Antibiotic sensitivity tests in the clinical laboratory are important in the evaluation of what antibiotic(s) and certain drug(s) called antimicrobials could be used in chemotherapy of bacterial infectious diseases. If the specimen is used in the test, it is considered to be a “direct” sensitivity test. Employing a pure culture isolated from the specimen, then it is called an “indirect” sensitivity test. Staphylococcus aureus - ATCC 25923, Escherichia coli - ATCC 25922, and Pseudomonas aeruginosa - ATCC 27853 are often used as quality control organisms since they are of known susceptibility to many antibiotics.
The antibiotic sensitivity of bacteria may be defined as the lowest test concentration (of the antimicrobial) which completely inhibits the growth of the bacteria; i.e., Minimum Inhibitory Concentration or MIC. The MIC will be determined by three methods:(1)disk, (2)agar plate, (3)broth tube method.
GENERAL INFORMATION - DISK METHOD
The disk method utilizing the disk method, is used extensively in clinical laboratories for the rapid evaluation of drug sensitivity of pathogens, but many factors affect the reliability*. A small disks of absorbent paper, impregnated with known concentrations of a specific antibiotic or antimicrobial, are spatially placed on the surface of an inoculated agar medium (specimen or pure culture). The drug diffuses into the medium during the incubation producing a concentration gradient as the microbial cells grow. The width of the “zone of inhibition” surrounding each disk is measure; thus a qualitative determination.
The Kirby-Bauer test is a standardized, quantitative test utilizing the disk method. A single disk of known (usually high) potency is placed on the surface of Mueller-Hinton agar medium inoculated with a standard inoculum. Using a special comparator that interpret the diameter of the zones of inhibition, consequently the organism can be described as resistant, intermediate, or sensitive. Tables are used to determine the breakpoint for each drug.
*The zone of inhibition around the antibiotic disk is influenced by the: (1) type of growth medium, (2) its cation content, (3) the antimicrobial concentration in the disk, (4) inoculum concentration, (5) temperature of incubation, and (6) atmosphere of incubation
GENERAL INFORMATION -
AGAR PLATE METHOD
The agar plate method allows (1) testing the sensitivity of a multitude of isolates; thus, (2) less time consuming and expensive than the tube method. Different concentrations of a specific drug are placed in individual tubes containing a specific volume of melted, cooled agar medium, mixed well, and poured into sterile plates. After solidification of the medium, standard inocula of organisms are placed on the agar surfaces including quality control organisms. Also a plate containing no antimicrobial is included as a control. The presence or absence of growth is observed after proper incubation.
NOTE: The
MIC is the lowest concentration of antibiotic tested that yields complete
inhibition of growth.
GENERAL INFORMATION - BROTH METHOD
In the quantitative broth method, a specific antibiotic concentration is the only variable in a series of suitable broth tubes (e.g., Mueller-Hinton broth). A standard inoculum of the pure culture is added to each tube of drug-broth including a tube of broth without antibiotic which serves as a control. Suitable quality control organisms may also be included. Again after proper incubation, the MIC is determined.
There are several disadvantages
of this technique. For example, (1) only one isolate may be
tested per series of tubes; (2) expensive and time consuming;
(3) contamination may occur without detection.
MATERIALS NEEDED FOR ALL THREE METHODS:
sterile distilled
water
assorted sterile
test tubes
sterile saline
sterile cotton-tipped
applicators
sterile 10-ml. and
1-ml. pipettes
pipetting devices
sterile petri plates
deep tubes of M-H
sensitivity agar, 20.0 ml. per tube
antibiotic sensitivity
disks and dispensers
L-glass spreading
rods
beakers of 95% ethanol
McFarland nephelometry
standard
rulers, in mm
stock cultures of
quality control organisms*:
Escherichia coli ATCC 25922
Staphylococcus aureus ATCC 25923
Pseudomonas aeruginosa ATCC 27853
Enterococcus fecalis ATCC 29212
stock solutions of antibiotics:
amoxicillin 1000 units/ml
ciprofloxacin 1000 ug/ml
other antibiotics 10,000 ug/ml
The agar deeps which
you prepared in a previous lab will be melted before lab time and placed
in a
50O
C water bath. Be very careful that agar in the bottom of the deeps
does not solidify when you are adding the antibiotic solutions prior to
pouring the plates.
* For the disk method, agar plate method, and broth method, the quality control organisms will consist of overnight Mueller-Hinton broth cultures which will be furnished to you.
You will be responsible
for the overnight culture of your isolate. This will be accomplished
by inoculating 5 mls of your M-H broth in a screw capped tube with a small
bit of your stock isolate.
EXERCISE - DISK METHOD
Work individually with your isolate and a group of four individuals for the four quality control organisms.
1. Pour two tubes of 20.0 ml. of Mueller-Hinton agar into sterile plates, allow them to solidify and dry.
2. Prepare light, faintly turbid suspension of the test organisms in sterile screw-capped tubes containing 5.0 ml of sterile saline to a turbidity which matches that of a McFarland 0.5 nephelometry standard. Depending on the turbidity of your overnight culture, approximately 0.5 ml of the stock into the 5.0 ml of saline should result in the faintly turbid suspension. The test organisms will include your isolate plus the three quality control organisms. The three quality control suspensions can be prepared on a group basis.
3. Pipette 0.1 ml of your diluted isolate onto one M-H plate and one of the four quality control broth cultures (the other three will be done by your partners) to the agar surface of another plates. Spread each with an alcohol-flamed L-rod. DRY.
4. Using the automatic disk dispenser to apply eight disks to the individual plates.
5. Incubate plates at 35O C overnight .
6. For interpretation, the diameter of the inhibitory zone is measured with millimeter ruler on the underside of the plate. The zone size around the individual antimicrobial disk can be converted to susceptible, intermediate, or resistant category for the organism being tested using the tables provided.
NOTE:You will be responsible
for the results of your isolate and the four quality control cultures.
EXERCISE - AGAR PLATE METHOD
Work in groups of 4, each person with one antibiotic.
1. Pipette 9.0 ml of sterile water to the required number of sterile tubes (three in some cases and two in others) to prepare dilution blanks to be used for diluting your antibiotic. Each table will have four different antibiotics.
2. Prepare the
following dilutions (below protocol) of antibiotics, from the stock solutions
supplied to you, in the water blanks you previously prepared (each individual
will dilute one antibiotic and prepare a series of plates):
Solutions to be Prepared
Supplied
Antibiotic
Concentrations of Antibiotics
Stock Solution
Amoxicillin 1, 10, and 100 units/ml 1000 units/ml
Ciprofloxacin 10, and 100 ug/ml 1000 ug/ml
Other antibiotics
10, 100, and 1000 ug/ml
10,000 ug/ml
3. Label plates with the plate number corresponding to the specific concentration of antibiotic (ug/ml or units/ml) to be tested (table next page).
4. Add the calculated volume of the proper antibiotic dilution to each agar deep previously melted, and cooled to 50 C; mix well and pour into the labeled plates. Be sure to work with only one agar deep at a time.
5. After the plates have solidified and dried, then divide each plate into eight (8) sectors with marker on the back of the plate. Number the sectors 1 to 8. Use a clockwise numbering pattern. Each number will correspond to the sector for one test organism. Sectors 1 through 4 are to be used for the four isolates from individuals in your group. The four sectors, 5 through 8 are assigned the quality control organisms.
6 Prepare
a dilution of each culture by adding the overnight broth culture to one
ml of saline until the turbidity approximately matches that of a McFarland
0.5 nephelometry standard. (These suspensions have already
been prepared for the disk method and can be used here.)
Protocols for Antibiotic
Agar Plate Series
Amoxicillin (0.6 ug / Unit )
Plate
Units
Units
Volume to be
Stock Solution
No.
per ml
per 20 ml
added in ml units/ml
1 0 0 - -
2 0.01 0.2 0.2 1.0
3 0.03 0.6 0.6 1.0
4 0.06 1.2 1.2 1.0
5 0.1 2.0 0.2 10.0
6 0.3 6.0 0.6 10.0
7 0.6 12.0 1.2 10.0
8 1.0 20.0 0.2 100.0
9 3.0 60.0 0.6 100.0
10
6.0
120.0
1.2
100.0
OTHER ANTIBIOTICS
Plate
Micrograms Micrograms
Volume to be
Stock Solution
No.
per ml
per 20 ml
added in ml
micrograms/ml
1 0 0 - -
2 0.1 2.0 0.2 10.0
3 0.2 4.0 0.4 10.0
4 0.4 8.0 0.8 10.0
5 1.0 20.0 0.2 100.0
6 2.0 40.0 0.4 100.0
7 4.0 80.0 0.8 100.0
8 6.0 120.0 0.12 1000.0
9 8.0 160.0 0.16 1000.0
10
10.0
200.0
0.20
1000.0
7. To actually carry out the procedure, dip a sterile cotton-tipped applicator into the bacterial suspension and squeeze out the excess fluid against the inside of the tube. Then make a single radial streak no more than an inch in length to the corresponding sector of each plate of the series, beginning with the control plate (no antibiotic) and progressing through the increasing concentration plates, in other words, starting with plate #1 and continue through plate #10. Repeat this procedure with each of the organisms.
8. You will be responsible for applying your isolate(s) to each of the other members of the group plates (four different antibiotics).
NOTE: Each individual will need 8 swabs. Four swabs for your isolate to be applied to four different antibiotic series plates (your partners plates). The four other swabs are used to inoculate the quality control cultures to your own series plates. Be careful to use only the needed amount.
9. Be sure all of the inocula have dried or have been absorbed into the agar plate medium before closing and incubation.
10. Incubate
for 24 hours at 35 C and observe the growth, then record the results using
the scale below on the prepared report sheet.
growth equivalent to control ++++
moderate growth +++
intermediate growth ++
scant growth +
no growth
-
11. Do not confuse marks of the applicator on the agar with trace growth. Record the results for all of the organisms that were inoculated on your series of plates. You will need to share the results of the isolates and the quality control cultures with individuals of your group. Record the MIC (minimal inhibitory concentration) of each culture in the last column of your record sheet table. Do not discard your plates until all of the individuals your group have had a chance to observe their actual results on your plates. After all this has been taken care of, discard the plates in designated area.
Be sure to save the
supplied antibiotic stocks and also your isolate stock slant, in
that you will need them again next week.
EXERCISE - BROTH METHOD (following week)
You will need an overnight broth culture of your isolate using 5 ml. of your Mueller-Hinton broth.
1. After the sensitivity of your isolate has been determined by the agar plate method, retest it in broth containing concentrations of the antibiotic which are to be selected on the basis of the agar plate results. You need to calculate the varying concentration of antibiotic needed for each of the tubes of the broth method. Each tube will have a specific concentration of antibiotic that is greatest in tube #10 and lowest in tube #1. This is used to determine the minimum concentration by broth method. By the general rule the MIC by the broth method is usually lower than by the plate method. Therefore, the lowest concentration tested should be about 1/10th of the MIC that you determined in the agar plate method and the highest concentration about double or 2X that of the MIC . For example, if the culture grew at 2.0 but not at 4.0 micrograms of antibiotic per ml of agar, therefore the MIC would be 4.0 ug/ml (or 4.0 U/ml). A satisfactory broth series would be 0.4, 0.8, 1.6, 2.4, 3.2, 4.0, 5.0, 6.0, 7.0, 8.0 ug/ml respectively. Also notice that your agar determined MIC concentration should be in tube #6. Refer to the attached example protocol for further information. Prepare a protocol similar to this one using your specific MIC for your organism and selected antibiotic. Also, show diagrammatically your dilutions (as shown in stock prep. example on last page) that will needed to make from the stock of 1000 U/ml or 1000 ug/ml or 10,000 ug/ml respectively of your selected antibiotic.
* Refer to the example protocol and further explanation on the following pages.
NOTE: I will check your protocol, calculations, and diagrammatic representations for stock dilutions of the antibiotic before proceeding with the exercise. This can be done prior to the lab period if time allows.
2. Set up and number 12 sterile screw capped tubes.
3. Begin by adding the needed volume of M-H broth to each tube that is to be made up to a volume of 5.0 ml. This information can be gotten from your protocol column “broth added”. This procedure can be best accomplished by using a 5.0 ml pipette, add 5.0 ml to the tubes #11 and #12. Also add 4.0 ml to each of the tubes #1 - 10. The fraction of a ml should be added with a 1 ml pipette.
4. Add the specific volume of diluted antibiotic to each of the designated tubes. This information can be found in your protocol column “volume to be added” from the specifically diluted antibiotic “stock” column.
5. No antibiotic is added to tubes #11 and #12. One of these is the inoculated control - #11, the other the uninoculated control - #12 (blank for standardization of the spectrophotometer).
6. Prepare a 1:100 dilution of a 24-hour broth culture of your test organism by adding 0.1 ml of the culture to the 9.9 ml sterile saline. Add 0.05 ml of this diluted suspension to each tube, excepting the uninoculated control - tube #12.
7. Incubate the test tubes for 18 to 24 hours at 35 C. Steam the culture in a boiling water bath for 10 minutes to kill the organisms. The culture should be cooled to room temperature before proceeding.
8. Measure the turbidity in each tube with the Spectronic 20 spectrophotometer at 550 nm (Follow the directions for the operation of the “Spec 20” given to you in a previous lab). First set the machine to 0 (zero) OD by using the uninoculated blank - #12 as the blank . Then read the tubes in reverse order starting with tube #10 and proceeding to tube #1 and lastly reading tube # 11. Use only a single cuvette for all your turbidity readings. Also use the entire 5 ml in your cuvette. If you follow the above prerscribed procedure you will not have to rinse the cuvette between each reading.
9. Use the % Transmittance reading for your inital record. Then convert the % T reading to OD by using the conversion tables given to you in a previous lab handout.
10. Plot on semi
log graph paper the scale reading (OD550) against the concentration of
antibiotic in micrograms or units per ml. Determine the concentration
required to effect 50 percent inhibition of growth. The OD
scale reading of the inoculated control (tube # 11) is taken to be the
growth maximum or 100%.
*Example Protocol For Broth Method With Explanations
1. Referring to the EXAMPLE PROTOCOL (two pages down), in the second column (ug/ml), the first tube should be 1/10th of your MIC, tube # 6 should be the same as your MIC, and tube #10 double your MIC.
To calculate the ug/ml in each of tubes #2-5, multiply the MIC by 0.2, 0.4, 0.6, 0.8, for each tube respectively. For example:
tube # 2 4 X 0.2 = 0.8 ug/ml
tube # 3 4 X 0.4 = 1.6 ug/ml
To calculate the ug/ml in each of tubes #7-10, subtract the ug/ml in tube # 6 from the ug/ml in tube #10 and divide by four (the number of tubes). This number will indicate the approximate increment of antibiotic to be added to each successive tubes (#7-10). For example if tube #6 has 4.0 ug/ml and tube #10 has 8.0 ug/ml, then the difference is 4.0. Divide this number (4) by the number of tubes (which is 4) and we find this equals 1. So we will add 1 ug increment to each successive tube. So tube #7 will be 5.0 ug/ml, tube #8 will be 6.0 ug/ml, and so on.
2. To determine the ‘ug/5 ml’ in column 3, just multiply the ug/ml for each tube by 5.
3. To calculate the “volume to be added” of a specific stock concentration use formula below:
stock ug(/ml)
ug/5 ml - needed
______________ =
_____________
per 1 ml x - (volume to be added)
To calculate the specific volume to be added , initially arbitrarily pick a stock concentration to plug it into the formula. It is best to start with a stock concentration at 10 ug/ml or less. If X is between 0.1 and 0.5 ml, we can use this stock concentration. Check specific examples on last page.
Keep the volume to be added as close to 0.5 ml as possible, but do not exceed the 0.5 ml volume. Additionally, do not use excessively small volumes such as 0.01. These are impossible to measure with a 1.0 ml pipette.
Also each “stock” (in column 5) should be used more than once. Also you should try to use the smallest number of stocks as possible, but keeping in mind the volume rules stated above.
4. Finally, the “broth added” in ml column (right side-column 6) is determined by subtracting the volume to be added column from 5 ml.
For example:
tube # 1 5.0 - 0.2 = 4.8 ml broth to be added
EXAMPLE PROTOCOL FOR BROTH METHOD
MIC = 4.0 ug/ml (could
also be units/ml)
Tube #
ug/ml ug/5 ml
*vol of stock
Stock
Broth added
OD550
be added
ug/ml
to get 5 ml
(not to exceed
0.5 ml)
1 0.4 2.0 0.2 10 4.8 1.2
2 0.8 4.0 0.4 10 4.6 1.2
3 1.6 8.0 0.2 40 4.8 1.0
4 2.4 12.0 0.4 40 4.6 0.2
5 3.2 16.0 0.2 80 4.8 0.05
6 4.0 20.0 0.25 80 4.75 0.01
7 5.0 25 0.31 80 4.69 0
8 6.0 30 0.38 80 4.62 0
9 7.0 35 0.44 80 4.56 0
10 8.0 40 0.50 80 4.50 0
11 0 0 0 - 5.0 1.2
12
0
0
0
-
5.0
0
*Calculation for volume to be added of a specific concentration stock to obtain a specific concentration of antibiotic on next page:
THE STOCK SOLUTION SOLUTION TO PREPARED
Concentration of stock (ug)
Concentration needed (ug/5 ml)
____________________
= _______________________
Volume (per 1 ml)
Volume (X) to be added (ml)
For tube #1
10 ug
2.0 ug
________ =
_________
1.0 ml
X
X =
0.2 ml
For tube #7
80 ug
25 ug
________ =
_________
1.0 ml
X
X = 0.31 ml
Antibiotic Stock Solution
Preparation:
10,000ug/ml
1000ug/ml
100ug/ml
10ug/ml
80ug/ml
40ug/ml