and the molecular weight of the antimicrobial compound. Larger molecules will diffuse at a slower rate than lower molecular weight compounds. These factors, in combination, result in each antimicrobial having a unique breakpoint zone size indicating susceptibility to that antimicrobial compound.

If the agar plate has been inoculated with a suspension of the pathogen to be tested prior to the placing of disks on the agar surface, simultaneous growth of the bacteria and diffusion of the antimicrobial compounds occurs. Growth occurs in the presence of an antimicrobial compound when the bacteria reach a critical mass and can overpower the inhibitory effects of the antimicrobial compound. The estimated time of a bacterial suspension to reach critical mass is 4 to 10 hours for most commonly recovered pathogens, but is characteristic of each species, and influenced by the media and incubation temperature (7). The size of the zone of inhibition of growth is influenced by the depth of the agar, since the antimicrobial diffuses in three dimensions, thus a shallow layer of agar will produce a larger zone of inhibition than a deeper layer.

The point at which critical mass is reached is demonstrated by a sharply marginated circle of bacterial growth around the disk. The concentration of antimicrobial compound at this margin is called the critical concentration and is approximately equal to the minimum inhibitory concentration obtained in broth dilution susceptibility tests. 

Zone size observed in a disk diffusion test has no meaning in and of itself (7). The interpretation of resistance and susceptibility to antimicrobials is determined through in vivo testing of blood and urine to calculate the obtainable level of a given antimicrobial that results in resolution of an infection. This information is correlated with zone sizes resulting in the interpretive standards. The current interpretation standards can be found in the Clinical Laboratory Standards Institute Performance Standards for Antimicrobial Disk Susceptibility Tests: Approved Standards 9th Edition (3).

RECIPE

Sterile saline in 2-ml tubes

0.5 McFarland standard

Wickerham card

Mueller-Hinton agar plates, 100 mm or 150 mm

Caliper or ruler

Antibiotic disksb

Forceps

Antibiotic disk dispenser (optional)

18- to 24-ho

Vortex

Sterile swabs

Inoculating lo

Bact-cinerato

Alcohol pads

35°C to 37°C

a. Recommended organisms for quality assurance purposes are Staphylococcus aureus ATCC 25923 (Biosafety level (BSL) 2), Escherichia coli ATCC 25922 (BSL 1), and Pseudomonas aeruginosa ATCC 27853 (BSL 2) (www.atcc.org), as the zone of inhibition for these organisms is known. Because the zone sizes are known for these organisms, they are recommended for use in the educational setting, although the use of unknowns should also be incorporated into the educational experience. For quality control testing, the zone sizes for these three organisms can be found on the package insert from any antimicrobial disk you purchase.

b. Selection of antimicrobial is based on the type of organism being tested and source of the isolate (blood, urine, wound, etc.). See the Interpretative Standards Tables for suggested antimicrobials to use inthis exercise.

Additional Notes

Mueller-Hinton agar

MH agar is considered the best medium to use for routine susceptibility testing of nonfastidious bacteria for the following reasons:

· It shows acceptable batch-to-batch reproducibility for susceptibility testing

· It is low in sulfonamide, trimethoprim, and tetracycline inhibitors

· It supports satisfactory growth of most nonfastidious pathogens

· A large body of data and experience has been collected concerning susceptibility tests performed with this medium (6).

Please note that the use of media other than Mueller-Hinton agar may result in erroneous results. Also note that only the aerobic or facultative bacteria that grow well on unsupplemented MH agar should be tested using this protocol. Fastidious organisms require MH agar supplemented with additional nutrients and require that modification to this protocol be made. Neither the supplements nor the procedural modification are discussed in this basic protocol.

MH agar may be purchased as prepared agar plates from Remel (Lenexa, KS), BD BBL (Franklin Lakes, NJ ), or any other supplier of prepared agar plates. Follow the manufacturer’s recommendation for storage of prepared plates. MH agar can also be prepared from dehydrated media available from companies such as Remel, BD BBL, or any other supplier of dehydrated media. Be sure to prepare the media according to the manufacturer’s directions.

Formula for Mueller-Hinton agar per liter of purified water (4)

Beef, Infusion from            300.0 g

Casamino acid, technical   17.5 g

Starch                                 1.5 g

Agar                                   17.0 g

Suspend the components listed above in 1 liter of purified water. Mix thoroughly. Heat with frequent agitation and boil for 1 minute to completely dissolve the components. Autoclave at 121°C for 15 minutes. Dispense as desired. Allow to solidify at room temperature, then store at 4 to 8°C. Mueller-Hinton agar is stable for approximately 70 days (per Remel Technical Services, 1 September 2009) from the date of preparation. Each lab should verify the quality and functionality of each batch of prepared media by testing known strains of organisms against each antimicrobial compound being used as the 70-day expiration date approaches.

· If you prepare the MH agar plates from dehydrated media, the plates must be poured to a depth of 4 mm (approximately 25 ml of liquid agar for 100-mm plates and 60 ml of liquid agar for 150-mm plates, but in any case to a measured depth of 4 mm). Plates that are too shallow will produce false susceptible results as the antimicrobial compound will diffuse further than it should, creating larger zones of inhibition. Conversely, plates poured to a depth >4 mm will result in false resistant results.

· pH of the MH agar should fall between 7.2 and 7.4 at room temperature after solidification and should be tested when the media is first prepared. If the pH is <7.2 certain drugs will appear to lose potency (aminoglycosides, quinolones, macrolides), while other agents may appear to have excessive activity (tetracycline). If the pH is >7.4, the opposite results may occur.

· Excessive thymidine or thymine can reverse the inhibitory effects of sulfonamides and trimethoprim resulting in smaller and less distinct zones of inhibition, or no zones at all.

· The incorrect concentration of divalent cations (calcium and magnesium) will affect the results of aminoglycoside and tetracycline tests against Pseudomonas aeruginosa. Excess cation concentration will result in reduced zone sizes and low concentration will increase zone sizes. Excess calcium will increase the zone size of P. aeruginosa against daptomycin. Excess zinc ions may reduce the zone size of carbapenems against P. aeruginosa.

· MH agar should be tested with known strains of organism at least weekly in order to verify that the media and disks are working as expected.

Antibiotic susceptibility disks

Antimicrobial disks can be purchased from any reputable suppliers, such as Remel or BD BBL. They are packaged in spring-loaded cartridges containing 25 or 50 disks and can be ordered as individual cartridges or in packages of 10 cartridges. Proper storage of these disks is essential for reproducible results.

Sealed cartridges containing commercially prepared paper disks should be stored at either 8°C or frozen at -14°C in a non-self-defrosting freezer. Allow disks to come to room temperature prior to removing the protective plastic packaging. Once opened, store the cartridges in a storage container containing desiccant for no more than 1 week.

Semiautomatic disk dispensers are available from companies such as Remel and BD BBL. Be aware that disk cartridges from one company may not fit the dispenser of another company.

McFarland standard

McFarland standards are suspensions of either barium sulfate or latex particles that allow visual comparison of bacterial density (Fig.

1). Commercially prepared standards are available for purchase from companies such as Remel or BD BBL. These often include a Wickerham card, which is a small card containing parallel black lines. 

A 0.5 McFarland standard is equivalent to a bacterial suspension containing between 1 x 108 and 2 x 108 CFU/ml of E. coli.

A 0.5 McFarland standard may be prepared in-house as describe below.

1. Add a 0.5-ml aliquot of a 0.048 mol/liter BaCl2 (1.175% wt/vol BaCl2 • 2H20) to 99.5 ml of 0.18 mol/liter H2SO4 (1% vol/vol) with constant stirring to maintain a suspension.

2. Verify the correct density of the turbidity standard by measuring absorbance using a spectrophotometer with a 1-cm light path and matched cuvette. The absorbance at 625 nm should be 0.08 to 0.13 for the 0.5 McFarland standard.

3. Transfer the barium sulfate suspension in 4- to 6-ml aliquots into screw-cap tubes of the same size as those used in standardizing the bacterial inoculums.

4. Tightly seal the tubes and store in the dark at room temperature.

Use of the McFarland standard in the Kirby-Bauer procedure.

1. Prior to use, vigorously agitate the barium sulfate standard on a mechanical vortex mixer and inspect for a uniformly turbid appearance. Replace the standard if large particles appear. If using a standard composed of latex particles, mix by inverting gently, not on a vortex mixer.

2. As the student adds bacterial colonies to the saline in the “preparation of the inoculum” step of the procedure, he or she should compare the resulting suspension to the McFarland standard. This is done by holding both the standard and the inoculum tube side by side and no more than 1 inch from the face of the Wickerham card (with adequate light present) and comparing the appearance of the lines through both suspensions. Do not hold the tubes flush against the card. If the bacterial suspension appears lighter than the 0.5 McFarland standard, more organisms should be added to the tube from the culture plate. If the suspension appears more dense than the 0.5 McFarland standard, additional saline should be added to the inoculum tube in order to dilute the suspension to the appropriate density. In some cases it may be easier to start over rather than to continue to dilute a bacterial suspension that is too dense for use.

FIG. 1. McFarland standards (left to right) 0.5, 1.0, 2.0, 3.0, positioned in front of a Wickerham card. McFarland standards are used to prepare bacterial suspensions to a specified turbidity. In the Kirby-Bauer disk diffusion susceptibility test protocol, the bacterial suspension of the organism to be tested should be equivalent to the 0.5 McFarland standard.

PROTOCOL

Preparation of Mueller-Hinton plate

1. Allow a MH agar plate (one for each organism to be tested) to come to room temperature. It is preferable to allow the plates to remain in the plastic sleeve while they warm to minimize condensation.

2. If the surface of the agar has visible liquid present, set the plate inverted, ajar on its lid to allow the excess liquid to drain from the agar surface and evaporate. Plates may be placed in a 35°C incubator or in a laminar flow hood at room temperature until dry (usually 10 to 30 minutes).

3. Appropriately label each MH agar plate for each organism to be tested.

Preparation of inoculum

1. Using a sterile inoculating loop or needle, touch four or five isolated colonies of the organism to be tested.

2. Suspend the organism in 2 ml of sterile saline.

3. Vortex the saline tube to create a smooth suspension.

4. Adjust the turbidity of this suspension to a 0.5 McFarland standard by adding more organism if the suspension is too light or diluting with sterile saline if the suspension is too heavy.

5. Use this suspension within 15 minutes of preparation.

Additional Notes

Inoculum preparation

Organisms to be tested must be in the log phase of growth in order for results to be valid. It is recommended that subcultures of the organisms to be tested be made the previous day.

Never use extremes in inoculum density. Never use undiluted overnight broth cultures or other unstandardized inocula for inoculating plates.

If the organism is difficult to suspend directly into a smooth suspension, the growth method of preparing the inoculums should be used. However, the recommended organisms listed in this procedure all produce smooth suspensions with little difficulty. See the Clincial Laboratory Standards

Institute document (3) for the growth procedure method for preparing the inoculums, if needed.

Inoculation of the MH plate 

1. Dip a sterile swab into the inoculum tube.

2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure, to remove excess fluid. The swab should not be dripping wet (Fig. 2).

3. Inoculate the dried surface of a MH agar plate by streaking the swab three times over the entire agar surface; rotate the plate approximately 60 degrees each time to ensure an even distribution of the

inoculum (Fig. 3).

4. Rim the plate with the swab to pick up any excess liquid (Fig. 4).

5. Discard the swab into an appropriate container.

6. Leaving the lid slightly ajar, allow the plate to sit at room temperature at least 3 to 5 minutes, but no more than 15 minutes, for the surface of the agar plate to dry before proceeding to the next step.

FIG. 2. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the test plate. Step 2. Rotate the swab against the side of the tube while applying pressure to remove excess liquid from the swab prior to inoculating the plate.

FIG. 3. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the Mueller-Hinton agar plate. Step 3. (A) Inoculate the plate with the test organism by streaking the swab in a back-and-forth motion very close together as you move across and down the plate. Rotate the plate 60° and repeat this action. Rotate the plate once more and repeat the streaking action. This ensures an even distribution of inoculum that will result in a confluent lawn of growth. (B) Diagram illustrating the pattern the swab should follow as it is drawn across the plate.

FIG. 4. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the Mueller-Hinton agar plate. Step 4. After streaking the Mueller Hinton agar plate as described in Step 3, rim the plate with the swab by running the swab around the edge of the entire the plate to pick up any excessive inoculum that may have been splashed near the edge. The arrow indicates the path of the swab.

Placement of the antibiotic disks

1. Place the appropriate antimicrobial-impregnated disks on the surface of the agar, using either forceps to dispense each antimicrobial disk one at a time, or a multidisk dispenser to dispense multiple disks at one time. (See steps a. through d. for the use of the multi-disk dispenser or steps e. through g. for individual disk placement with forceps.

a. To use a multidisk dispenser, place the inoculated MH agar plate on a flat surface and remove the lid (Fig. 5).

b. Place the dispenser over the agar plate and firmly press the plunger once to dispense the disks onto the surface of the plate.

c. Lift the dispenser off the plate and using forceps sterilized by either cleaning them with an alcohol pad or flaming them with isopropyl alcohol, touch each disk on the plate to ensure complete contact with the agar surface. This should be done before replacing the petri dish lid as static electricity may cause the disks to relocate themselves on the agar surface or adhere to the lid (Fig. 6d).

d. Do not move a disk once it has contacted the agar surface even if the disk is not in the proper location, because some of the drug begins to diffuse immediately upon contact with the agar.

e. To add disks one at a time to the agar plate using forceps, place the MH plate on the template (Fig. 7) provided in this procedure (Fig. 6a). Sterilize the forceps by cleaning them with a sterile alcohol pad and allowing them to air dry or immersing the forceps in alcohol then igniting.

f. Using the forceps carefully remove one disk from the cartridge (Fig. 6b).

g. Partially remove the lid of the petri dish. Place the disk on the plate over one of the dark spots on the template and gently press the disk with the forceps to ensure complete contact with the agar surface. Replace the lid to minimize exposure of the agar surface to room air (Fig. 6c, d).

h. Continue to place one disk at a time onto the agar surface until all disks have been placed as directed in steps f. and g. above.

2. Once all disks are in place, replace the lid, invert the plates, and place them in a 35°C air incubator for 16 to 18 hours. When testing Staphylococcus against oxacillin or vancomycin, or Enterococcus against vancomycin, incubate for a full 24 hours before reading.

FIG. 5. Kirby-Bauer disk diffusion susceptibility test protocol, placement of antibiotic disks using an automated disk dispenser. Step 1, a. through d. An automatic disk dispenser can be used to place multiple disks simultaneously on a MH agar plate. () Set the dispenser over the plate.

(B) Place the palm of your hand on the top of the handle. (C) Press down firmly and completely to dispense the disks. The spring loaded handle will return to the original position when pressure is removed.

FIG. 6. Kirby-Bauer disk diffusion susceptibility test protocol, placement of antibiotic disks using forceps to manually place the disks. Step 1, e. through h. Antibiotic disks can be manually placed on the MH agar plate if desired. (A) Place the Mueller-Hinton agar plate over the disk template. (B) Remove one disk from the cartridge using forceps that have been sterilized. (C) Lift the lid of the plate and place the disk over one of the positioning marks. (D) Press the disk with the forceps to ensure complete contact with the agar surface. Replace the lid of the plate between disks to minimize exposure to air-borne contaminants.

Additional Notes

Disk placement

Disks should not be placed closer than 24 mm (center to center) on the MH agar plate. Ordinarily, no more than 12 disks should be placed on a 150-mm plate or more than 5 disks on a 100-mm plate. However, the semiautomatic disk dispensers hold 16 and 8 disks respectively and may not maintain the recommended 24 mm center to center spacing. The template provided in this protocol (Fig. 7) maintains the recommended 24 mm center to center spacing and allows the placement of up to 8 disks on the plate.

You should avoid placing disks close to the edge of the plate as the zones will not be fully round and can be difficult to measure.

Each disk must be pressed down with forceps to ensure complete contact with the agar surface or irregular zone shapes may occur.

If the surface of the agar is disrupted in any way (a disk penetrating the surface, visible lines present due to excessive pressure of the swab against the plate during inoculation, etc.) the shape of the zone may be affected.

When printing the template for use in your microbiology lab, be sure that the diameter of the circle on the template is the same size as the Mueller-Hinton agar plates that you use in lab (100 mm). The "reduce" or "enlarge" function on a photocopier can be used to change the size of the template if needed. You may also make your own template by drawing a circle around a MH agar plate on a sheet of paper. Add the placement marks based on the number of disks you plan to use in your lab session, maintaining the recommended spacing as indicated above.

Incubation of the plates

A temperature range of 35°C ± 2°C is required.

Note that temperatures above 35°C may not allow the detection of methicillin-resistant Staphylococcus.

Do not incubate plates in CO2 as this will decrease the pH of the agar and result in errors due to incorrect pH of the media.

Results can be read after 18 hours of incubation unless you are testing Staphylococcus against oxacillin or vancomycin, or Enterococcus against vancomycin. Read the results for the other antimicrobial disks then reincubate the plate for a total of 24 hours before reporting vancomycin or oxacillin.

FIG. 7. Manual disk placement template for eight disks on a 100-mm plate. Place the MH agar plate on the figure above so that the edge of the plate lines up with the outer circle. Remove the lid from the plate and place one antibiotic disk on each dark gray circle. If fewer than eight antibiotics are used, adjustments can be made to the spacing of the disks. See "Additional Notes" disk placement for suggestions for printing this template.

Measuring zone sizes

1. Following incubation, measure the zone sizes to the nearest millimeter using a ruler or caliper; include the diameter of the disk in the measurement (Fig. 8, 9b).

2. When measuring zone diameters, always round up to the next millimeter.

3. All measurements are made with the unaided eye while viewing the back of the petri dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with reflected light (Fig. 9a).

4. View the plate using a direct, vertical line of sight to avoid any parallax that may result in misreading.

5. Record the zone size on the recording sheet.

6. If the placement of the disk or the size of the zone does not allow you to read the diameter of the zone, measure from the center of the disk to a point on the circumference of the zone where a distinct edge is present (the radius) and multiply the measurement by 2 to determine the diameter (Fig. 10).

7. Growth up to the edge of the disk can be reported as a zone of 0 mm.

8. Organisms such as Proteus mirabilis, which swarm, must be measured differently than nonswarming organisms. Ignore the thin veil of swarming and measure the outer margin in an otherwise obvious zone of inhibition.

9. Distinct, discrete colonies within an obvious zone of inhibition should not be considered swarming. These colonies are either mutant organisms that are more resistant to the drug being tested, or the culture was not pure and they are a different organism. If it is determined by repeat testing that the phenomenon repeats itself, the organism must be considered resistant to that drug.

FIG. 8. Measuring zones of inhibition. Gray shading represents a confluent lawn of bacterial growth.

The white circle represents no growth of the test organism.

B

                              

FIG. 9. Kirby-Bauer disk diffusion susceptibility test protocol, measuring zone sizes. (A) Using a ruler or caliper measure each zone with the unaided eye while viewing the back of the petri dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with reflected light. (B) The size of the zone for this organism-antibiotic combination is 26 mm.

FIG. 10. Kirby-Bauer disk diffusion susceptibility test protocol, measuring zone sizes; an alternate method for measuring zones. If the zones of adjacent antibiotic disks overlap, the zone diameter can be determined by measuring the radius of the zone. Measure from the center of the antibiotic disk to a point on the circumference of the zone where a distinct edge is present. Multiply this measurement by 2 to determine the diameter of the zone of inhibition. In this example, the radius of the zone is 16 mm. Multiply this measurement by 2 to determine the zone size of 32 mm for this organism-antibiotic combination.

Measuring zone sizes

If the plate was properly inoculated and all other conditions were correct, the zones of inhibition should be uniformly circular and there will be a confluent lawn of growth.

If individual colonies are apparent across the plate, the inoculum was too light and the test must be repeated.

The zone margin should be considered the area showing no obvious, visible growth that can be detected with the unaided eye. Do not use a magnification device to observe zone edges.

When measuring the zone of inhibition for organisms that swarm (e.g., Proteus sp.), ignore the thin veil of swarming growth in an otherwise obvious zone of inhibition.

With trimethoprim and the sulfonamides, antagonists in the medium may allow some slight growth; therefore, disregard slight growth (20% or less of the lawn of growth) and measure the more obvious margin to determine the zone diameter (3).

Interpretation and Reporting of the Results

1. Using the published CLSI guidelines, determine the susceptibility or resistance of the organism to each drug tested (Tables 1, 2, 3). Note that there are different charts for different organisms. Abbreviated charts specific for the author’s suggested organisms and antimicrobial disks to use are provided below.

2. For each drug, indicate on the recording sheet whether the zone size is susceptible (S), intermediate (I), or resistant (R) based on the interpretation chart.

3. The results of the Kirby-Bauer disk diffusion susceptibility test are reported only as susceptible, intermediate, or resistant. Zone sizes are not reported to physicians.

Recommended Antimicrobial Disks and Interpretative Zone Sizes (3)

This is a suggested battery of disks to use in the educational setting as it minimizes the different antimicrobials that would need to be purchased to test all three groups of organisms. Educators may select other drugs from the CLSI charts, as appropriate for their situation.

TABLE 1. Zone diameter interpretative standards for Staphylococcus species (3)

TABLE 2. Zone diameter interpretative standards for Pseudomonas aeruginosa and other nonfermenting gram-negative rods (3)

TABLE 3. Zone diameter interpretative standards for E. coli and other enteric gram-negative rods (3)

SAFETY

The ASM advocates that students must successfully demonstrate the ability to explain and practice safe laboratory techniques. For more information, read the laboratory safety section of the ASM Curriculum Recommendations: Introductory Course in Microbiology and the Guidelines for Biosafety in Teaching Laboratories.

REFERENCES

1. Bauer, A. W., D. M. Perry, and W. M. M. Kirby. 1959. Single disc antibiotic sensitivity testing of Staphylococci. A.M.A. Arch. Intern. Med. 104:208–216.

2. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 36:493-496.

3. Clinical Laboratory Standards Institute. 2006. Performance standards for antimicrobial disk susceptibility tests; Approved standard—9th ed. CLSI document M2-A9. 26:1. Clinical Laboratory Standards Institute, Wayne, PA.

4. Difco. 1984. Difco manual, 10th ed. Difco Laboratories, Detroit, MI.

5. Kirby, W. M. M., G. M. Yoshihara, K. S. Sundsted, and J. H. Warren. 1957. Clinical usefulness of a single disc method for antibiotic sensitivity testing. Antibiotics Annu. 1956-1957:892.

6. Winn, Jr., W., et al. 2006. Konemann’s color atlas and diagnostic text of microbiology, 6th ed., p. 945–1021. Lippencott Williams & Wilkins Publishers, Philadelphia, PA.

7. Jorgensen, J. H., and J. D. Turnidge. 2007. Susceptibility test methods: dilution and disk diffusion methods, p. 1152–1172. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry, and M. A. Pfaller

(ed.), Manual of clinical microbiology, 9th ed. ASM Press, Washington, D.C.

REVIEWERS

This resource was peer-reviewed at the ASM Conference for ndergraduate Educators 2009.

Participating reviewers:

Farah Bennani

Front Range Community College, Westminister, CO

Louise A. Brown

Front Range Community College, Westminister, CO

Rebecca Buxton

University of Utah, Salt Lake City, UT

Laura Cathcart

University of Maryland, College Park, College Park, MD

Michel J. Cloutier

St. Louis College of Pharmacy, St. Louis, MO

Karen Dalton

Community College of Baltimore County, Baltimore, MD

Kevin Dixon

East Central College, Union, MO

Cori Fata-Hartley

Michigan State University, East Lansing, MI

Becky Graham

Ohio Dominican University, Columbus, OH

Debora V. Harbor

College of Southern Nevada, Las Vegas, NV

Jeffrey Henriksen

Bellevue University, Bellevue, NE

Carol Hurlburt

Lansing Community College, Lansing, MI

Maria Isaza

County College of Morris, Dover, NJ

Ian Johnston

Bethel University, St. Paul, MN

Jacqueline Krueger

Olive-Harvey College, Chicago, IL

Dorothy Matthews

The Sage Colleges, Voorheesville, NY

Carl McAllister

Georgia Perimeter College, Stone, GA

Liza Mohanty

Olive-Harvey College, Chicago, IL

Rita Moyes

Texas A & M University, College Station, TX

Maria Niswonger

York County Community College, Wells, ME

Juana Ortellado

University National Asuncion, Paraguay

Steven C. Roschke

Good Samaritan College of Nursing and Health Science, Cincinnati, OH

Elizabeth Rozema

East Central College, Union, MO

Janie Sigmon

York Technical College, Rock Hill, SC

Sherry Stewart

Navarro College, Corsicana, TX

Lori Threlkeld

Henderson Community College, Henderson, KY

Igor Zaitsev

Borough of Manhattan Community College, New York, NY

By. SUYATNO RINDANG

30. Dillenia suffruticosa (Griff. 1854) Martelli 1886

Wormia suffruticosa G-riff., Not. 4, 1854, p. 706 & *Ic. PI. As., 1854, t. 649; Hook.f. & Thorns., Fl. Br. Ind. 1, 1872, p. 35; Villar, Nov. App., 1880, p. 347; King, J. As. Soe. Beng. 58, II, 1889, p. 364; Ridl, J. Str. Br. R. A. S. 54, 1910, p. 5; Back., Schoolfl. Java, 1911, p. 10; Koord., Exk. Fl. Java 2, 1912, p. 601; "Blaauw, Trop. nat. in sc.hotsen on kleuren, 1913, p. 17, t. 12; Rid]., Fl. Mai. Pen. 1, 1922, p. 8; Burk., Diet. Eeon. Prod. Mai. Pen., 1935, p. 2265; Corn., Gard. Bull. S. S. 10, 1939, p. 9; 'Corn., Wayside Trees Malaya, 1940, p. 207, pi. 53; Back., Bekn. Fl. Java (em. ed.) 4, 1942, fana. 80, p. 4.

Wormia excelsa Auct. non Jack; Hook.f. & Thorns., Fl. Ind. 1, 1855, p. 67. Wormia subsessilis Miq., Fl. Ind. Bat. Suppl., 1860, p. 619; Teysm. & Binn., J. Bot. Nderl. 1, 1861, p. 364; "Miq., Ann. Mus. Bot. Lugd. Bat. 1,"l864, p. 315, t. 9; Miq., Ann. Mus. Bot. Lugd. Bat. 4, 1868, p. 77; Ridl., J. Str. Br. R. A. S. 54, 1910, p. 4; Ridl., Saraw. Mus. .T. 1, 1913, p. 71; 'Ridl., Fl. Mai. Pen. 1, 1922, p. 7.

Wormia revoluta Teysm. Sc Binn., J. Bot. Neerl. 1, 1861, p. 364, in syn.

Wormia burbidgei '"Hook.f., Bot. Mag., 1880, t. 6531.

Dillenia suffruticosa (Griff.) Martelli in Becc., Malesia 3, 1886, p. 163; Fin. & Gagnep., Bull. Soc. Bot. Fr. Mem. 4, 1906, p. 10; Merr., Bibl. En. Bom. PI., 1921, p. 384; de Wit, Bull. Bot, Gard. Btzg III, 18, 1949, p. 208; 'Hoogl., Fl. Mai. I, 4, 1951, p. 162.

Dillenia burbidgei (Hook.f.) Grig in Engl. & Prantl, Nat. Pfl. Fam. 3, 6, 1893, p. 123.

Dillenia suffruticosa var. borneensis (Ridl.) Ridl., Saraw. Mus. J. 1, 1913, p. 71; Merr., Bibl. En. Born. PI., 1921, p. 384.

Type specimens: Wormia suffruticosa: Griffith Kew Distr. 55, Malacca, 1845; holotype in K, isotvpes in C, OGE, PI, GH, K, L, M, NY, P. — Wormia subsessilis: Tcysmann 3203 HE, Banka; liolotypo in TJ, isotypes in BZ, CAL, K, L, MEL. — Wormia burbidgei: Burbidge, Borneo, 1877—8; holotype in K. — Wormia subsessilis var. borneensis: Hewitt, Kuching, 1909; holotype in SING, isotypo in K.

Large shrubs or small trees, evergreen, up to 10 m high, with stout, brown trunk, often forming thickets. Branches sympodial, younger ones ca 3 mm thick, glabrous to densely villose with 2—3 nun long hairs, more or less glabrescent. Leaf-scars amplexicaul, for about 1 /2 single line, for Y, subfalcate with ca 20 leaf-traces near lower margin. Leaves elliptic to" ovate, (10—) 15—25(—45) X (5—)8—12(—26) cm, with (7—)12—20 (—27) nerves on either side ; rounded to obtuse at apex, obtuse at base, decurring into petiolar wings; margin entire to dentate or doubly dentate, nerves ending in apex of teeth, in larger leaves often 1—3 rather strong secondary nerves directed downward near margin, ending in apex of Smaller teeth; glabrous, rarely slightly villose on intervenium in younger leaves only above, slightly to densely villose on nerves, on both sides along midrib (continuing on petiole), and along line which delimits budenclosing part of leaf-base beneath. Petiole 2—6 cm, with amplexicaul wings; wings 4—40(—15) mm broad, nervation of blade continuing, birt

less marked, on wings, wings and base of blade below line where both sides cohere in young leaf of different colour (darker when dry) ; wings usually persistent. Inflorescences terminal, (4 —)6—10 (—18)-flowered, up to 30 cm long, simple racemes or composed by having lateral branch at place of second, sometimes also third flower; axis ca 3 mm thick, glabrous to, particularly when young, densely villose; bracts caducous, triangular, 6—15 X 3—5 mm. Flowers 8—12 cm across. Pedicel 0.8—3 cm long, 2— 3 mm thick, thickened to 3—4 mm at apex, without bracteoles. Sepals 5, obovate, 15—22 X 8—12 mm, glabrous inside, glabrous to rather densely villose outside. Petals 5, bright yellow, obovate, 40—50 X 25—30 mm, rounded at apex, narrowed towards base. Androecium with distinct group of staminodes on outer side. Staminodes ca 100, linear, ca 4—6 X 0.3 mm, yellow, obtuse at apex. Stamens ca 175, outermost ones slightly curved in bud, ca 8 mm long, innermost ones with apical part reflexed outward in bud, ca 13 mm long, with stamens of intermediate lengths between; filament of outermost stamens ca 3 1 / 2, of innermost ones ca 2 mm long; anther ca 0.5 mm broad, obtuse at apex; thecae linear, opening with pore near apex on outer side. Carpels 5—8, usually 7, arranged around sharp conical receptacle, light green, elliptic, ca 5 X 2 mm, glabrous, each with 7—10 ovules; styles spreading, filamentous, ca 10 mm long, 0.5 mm thick, yellowish white. Pseudocarps dehiscent, when ripe sepals enlarged to 18—25 X 10—15 mm; carpels red, 20—25 X 10—16 mm, each with 1—4 seeds. Seeds obovoid, ca 3 X 2 mm, brown or black, enclosed by scarlet, membranous aril.

Sumatra: Palembang: Pladju, Polak 140, fl. & fr. Oct. 1030 (BZ). — Banka: H ors field, fl. (BM); Kurz 425, fl. (K) ; Baturasak, Amand, fl. Juno 1858 (U) ; Ploem, fl. & fr. (L) ; Djcbus, Berlchowt 7g, fl. Aug. 1886 (BZ) ; Berkhout (BZ) ; G. Menumbing, near Muntok, Biinnemeyer 1S51, fl. & fr. Oct. 1917 (BZ, L) ; Bakem, Sungai Liât, Biinnemeyer 1731, fl. Oct. 1917 (BZ) ; Sungai Lajang, Sungai Liât, Biinnemeyer 1807, fl. & fr. Oct. 1917 (BZ, L) ; Sungai Liât, Rcbo, Biinncmeyer 2510, fl. Nov. 19.17 (B'Z) ; near Pangkalpinang-Belinju, Huitema 24, fl. 1932 (IIZ)'; Bakit, Belinju, Coert 1630, fl. Sept. 1941 (L). — Billiton: Riedel, fl. (FI); S of Manggar,, n<wi 32, fr. March 1907 (BZ). — Riouw-Lingga Arch.: Pulau Tudjuh, Ajer Suar, Biinnemeyer 5966, fl. & fr. May 1919 (BZ, L) ; Sungai Tanda, Pulau Liugga, Biinnemeyer 6933, fl. July 1919 (BZ).

Malay Peninsula: Walker 224, fr. (GL, P) ; Suugai Iwong, Goodenough, fl. May 1892 (RM). — Pcrak: Maxwell's Hill, Taiping, Henderson SF 10014, fl. & fr. 1922 (SING) ; Taiping, Henderson SF 10358, fr. Jan. 1923 (SING) ; Tinmines, Taiping, llaniff SF 13130, March 1924 (SING). — Pahang: Balok, Teop CF 3616, fr. Jan. 1920 (K, SING). — Selangor: Rawang Bistr., Goodenough 10470, fl. March 1809 (SING) ; Garcona Kuala Lumpur, Kalong CF 17479, fl. Aug. 1929 (SING); Fran ok 1115, fl. & fr. Sept. 1937 (C) ; Tinmines, Ranching, Nv.r SF 34430, fl. & fr. Nov. 1937 (MO, SING). — Negri Sembilan: Seremban, -A• Ivins 1783, fr. July 1885 (SING). - Malacca: fr. (SING) ; Gmidichaud 26, fl. & fr. Feb. 18137 (G, P) ; o _o_. i r> A C\ /n \ . A Ti N ... Cuming 2858, fl. 1840 (BM, CÖE, K) ; Delesscrt, fl. & fr. 1840 (G) ; Ayer Panas, x , , , ... v , , . , Griffith (Herb. E. I. Comp. 55) , fl. & fr. 184-5 (C, CGE, PI, GH, IC, L, M, NY, P) ; Griffith, fr. (OAL) ; Maingay 1020 (Kcw Dixtr.■. 4), fl. 1865—6 (K) ; Maingay lOSOA (Kew Distr. 4), fl. May 1868 (GAIL, K, L) ; A Ivins, fl. & fr. (SING); Bukit Braang, Uolmberg 710, fl. & fr. Apr. 1891 (MEL, SING); Ilervey, fl. 1S91 (BM, GAL, SING) ; Ilervey, fl. & fr. Apr. 1893 (CAL, P) ; Ilervey, fr. (A). — Joliore: Pinyerong, Ridley, fr. May 1889 (SING); Pulau Tekong, Eidley 3966, fl. 1890 (SING); Eidley, fl. 1890 (BM) ; Kuala Sedili Besar, Feilding, fl. 1892 (SING); Sedili Kechik, Yeob CF 5832, fl. & fr. July 1921 (SING) ; Mawai, Sedili, Corner SF 21189, fl. & fr. May 1932 (K, NY, SING) ; Sungai Sedili, Corner SF 32984, fl. & fr. May 1937 (L, SING) ; Sungai Sedili, near Mawai, Corner SF 33546 $• 33547, fl. & fr. June 1937 (L, SING). — Pulau Penan g : Delessert, fl. & fr. 1835 (G). — Singapore: Thomson 10, fl. (K, P) ; Lobb 329, fl. (OGiE, FI, G, GH, GL, K, MEL, OX) ; Anderson 2, fl. Oct. 1861 (OAL,- MEL) ; Kurz 2961, fl. (CAL); Maingay 2624A ((Kew Distr. 4), fl. & fr. 1867 (BM) ; Maingay 2624 (Kew Distr. 4), fl. 1867—8 (K) ; Kuntze' 6094, fl. Oct. 1875 (NY) ; millett 61, fl. 1884 (K) ; Ihwilund (CAL) ; Jurong, Ridley 435, fl. Oct. 1889 (CAL, SING) ; Eidley, fl. 1S90 (UC) ; Tangkei, Eidley, fl. & fr. Jan. 1905 (MO) ; Eidley, fl. & fr. (CAL, SING); Pulau Ubin, Ridley, fl. (SING).

Java: W. Java: Floem, fl. (L) ; Regantang, Boerlage, fr. June 1888 (L) ; Waning Manga, Kedunghalang, Boerlage 157, fr. Oct. 1888 (L) ; Buitenzorg, Boerlage, fl. & fr. Dee.. 1888 (L) ; ibidem, lldtticr Sa, fl. & fr. Apr. 1893 (BZ) ; Djasinga, Baclcer 9903, fl. Nov. 1913 (BZ) ; ibidem, Backer 10056, fl. & fr. Nov. 1913 (BZ) ; l-\ _ 1 T k !.. sn m -4 et ti ei T i , i / r*.r# \ n e r\ • • û i ,n et s\ /n .r* /• i n « n E of Dcpok, Backer 23168, fl. Jan. 1918. (®Z) ; S of Djasinga, Backer 26022, fr. 1918 (BZ) ; E of Depok, Baoker 26285, fl. Oct. 1918 (BZ) ; Dcpok, „ ,, _ ... _ _ r , , ... ... v ,, r , Bakhuizen van den Brink Jr 890, fl. & fr. Nov. 1921 (BZ, CAL, Gr, K, L, P, SING); Tegal Sapi, Bakhuizen van den Brink Jr 1568, Aug. 1922 (BZ) ; Djambu near Leuwiliang, Bakhuizen van den Brink 7830, fl. & fr. Aug. 19.11 (BZ, IC, L, U) ; Tendjoleat near Bunar, de Voogd 5, fl. Oct. 1940 (BZ) ; Land Bolang near Bunar, van Stccnis 12681, fl. Nov. 1941 (BZ) ; Parung Pandjang, Broekhuizen 15, fl. & fr. Dec. 1944 (RZ) ; ibidem, Broclchuizen 9, Feb. 1945 (BZ, L) ; ibidem, Broekhuizen, fr. Feb. 194i5 (BZ).

Borneo: (OAL, U) ; Korthals, fl. & fr. (CAL, K, L, MEL, S) ; Ü. S. S. Pac. Expl. Bxped. 18S8—13, fl. (NY) ; Burbidge, fl. (IC). — NU'. Borneo: Kuching, Bcccari PB 175 4- 193, fl. & fr. July 1865 (Fl, K, P) ; Brunei, Beeoari PB 4067, fl. Aug. 1867 (Fl) ; Rajang, Sibu, Ifavilatid = 2100, fl. Nov. 1892 (UC) ; near Kuching, lin Vila nd 2100, fl. & fr. Doc. 1892 (K) ; ibidem, JIaviUmd if- Hose = 2160, fl. & fr. Oct. 1894 (DM, DZ, CAL, CGE, GH, K, L, P) ; Kuehing, Eidley, fr. Aug. 189!) (SING) ; Sarawak, A nt. Coll. 557, f!. (A) ; Kapit, Upper Rajang Riv., Clemens 21167, fl. May 1929 (A, DZ, K, MO, NY). — IV. Borneo: Chaper, fr. (P) ; Sintang, Langlassê 68, fl. Juno 1894 (P) ; ibidem, Langlassé 87, fl. June 1894 (G, P) ; Acr Itam N of Pontianak, Folate 680, fl. Oct. 1940 (BZ, L) ; Pontianak, Enoh 200, fl. Sept. 1948 (BZ, K, L). — S. and SE. Borneo: Martapura, Korthals, fl. (L) ; Pulau Lampei, Korthals, fl. & fr. (L) ; Tewingan near Martapura, Labohm 1180, fl. Juno 1SHS (BZ) ;

Asem near Pleihari, Lahohm 1056, f 1. May 1!U!> (BZ) ; Bantau, Antasan Mastam, -*— — 7 "7 "*• V— y I J-WIU tau, XX41IUIVOIWI XliUOl/CWH, DG oh" lan 17, fl. Aug. 1922 (BZ). — E. and NE. Borneo: Sungai Rontanan, Bontang, Kutten 417, fr. March 1911 (BZ, U) ; Sungai Berbas, Bontang, Kutten 496, fl. & fr. May 1911 (U); Tikung, A m d jali 917, fl. & fr. Nov. 1912 (BZ, L, UC) ; Amdjali 1017, fl. 1912 (BiZ); near Sadjan Riv., Bulongan, Kutten 88, fr. 1914 (U). — Br. N. Borneo: Kudat, Fraser 120, fl. June 188ü (K) ; Sandakan, Creagh, fl. Apr. 1895 (K) ; Kinabalu Region, Gib bs 2930, fl. 1910 (BMj; Sandakan, Clemens 9504, fl. Doc. 1915 (A); TVood 458, fl. May—Aug. 1917 (K) ; Yates 56, fr. Oct. 1917 (A, US); Sandakan & Vicin., Wood 794, fl. & fr. Feb.—March 1920 (A, BZ, L, US); Sandakan, Mvburgh Pro v., Elmer 20104, fl. & fr. Oct.—Dec, 1921 (A, BM, BZ, C, OAB, X, L, M, MO, NY, P, S, SING, U, UC); Kg Mengalong, Weston, Suleiman BNB 2221, fr. Apr.' 1932 (BZ, IFI, K) ; Talaga, BalajaMa BNB 2561, fl. Aug. 1932 (A, BZ, IFT, K) ; Mt Kalawat, Kinabalu, Clemens 51317, fl. Jan. .1934 (BiM) ; Kabili For. Res., Sandakan, Puasa BNB 4846, fl. & fr. June 1935 (SING) ; Jambongaii Isl., Cabilmg BNB 3776, fl. (U-C) ; Tiaggau Ri»v., Keith BNB 9091, fl. June 1988 (SING) ; Marudu, Kudat, Kitaku Fior. Rtes., Austin BNB A1182, fl. Feb. 1948 (SINÎG) ; Elopura, Sandakan, Anthony BNB A 788, fr. March 1948 (SING) ; ibidem, Kadir BNB A[ 901, fl. Dec. 1948 (SING); ibidem, Kadir BNB A @691, fl. & fr. (L). — Baiiguey Isl. : „ 7 , __ t . Castro $■ Mele- (jrito 1502, fl. Sept. 1923 (A, UC, US). — Labuan: Low 192, fl. & fr. 1867 (Fl) ; Treacher, 1880 (S). — Anambas & Natuna Isls: near Genting, Sedanau Isl., van Stcenis 1055, fr. Apr. 1928 (BZ, L).

Philip piffles: hoher 2O , fl. (K). — Luzon: Mt Makiling, Laguna Prov., Quisumhmg, fr. Sept. 1925 (UC).

Cultivated: Bot. Gard. Bogor (origin : Banka) : Teysmawa, fl. & fr. 1800 (10; Kars 425, fl. (OAL) ; Kurz, fl. 1863 (P) ; Teysmamn, fr. 1867 (L) ; Beecari, fl. 1876 (Fl) ; Boerlage, fl. (L); no IV-G-18, fl. Nov. 1889 (BZ) ; J arise, 1899 (GRO) ; no IV-G-18, fr. 1903 (GH, NY, US) ; van Ilarreveld, fl. & fr. Sept. 1907 (GRO, L) ; Backer, fl. 1908 (BiZ) ; fl. & fr. Apr. 1912 (BZ) ; no IV-G-18a, fr. 1916 (BZ) ; Fevrell tf- Heide, fl. & fr. Dee. 1921 (S). —• ICultuurtuin Bogor: fl. & fr. (U) ; Giesmfiagen, fl. Feb. 1900 (M) ; no A-III-738, Idris, fl. & fr. May 1924 (WAG). — Victoria Garden, Bombay : Land, fl. Dee. 1935 (CAL). — Jamaica: Castleton Distr. (escape from Bot. Gard.), Harris 10889, fl. March 1910 (K, NY, U). — Public Gardens, Kuala Lumpur: Strugnell CF 126%%, fl. Jan. 1927 (SING). — Manila: Manila Gardens, Quimmbing BS 84737, fl. (NY); Fcnix 92, fl. & fr. Apr. 1938 (A) ; College of Agric., Laguna, Sulit PNI1 6.977, fl. & fr. Dee. 11)47 (A). — Panama Canal Zone Èxper. Gard.: JVetmore <)• Abbe 228, fl. Jan. 1932 (A). — Bot. Gard., Penang: Niir, fl. S;ept. 1918 (SING). — Bot. Gard"., Peradeniya: Faircliild Dorsett 262, fl. Feb. 19i26 (UC). — Royal Lakes Gard., Rangoon: Parkinson 14029, fl. March 1932 (DD) ; Parkinson 14455, fl. Juno 193:2 (DP). — School Gard., Semarang: Dooiers van Leeuwen-Eeynvami, fl. June 1910 (BZ). — Sierra Leone: Lanc-Poole 118bis, fl. June 1912 (K). — Bot. Gard., Singapore: fl. (A) ; llullett, fl. & fr. Aug. 1885 (SING) ; Curtis, ƒ]. Sept. 1900 (SING) ; Sargent, fl. Oct. 1003 (A) ; Lawn T, Nur, fl. Aug. 1918 (UC) ; Nur, fl. May 1919 (UC) ; Lakeside, N'ur, fl. & fr. Aug. 1920 (CAL, SING, UC) ; Arboretum, Nur, fl. June 1924 (SING) ; Kiah, fl. May 1929 (IFI) ; Lawn G, Nur, fl. Oct. 192,9 (BRI, BZ, SING) ; Chiliens M50&, fl. & fr. Nov. 1929 (NY, P) ; Furtado SF 34822, fl. Feb. & Sept. 193-8 (A, DO, IFI, L, SING). — Bot. Gard., Trinidad: Broadway, fl. May 1927 (IFI).

Distribution. Sumatra, Malay Peninsula, Riouw and Lingga Archipelagos, Banka, Billiton, Borneo, W. Java, Philippines. Prom Sumatra the species is known only from Palembang. The two collections from the Philippines may represent cultivated or naturalized specimens; I strongly suspect the species not to be indigenous in the Philippines. De Wit (1949) has pointed out that the species would be indigenous in Java. I do not agree with him for the following reasons: The first collecting of the species outside the Botanic Garden was done in 1888, i. e. about 30 years after its introduction. The first collections are all close near Bogor and later collections gradually come to farther distances. It is difficult to understand that a species like the present one with large and conspicuous flowers would have escaped the attention of earlier collectors, particularly as it flowers throughout the whole year in Bogor.

Ecology. In marshes, along streams, and on the margin of forests, sometimes immediately behind the mangrove, up to 500 m altitude Flowering continuously, each flower open for one day only; between the flowers of one raceme a difference in flowering-time of about 3—4 days. The fruit ripens after 36 days (Corner, 1940) ; the arillate seeds are eaten by birds, what may be a means of dispersal.

Vernacular names. Sumatra: Sempur ajor, S. rawah (Palembang) Simpong, Kaju sipur (Riouw and Lingga Arch.); Kembang masimpur (mesimpur), Minipor, Simpur, Simpor prampuan, Sipor (Banka) ;

Simpur (Billiton). Malay Peninsula: Champurna, Simpoh ayer (= water simpoh), S. gajah (i= elephant or big s.), S. pasir, S. paya (<= marsh s.). Borneo : Simpur (Mai., SE. Borneo), Simpor ayer (Mai., Br. N. Borneo), Simpor bini (Mai., Brunei), Simpor rimba (Kedayan, Br. N. Borneo), Tambakau (Tengara, Br. N. Borneo).

Uses. Because of its beautiful foliage and abundant flowering the species is often planted as an ornamental shrub.

Notes. 1. Wormia suffruticosa and W. subsessilis differ only in the indûment of the lower side of the leaves and petioles, this being villose in W. suffruticosa, glabrous in W. subsessilis. As several intermediates are known, the distinction of two separate taxa does not appearto be justified.

2. Wormia subsessilis var. borneensis differs from the other specimens by the large leaves and the different colour of the pseudocarp, this being white. As to the latter difference, I do not think it to be of any importance., whereas as to the other characters the pseudocarps in the type specimen of var. borneensis agree with those in other specimens. As to the size of the leaves, these are really large in the specimen cited, but intermediates as to size are found in a number of collections.

Asian Pacific Journal of Tropical Biomedicine

Ethnobotanical review and pharmacological properties of selected medicinal plants in Brunei Darussalam: Litsea elliptica, Dillenia suffruticosa, Dillenia excelsa, Aidia racemosa, Vitex pinnata and Senna alata

3. D. suffruticosa

Locally known as “Simpor bini”, D. suffruticosa is a medium sized tree characterized by its large bright flowers with five thin yellow petals around its white stamen, and dark-pink star-shaped fruits all of which are surrounded by large oval leaves (Figure 2). This plant species typically grows in wastelands, swamps, poor soil, white sands, secondary forests, along roadsides or the edge of forests [11–13]. Its leaves have been traditionally used for different treatments such as to promote wound healing, relieve rheumatism and treat fever while the fruit was claimed to be able to treat cancerous growths [12–16].

The methanolic extract of the roots of this plant have displayed significant antioxidant and cytotoxic activities particularly towards the HeLa cervical cancer cell line [17]. Studies conducted by Armania et al. have indicated that the phenolic content was an important contributor to the high antioxidant activity observed in the methanolic root extract of this plant species [17,18]. Although this extract showed the highest antioxidant and cytotoxic activities in the Hela cell line, it was found that the dichloromethane and ethyl acetate extracts exhibited higher cytotoxicity in the breast cancer cell lines, MCF7, MDA-MB-231, the A549 lung cancer cell line and the HT29 colon cancer cell line [17]. Further mechanistic investigation demonstrated that the plant extract inhibited the proliferation of the HeLa cervical cancer cell line as well as the MCF7 and MDA-MD-231 breast cancer cell lines via the

induction of apoptosis and the G2/M cell cycle arrest [17,18]. An in vivo study conducted by Yazan et al. showed that oral intake of aqueous root extracts have successfully reduced breast cancer induced in rats and also inhibited metastasis of the cancer to the heart [19]. The study further demonstrated that the extract was not toxic at the acute toxicity level up to a high dose of 500 mg/kg, however, mild focal hemorrhage was observed when a dose of 1000 mg/kg of the extract was used for treatment [19]. The cytotoxic activities of this plant species could be attributed to the presence of phytochemicals such as saponins, triterpenes, sterols, and polyphenolic compounds [17,20–26].

Saponins is a collective term for triterpenoid and steroidal glycosides [27], which consists of at least 150 kinds of natural saponins that have displayed significant anti-cancer properties [28]. Moreover, they have been recognized for their ability to reduce cholesterol level in the blood [29]. Dietary and endogenous cholesterols pass through the bile or desquamatedintestinal cells and reach the intestine before they are absorbed into the blood stream [29]. Saponins, being poorly absorbable from the intestine into the blood stream, interact with cholesterols and other sterols, and thus interfere and prevent them from being absorbed into the blood stream [29]. Additionally, saponins have the ability to stimulate the immune system and enhance antibody production [30].

Interestingly, multiple studies have reported reduction in bone loss with diets that are high in saponins [31,32]. One of the many saponins in particular, called asperosaponin VI, was able to induce the differentiation and maturation of osteoblasts and thus increase bone formation via the bone morphogenetic protein-2/p38 synthesis, and activation of the extracellular signal-regulated kinase 1/2 pathway [32].

On the other hand, polyphenols, which include phenolic acids, flavonoids, stilbenes and lignans, have been recognized for their multiple health-benefiting properties [33]. Polyphenols such as resveratrol and quercetin were found to exhibit significant cardio-protective effects by preventing platelet aggregation, disrupting atherosclerotic plaques and inhibiting protein expressions [33]. Other than that, anti-cancer, anti-viral, anti-diabetic, anti-aging and neuro-protective effects were also noted for the phytochemicals in the polyphenol group. They also showed beneficial effects towards asthma, osteoporosis, bone loss, skin damage and mineral absorption in intestines [33].

The phytochemical contents within D. suffruticosa play a significant role in relieving and alleviating illnesses. Perhaps it is a combination of their effects that promote wound healing, relieve rheumatism, and treat fever and cancerous growth traditionally. However, more studies should be performed to fully validate their traditional uses for such diseases.

ABSTRAK

Nama                   :    Suyatno

Program Studi     :    S1 Farmasi

Judul                    :    Uji Aktivitas Antibakteri Ekstrak Etanol 70% Daun Sempur (Dillenia suffruticosa (Griff.) Martelli)  Terhadap Bakteri Staphylococcus aureus Dan Escherichia coli.

Telah dilakukan penelitian tentang uji aktivitas antibakteri ekstrak etanol 70% daun sempur (Dillenia suffruticosa (Griff.) Martelli) terhadap bakteri Staphylococcus aureus dan Escherichia coli. Penelitian ini bertujuan untuk mengetahui aktivitas antibakteri dan nilai konsentrasi hambat minimun dari ekstrak etanol 70% daun sempur. Bahan uji yang digunakan adalah daun sempur yang diperoleh dari Desa Pedindang, kecamatan Pangkalanbaru, kabupaten Bangka Tengah, Propinsi Kepulauan Bangka Belitung untuk selanjutnya diproses menjadi serbuk halus. Pembuatan ekstrak dilakukan secara maserasi dengan etanol 70%, kemudian di evaporasi menjadi ekstrak kental dengan hasil rendemen sebesar 24,74%, hasil ekstrak dilakukan pengujian aktivitas antibakteri dengan konsentrasi 5%, 10%, dan 15% terhadap bakteri uji. Selanjutnya dilakukan pengukuran diameter daya hambat, berdasarakan pengukuran DDH  terhadap Staphylococcus aureus dengan hasil 5%  10,52 mm , 10% 11,73 mm, dan 15% 12,67 mm sedangkan terhadap Escherichia coli 5% 11,29 mm, 10% 12,89 mm, dan 15% 13,84 mm. Nilai KHM pada Staphylococcus aureus 3% dan pada  Escherichia coli 5% .

Kata Kunci : Ekstrak Etanol 70% Daun Sempur, Staphylococcus aureus, Escherichia coli, DDH, KHM.

Dilleniae radix (Dillenia suffruticosa, L. Dilleniaceae) the cytotoxic effect of dichloromethane extract at the maximum concentration of 100 μg/ml was applied to the breast cancer cell line (MCF-7). The experiment was conducted using the MTT assay in the presence of tamoxifen as the positive control. The extract showed the time and dose dependent strong cytotoxic activity with an IC50 value of 15.5 ± 0.5 μg/mL after 72 hours of exposure. This significant result indicates that the Dilleniae radix extract has a potential for developing a new cytotoxic agent. In addition, the authors explained how the investigated extract exhibited the cytotoxic effect. The dichloromethane extract of Dilleniae radix induced the G0/G1 and G2/M phase cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 cells probably via the up-regulation of NF-κB, JNK1 and down regulation of the anti apoptotic genes AKT1 and ERK1 (70). Additionally, other studies confirmed the signalling pathway responsible for cytotoxicity of Dillenia suffruticosa extract on breast cancer cells (71, 72). Qualitative phytochemical screening of Dillenia suffruticosa extracts indicated the presence of saponins, triterpenes, sterols, and polyphenolic compounds, which contribute to the cytotoxic activities (72).

Journal of BIOLOGICAL RESEARCHES

ISSN: 08526834 | E-ISSN:2337-389X

Volume 22| No. 2| June | 2017

http://dx.doi.org/10.23869/bphjbr.22.2.20177

Published by © PBI East Java. Open Access  www.berkalahayati.org 74

 Corresponding Author:

Ridesti Rindyastuti

Purwodadi Botanic Garden, Indonesian Institute of Sciences.

Jl. Raya Surabaya-Malang Km. 65, Purwodadi, Pasuruan, Jawa Timur Phone: 0343-615033 Fax: 0341-426046

e-mail: ride17@gmail.com

Original Article

Carbon storage of medium-sized tree: a case study on Dillenia collection in Purwodadi Botanic Garden

Ridesti Rindyastuti

Purwodadi Botanic Garden, Indonesian Institute of Sciences Abstract

Abstract

Dillenia is a medium-sized tree which has high species diversity in tropical regions especially in Southeast Asia. Dillenia in Purwodadi Botanic Garden are collected from native habitats in Java, Kalimantan, Sulawesi and Papua which planted on the area of 17 x 55 m2. The purpose of this re-search is to study the above ground carbon storage in Dillenia collection in Purwodadi Botanic Garden. Carbon storage estimation was established by measuring stem carbon stocks from plant collections with plant age ranging between 12-30 years. Twelve years old collection contributed carbon storage of 7.35 tonnes/ha for D. sumatrana. Twenty years old species had the lowest carbon storage of 2.17 kg/plant for D. serrata and the highest of 51.9 kg/plant for D. auriculata with a range of carbon storage of 3.47 to 41.072 tonnes/Ha. Thirty years old plant contributed 39.465 kg/plant and carbon storages of 63.14 tonnes/ha for D. serrata and 135.59 kg/plant and 216.94 tonnes/ha for D. philipinensis. Overall, Dillenia collections in Pur-wodadi Botanic Garden contributed 793.94 kg carbon storages, store carbon on average of 30.54 kg/plant and 46.46 tonnes/ha. The increase of carbon storage in the second 10 years was higher than in the first 10 years. It indicated that Dillenia had growth strategy in the early growth then alocated more mass after 10 years. Carbon storage of Dillenia was high and different in age. D. serrata, D. papuana and D. auriculata are recommended species as a priority in planting trees based on carbon sink.

Keywords: Dillenia, carbon storage, medium-sized, tree, Purwodadi 

Received: 09 March 2017 Revised: 28 April 2017 Accepted: 08 June 2017

Introduction

According to Hairiah et al. (2011), carbon sequestra-tion is the ability of a system to store carbon from the atmosphere during a certain period. Plants through the process of photosynthesis have a function as an absorber of carbon emissions in the atmosphere and store it in the form of biomass. Generally, there are two kinds of carbon sequestration, which is below ground such as by soil microbes and roots, and above ground plant biomass mainly by tree stems (De Jong dkk., 1995; Cannell, 2003; IPCC, 2006). Climate change mitigation innitiatives recommend the reforestation programs and diversification of plant species for improving the ecosystems quality of degraded areas and for greening urban areas (IPCC, 2006; UN-FCCC, 2007). Selection of plant species in agroforestry programs based on carbon sink is important because plants could run various ecological functions and increase plants diversity as well (Diaz, 2009). Native species was proved of running more diverse environmental services compared to non-native species. In addition to its function as a carbon sink, native species are able to maintain the hydrology of an area, restore the food chain and native vegetation by associating with other local species.

Researches on carbon stocks of diverse plant species or groups, both native and non-native species in their nat-ural habitat have been carried out. Siregar and Darmawan (2011) examined the carbon sequestration of Dipterocar-paceae in Central Kalimantan, and argued that the dipter ocarp forests can store carbon 928.86 tonnes C/ha or 20.64 tonnes/plant. In the dipterocarp family, the varia-tions of their carbon storages are quite high. This can be caused by different species constituent, genetic factors that influence the allocation of nutrient storages and allocations, plant age and environmental factors (Diaz, 2009). Imiliyana et al. (2012) reported that mangrove is capable of storing 232.59 tonnes C/ha while according to Pramudji (2011) Acacia is capable of storing 56.05 tonnes C/ha.

Dillenia is one of medium-sized tree which has high species diversity in the tropics. This plant group has about 60 species distributed from Madagascar to Australia and is one of a vegetation component of tropical forest in low land areas. The leaves are oval to elliptical with promi-nent leaf midrib. The flowers are large with five petals and many stamens (Lemmens and Wong, 1995). Dillenia is utilized for many economic purposes such as wood products (most of Dillenia species), craft, and medicine such as Dillenia suffruticosa for anti-inflamatory (Shah et al., 2015), D. philiphinensis and D. indica for antimicrobial (Ragasa, 2009; Apu et al., 2010). Many previous studies on tree carbon storage did not use the growth size as a diagnostic characters to distinguish carbon stored in plants. Growth size of trees is controlled by gene and it influences common maximum tall and large of tree stem in which carbon stored. A group of trees which have a characteristic as medium-sized tree commonly grow to around 10-40 m tall, while group of large trees can grow up to 50 m tall even hundred m tall (Lemmens and Wong, 1995). The study on carbon storages of local plant species based on its growth size can increase the understanding of biology of trees and reveal the significant contributions of diverse plant species to carbon sequestration in reducing global carbon rising in the atmosphere.

Heng and Onichandran (2014), reported that Dille-nia suffruticosa in degraded area in Sarawak, Malaysia has slightly low biomass and carbon storage in the early forest formation (5.2 tonnes/ha). However, the highest proportion of biomass on this species was on the stem, and it increased with tree size and age. Dillenia can be a good recommendation for afforestation for both conserva-tion and tree planting in urban areas, but its carbon storage have not been studied well. Therefore, the aim of this research is to study 1) carbon storage of a medium-sized tree in a case of Dillenia collection in Purwodadi Botanic Garden and 2) the effect of different age on the carbon storage of Dillenia collection in a garden system, especially in Purwodadi Botanic Garden.

Method

Plant Materials

Dillenia collection examined in this study comprised of 10 species. The collections were planted on an area approximately of 17 x 55 m2. List of plant species studied together with the origin of the collection and distribution of the species are presented in Table 1.

Table 1. Species list, origin of plant collection and plant distribution of Dillenia collection in Purwodadi Botanic Garden.

(Source : Catalog of plant collection in Purwodadi Botanic Garden-LIPI, 2012; Lemmens and Wong, 1995)

Biomass measurement

Biomass was measured using allometrics method which can be estimated from stem diameter at breast height of adult trees so called DBH and trees height of each Dillenia collection in Purwodadi Botanic Garden-LIPI. Three replications were used in the measurement for each species. Biomass was obtained by calculating using the formula of biomass for plants in dry climates habitat and allometric equation for Dillenia as follows:

(Hairiah et al., 2010)

Biomass (kg) = 0,122 (rD2H)0.916

Where r = wood density (Zanne et al., 2009)

D = diameter (cm)

H = plant height (m)

Carbon stock values were obtained by multiplying the values of biomass with allometric values for carbon stock i.e. 0.46 (Hairiah et al., 2010). Carbon storage per hectare was converted using the total of area and plant spacing of collection in Purwodadi Botanic Garden i.e. 2.5 x 2.5 m.

Data Analyses

Biomass and carbon stocks of 10 Dillenia species were analyzed using the variance test (ANOVA) at the 95% confidence level to determine the variation of bio-mass and carbon stocks among species in Dillenia collec-tions. The data analyses were also conducted on the bio-mass and carbon stocks among species in different age level (12, 20 and 30 years).

Results

Carbon Storage in Dillenia

Table 2 showed the carbon storage of Dillenia collection in Purwodadi Botanic Garden estimated from their DBH and biomass. The statistical analyses are also conducted to show the differences of carbon stocks among Dillenia species. Based on the ANOVA test on a 95% confidence level, DBH of 10 species of Dillenia were significantly different amongs species with a P value of 0.004 (P <0.05). The statistical tests were conducted on the DBH of individuals in various age ranges (12-30 years). The data of biomass and carbon stocks were ab-normal based on the normality test on a confidence level of 95%. Therefore, the data only can be read descriptive-ly. The 30 years old D. philipinensis have the highest DBH while the twelve years old D. sumatrana has the lowest DBH. The species which are 20 years old have a range of DBH between 18.15 cm for D. ovalivolia to 54 cm for D. papuana. It showed that in the same age, plant species within genus may have different DBH and carbon storage.

Table 2. The value of DBH, biomass, per plant and per ha carbon stocks of 10 species of Dillenia in Purwodadi Botanic Garden.

*) Statictical analyses using ANOVA in the confidence level of 95%. Same letters in one column showed no significant  ifference among species 

**) These data are not normal and not random based on the normality and randomity test, thus the variance analysis can not be performed.

D. philipinensis has the highest biomass (29.47 kg) and contribute the total carbon storage of 135.58 kg C/plants or 216.94 tonnes C/ha. Twelve years old D. su-matrana has DBH 21.93 cm with 0.99 kg of biomass and carbon stocks of 4.59 kg/plant or 7.35 ton C/ha. This size is quite large compared to other 20 years old species (Fig 1 and Table 2). Figure 3 showed the carbon stored per hectare of Dillenia collection in Purwodadi Botanic Garden. Carbon storage of D. ovalifolia is the lowest among other species studied especially compared to other species at the same age (20 years old). At 20 years old, D. papuana has the highest carbon storage of 51.9 kg C/plant.