Look at the table for troubleshooting.
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Count colonies on plates (taking into account only plates with up to 500 colonies).
It is best to determine the relationship between OD600 and the microbial number basing the calculation on plates displaying a number of colonies between 100 and 400. Higher numbers do not accurately represent the original suspension as errors created by coincidence of colonies and nutrient limitation lead to numbers that are lower than expected. Relying on fewer than 100 colonies might, on the other hand, lead to incorrect conclusions due to the addition of statistical and methodological errors while performing the dilutions.
Incubation parameters:
\nCalculate the CFU per mL that were in the overnight culture according to the following formula, where N=(Cx10)/10-D (CFU/mL); C= number of colonies per plate; D= number of 1:10 dilution.
Average the results from three tests performed with the microbial isolate and correlate this number with the OD600 value obtained from the overnight culture. This relationship holds true for subsequent cultures of the same bacterium grown in the same way.
In order for the test to be valid, the agar plates for the cell count have to be checked to verify that the right number of CFU were used. The presence of around 100–200 colonies on the lower of the two dilutions (10-5) of the initial bacterial suspension is expected when using the correct bacterial suspension density of 1–2x108 CFU/mL. If the cell numbers are within the desired range, the test can be analyzed to determine the MIC. Also, check the antibiotic-free growth control plate. Visible growth needs to occur for the test to be valid.
The MIC is defined as the lowest concentration of the antimicrobial substance that inhibits visible growth of the tested isolate. The growth of a single colony or faint film caused by the inoculum should be disregarded. When growth of the tested organism occurs on all agar plates with antimicrobial agent, the MIC is recorded as greater than the highest concentration tested. The MIC is recorded as less than or equal to the lowest concentration when no growth occurs on any of the agar plates but the growth control.
Bacterial suspension preparation: Mix the bacterial suspension, adjusted to 1x108 CFU/mL from [#Growth method~tsk~YI] or [#Colony suspension~tsk~YH] by vortexing and dilute it 1:10 into a cavity of a sterile 96-well microtiter plate by pipetting 10 mL into a well containing 90 mL of sterile broth or saline. Repeat for each bacterial isolate to be tested. Be sure to make a note of the content of each well and to inoculate the microtiter plate in a way that the inocula can be transferred to the agar plates using a 48-pin replicator. For up to 48 tests, inoculate only within rows A–H, columns 1–6 of the microtiter plate. As an alternative to the replicator, a multichannel micropipette set at 1µL can be used to deliver the spots. Make sure that the required number of bacterial cells is going to be transferred. A 48-pin replicator with 1.5 mm pins delivers 1 µL. The final inoculum for a spot with a size of 5–8 mm should deliver the desired cell density of around 104 CFU per spot.
Inoculation: Sterilize the 48-pin replicator by soaking the pins in 95% ethanol and passing them through a Bunsen burner flame. Hold the pins in an upright position until the flame extinguishes. Let the pins cool in an inverted position to maintain sterility. Place the sterilized replicator into the microtiter plate to soak the pins and transfer it onto the agar plate. Start by inoculating a growth control plate without antibiotic. Make sure that each agar plate has the same orientation while inoculating and to make a note of the orientation so that spots on the agar plate can be assigned to the respective isolate tested. Inoculate the antibiotic-containing agar plates starting with the lowest concentration. Let the inoculum spots dry at room temperature before inverting the plates. Plate 100 µL of the last two 1:10 dilutions (10–4 to 10–5) evenly onto antibiotic-free nutrient-rich agar plates using a sterile cell spreader.
Broth dilutions preparation: Add 1 mL of each antibiotic dilution into one test tube for each isolate to be tested. Fill the control tubes with 1 mL sterile broth without an antimicrobial agent. Mix the bacterial suspension well. The suspension should be adjusted to 1x108 CFU mL/L from [#Growth method~tsk~YI] and [#Colony suspension~tsk~YH] by vortexing, and dilute it by a factor of 1:100 by adding 200 µL bacterial suspension to 19.8 mL sterile MHB in a sterile 50 mL Erlenmeyer flask to prepare a 20 mL inoculum. Adjust volumes if necessary.
Inoculation: Inoculate each test tube containing the antibiotic solution and one control test tube (growth control) with 1 mL of the bacterial suspension. This results in the final desired inoculum of 5x105 CFU mL/L.
Incubation:
Prepare antibiotic dilutions following steps bellow.
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Label control tubes for bacterial growth and a single tube for sterility control for the entire measurement.
Dilute the 10 mg/mL antibiotic stock solution 1:10 in sterile broth or water to achieve a 1 mg/mL solution.
Dilute the 1 mg/mL solution 1:10 in sterile broth or water to achieve a 0.1 mg/mL solution
Prepare agar plates following guidelines bellow.
", "position": 3, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-09T12:22:03.852Z", "updated_at": "2018-12-24T09:08:20.489Z", "last_modified_by_id": 202, "protocol_id": 3453 }, "checklists": [ { "checklist": { "id": 966, "name": "Guidelines:", "step_id": 4370, "created_at": "2018-12-21T11:52:37.302Z", "updated_at": "2018-12-21T11:52:37.302Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3975, "text": "Dispense appropriate amounts of antibiotic solution into the respective containers. Follow table steps.", "checked": false, "checklist_id": 966, "created_at": "2018-12-21T11:52:37.304Z", "updated_at": "2018-12-21T11:52:37.304Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3976, "text": "For each agar plate, add 25 mL agar (now at a temperature of 50°C) into the container, mix well (avoid bubbles) and pour 25 ml into a petri dish labelled with the respective antibiotic concentration.", "checked": false, "checklist_id": 966, "created_at": "2018-12-21T11:52:37.311Z", "updated_at": "2018-12-21T11:52:37.311Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3977, "text": "Pour a control agar plate without any antibiotic. Adjust the number if necessary.", "checked": false, "checklist_id": 966, "created_at": "2018-12-21T11:52:37.317Z", "updated_at": "2018-12-21T11:52:37.317Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3978, "text": "Allow agar to set.", "checked": false, "checklist_id": 966, "created_at": "2018-12-21T11:52:37.324Z", "updated_at": "2018-12-21T11:52:37.324Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3979, "text": "Dry the surface of the agar plates either in an incubator or in a laminar airflow hood for 30 min. Leave the lid ajar.", "checked": false, "checklist_id": 966, "created_at": "2018-12-21T11:52:37.330Z", "updated_at": "2018-12-21T11:52:37.330Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 }, { "id": 3980, "text": "Mark the bottom of the agar plates to define an orientation.", "checked": false, "checklist_id": 966, "created_at": "2018-12-21T11:52:37.336Z", "updated_at": "2018-12-21T11:52:37.336Z", "created_by_id": null, "last_modified_by_id": null, "position": 5 } ] } ], "step_comments": [], "step_assets": [], "assets": [], "step_tables": [ { "id": 545, "step_id": 4370, "table_id": 682 } ], "tables": [ { "id": 682, "created_at": "2018-12-20T13:07:47.021Z", "updated_at": "2018-12-24T09:08:20.452Z", "created_by_id": 202, "last_modified_by_id": 202, "name": "", "team_id": 1, "contents": "eyJkYXRhIjpbWyJBbnRpbWljcm9iaWFsIGNvbmNlbnRyYXRpb24gKG1nL0wp\nIiwiVm9sdW1lIG9mIGFudGliaW90aWMgc3RvY2sgc29sdXRpb24gKM68TCki\nLCJGaW5hbCBjb25jZW50cmF0aW9uIHdoZW4gYWRkaW5nIDI1bUwgb2YgYWdh\nciJdLFsiMTAiLCIzMjAiLCIxMjgiXSxbIjEwIiwiMTYwIiwiNjQiXSxbIjEw\nIiwiODAiLCIzMiJdLFsiMTAiLCI0MCIsIjE2Il0sWyIxIiwiMjAwIiwiOCJd\nLFsiMSIsIjEwMCIsIjQiXSxbIjEiLCI1MCIsIjIiXSxbIjAuMSIsIjI1MCIs\nIjEiXSxbIjAuMSIsIjEyNSIsIjAuNSJdLFsiMC4xIiwiNjIuNSIsIjAuMjUi\nXSxbIjAuMSIsIjMxLjI1IiwiMS4xMjUiXV19\n", "data_vector": "JzAuMSc6MzksNDIsNDUsNDggJzAuMjUnOjQ3ICcwLjUnOjQ0ICcxJzozMCwz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\nZW4nOjEz\n" } ] }, { "step": { "id": 5197, "name": "Preparation and autoclaving of media", "description": "\n\nPrepare MHA medium following manufacturer’s instructions. Autoclaving at 121.1°C for 15 minutes at 1 bar. Cool medium to 50°C after that. Around 25 mL is necessary to pour one 15 100 mm petri dish to produce the required depth of 3–4 mm.
As the medium cools down, calculate the amount of antibiotic solutions (10, 1 and 0.1 mg/L) needed. The table is an example of the volumes needed for a concentration range between 0.125 and 128 mg/L for one 25 mL agar plate per concentration. Adjust if a different concentration range is needed. The volumes of antibiotic and agar can also be varied depending on the number of plates to be poured. As each agar plate can be used for up to 48 tests, more than one plate might be necessary if a larger amount of isolates is going to be tested.
Label sterile containers appropriately (glass Erlenmeyer flasks closed with metal caps, tinfoil caps or cotton buds) with the final antibiotic concentration.
Follow the protocol in [#Cell count~tsk~YL]
", "position": 4, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-17T14:34:48.930Z", "updated_at": "2018-12-17T14:34:48.930Z", "last_modified_by_id": 202, "protocol_id": 3450 }, "checklists": [], "step_comments": [], "step_assets": [], "assets": [], "step_tables": [], "tables": [] }, { "step": { "id": 4357, "name": "Isolate preparation", "description": "\nFollow the guidelines below to prepare an isolate.
", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-09T09:26:34.638Z", "updated_at": "2018-12-24T08:59:10.687Z", "last_modified_by_id": 202, "protocol_id": 3450 }, "checklists": [ { "checklist": { "id": 962, "name": "Guidelines", "step_id": 4357, "created_at": "2018-12-21T11:09:29.266Z", "updated_at": "2018-12-21T11:09:29.266Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3957, "text": "For each isolate, select three to five morphologically similar colonies from the fresh agar plate from task", "checked": false, "checklist_id": 962, "created_at": "2018-12-21T11:09:29.268Z", "updated_at": "2018-12-21T11:09:29.268Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3958, "text": "Plate preparation", "checked": false, "checklist_id": 962, "created_at": "2018-12-21T11:09:29.276Z", "updated_at": "2018-12-21T11:09:29.276Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3959, "text": "Touch the top of each selected colony using a sterile loop or cotton swab.", "checked": false, "checklist_id": 962, "created_at": "2018-12-21T11:09:29.287Z", "updated_at": "2018-12-21T11:09:29.287Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3960, "text": "Transfer the growth into a sterile capped glass tube containing sterile broth or saline solution.", "checked": false, "checklist_id": 962, "created_at": "2018-12-21T11:09:29.300Z", "updated_at": "2019-02-07T09:10:36.760Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3961, "text": "Mix using a vortex mixer.", "checked": false, "checklist_id": 962, "created_at": "2018-12-21T11:09:29.307Z", "updated_at": "2018-12-21T11:09:29.307Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 } ] } ], "step_comments": [], "step_assets": [], "assets": [], "step_tables": [], "tables": [] }, { "step": { "id": 5195, "name": "Pure cultures of bacterial isolates", "description": "\n\nGrowth conditions:
\nTurbidity can be assessed visually by comparing the test and the McFarland Standard.
Mix the McFarland 0.5 BaSO4 standard vigorously using a vortex mixer. The turbidity is verified measuring the absorbance of the suspension spectrophotometrically. The absorbance should be in the same range as that of the McFarland standard 0.5 (OD625 nm should be at 0.08–0.13).
Adjust the suspension’s turbidity to that of a McFarland Standard 0.5 by adding sterile distilled water, saline or broth, if the turbidity is too high, or by adding more bacterial material if is too low.
Go back to step 3.
Take samples for cell count and follow protocol in [#Cell count~tsk~YL].
", "position": 5, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-17T14:33:52.646Z", "updated_at": "2018-12-17T14:33:52.646Z", "last_modified_by_id": 202, "protocol_id": 3452 }, "checklists": [], "step_comments": [], "step_assets": [], "assets": [], "step_tables": [], "tables": [] }, { "step": { "id": 4360, "name": "Adjustment of suspension turbidity", "description": "\nAdjust the suspension’s turbidity to that of a McFarland Standard 0.5 by adding sterile distilled water, saline or broth, if the turbidity is too high, or by adding more bacterial material if is too low.
Go back to step 4.
Follow steps bellow for isolate preparation.
", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-09T10:25:56.680Z", "updated_at": "2018-12-24T09:01:33.668Z", "last_modified_by_id": 202, "protocol_id": 3452 }, "checklists": [ { "checklist": { "id": 964, "name": "Guidelines:", "step_id": 4362, "created_at": "2018-12-21T11:14:19.776Z", "updated_at": "2018-12-21T11:14:19.776Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3965, "text": "For each isolate, select three to five morphologically similar colonies from the fresh agar plate from task .", "checked": false, "checklist_id": 964, "created_at": "2018-12-21T11:14:19.778Z", "updated_at": "2018-12-21T11:14:19.778Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3966, "text": "Plate preparation.", "checked": false, "checklist_id": 964, "created_at": "2018-12-21T11:14:19.785Z", "updated_at": "2018-12-21T11:14:19.785Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3967, "text": "Touch the top of each selected colony using a sterile loop or cotton swab.", "checked": false, "checklist_id": 964, "created_at": "2018-12-21T11:14:19.791Z", "updated_at": "2018-12-21T11:14:19.791Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3968, "text": "Transfer into a tube containing 3–4 mL of a suitable nutrient-rich medium.", "checked": false, "checklist_id": 964, "created_at": "2018-12-21T11:14:19.798Z", "updated_at": "2018-12-21T11:14:19.798Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3969, "text": "Mix using a vortex mixer.", "checked": false, "checklist_id": 964, "created_at": "2018-12-21T11:14:19.805Z", "updated_at": "2018-12-21T11:14:19.805Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 } ] } ], "step_comments": [], "step_assets": [], "assets": [], "step_tables": [], "tables": [] }, { "step": { "id": 4363, "name": "Pure cultures of bacterial isolates", "description": "\nFollowing guidelines below will get you to obtain bacterial isolates.
", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-09T10:25:56.700Z", "updated_at": "2019-02-20T07:38:26.679Z", "last_modified_by_id": 202, "protocol_id": 3452 }, "checklists": [ { "checklist": { "id": 963, "name": "Guidelines:", "step_id": 4363, "created_at": "2018-12-21T11:12:20.820Z", "updated_at": "2018-12-21T11:12:20.820Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3962, "text": "Prepare media (store at 4°C).", "checked": false, "checklist_id": 963, "created_at": "2018-12-21T11:12:20.822Z", "updated_at": "2018-12-21T11:12:20.822Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3963, "text": "Prepare antibiotic stock solutions.", "checked": false, "checklist_id": 963, "created_at": "2018-12-21T11:12:20.831Z", "updated_at": "2018-12-21T11:12:20.831Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3964, "text": "Streak the bacterial isolates to be tested onto nutrient-rich agar plates without inhibitor to obtain single colonies", "checked": false, "checklist_id": 963, "created_at": "2018-12-21T11:12:20.838Z", "updated_at": "2018-12-21T11:12:20.838Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] } ], "step_comments": [], "step_assets": [ { "id": 3087, "step_id": 4363, "asset_id": 3749 } ], "assets": [ { "asset": { "id": 3749, "created_at": "2019-02-20T07:37:48.184Z", "updated_at": "2019-02-20T07:38:26.671Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 48768, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Plate_Streaking.jpg" } } ], "step_tables": [], "tables": [] }, { "step": { "id": 4364, "name": "Incubate the broth", "description": "\n\nIncubation conditions:
\n