NL2030349B1 - Method for isolation and screening and evaluation of antibiotic resistance of e. coli and s. aureus in estuary and nearshore marine environment - Google Patents

Method for isolation and screening and evaluation of antibiotic resistance of e. coli and s. aureus in estuary and nearshore marine environment Download PDF

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NL2030349B1
NL2030349B1 NL2030349A NL2030349A NL2030349B1 NL 2030349 B1 NL2030349 B1 NL 2030349B1 NL 2030349 A NL2030349 A NL 2030349A NL 2030349 A NL2030349 A NL 2030349A NL 2030349 B1 NL2030349 B1 NL 2030349B1
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aureus
coli
filter membrane
colonies
antibiotic resistance
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NL2030349A
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Dutch (nl)
Inventor
Li Ruijing
Zhang Haibo
Na Guangshui
Gao Hui
Jin Shuaichen
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Nat Marine Environmental Monitoring Ct
Univ Hainan Tropical Ocean
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/14—Streptococcus; Staphylococcus

Abstract

The present disclosure discloses a method for isolation and screening and evaluation of antibiotic resistance of E. coli and S. aureus in an estuary and a nearshore marine 5 environment. In this method, an MI agar and a VJ agar are immersed with screening antibiotics (such as tetracycline, sulfa and quinolone) to prepare sensitive specific selection agars, and antibiotic-resistant E. coli and S. aureus in the estuary and the nearshore marine environment are quickly isolated and counted to evaluate a resistance level thereof. In the present disclosure, the method for isolation and screening and 10 evaluation of antibiotic resistance of E. coli and S. aureus in an estuary and a nearshore marine environment has simple and easy operations, high accuracy, safety and environmental protection. The method is suitable for rapid analysis and detection of antibiotic resistance levels of the E. coli and the S. aureus in water and sediment samples of coastal oceans, estuaries and sewage outlets into the sea.

Description

METHOD FOR ISOLATION AND SCREENING AND EVALUATION OF
ANTIBIOTIC RESISTANCE OF E. COLI AND S. AUREUS IN ESTUARY AND
NEARSHORE MARINE ENVIRONMENT
S TECHNICAL FIELD
[01] The present disclosure belongs to the technical field of environmental monitoring, and relates to a method for isolation and screening and evaluation of antibiotic resistance of I. coli and S. aureus in an estuary and a nearshore marine environment.
BACKGROUND ART
[02] In the field of environmental scientific research and monitoring, there is still a lack of detection methods for antibiotic resistance levels of typical pathogenic microorganisms £. coli and S. aureus.
[03] In the existing technology, the E. coli in environmental media is counted and detected mainly using a fermentation experiment combined with eosin-methylene blue agar or violet red bile agar; and the S. aureus is counted and detected using a Baird-Parker plate and a blood plate combined with Gram staining experiment. However, due to the cumbersome experimental process, long period and requirement of multiple detection and identification tests such as staining microscopy and plasma coagulase test, the above methods are difficult to truly and effectively reflect the true abundance levels of the above two pathogenic microorganisms in the environmental medium, let alone assess the antibiotic resistance levels of the two pathogenic microorganisms.
SUMMARY
[04] The present disclosure provides a method for isolation and screening and evaluation of antibiotic resistance of £. coli and S. aureus in an estuary and a nearshore marine environment.
[05] In this method, MI agar and VJ agar are immersed with screening antibiotics (such as tetracycline, sulfa and quinolone) to prepare sensitive specific selection agars, and antibiotic-resistant £. coli and S. aureus in the estuary and the nearshore marine environment are quickly isolated and counted to evaluate a resistance level thereof.
[06] The present disclosure employs the following technical solutions.
[07] A method for isolation and screening and evaluation of antibiotic resistance of £.
coli and S. aureus in an estuary and a nearshore marine environment specifically includes the following steps:
[08] (1) sterilization;
[09] before the experiment, putting a filter membrane between filter papers, and autoclaving the filter membrane together with a filter and a petri dish that are wrapped in a kraft paper at 121°C for 15 min, where before filtering different samples each time, a top of a funnel is scrubbed with alcohol and burned with a flame to eliminate interferences between the samples;
[10] (2) preparation of culture media:
[11] S. aureus: accurately weighing 60 g of a VJ medium (BectonDickinson, New
Jersey), adding 1 L of sterile water, heating to dissolve the VJ medium under constant stirring, boiling for 1 min and sterilizing (103 Kpa, 121°C for 15 min); put a sterilized VJ medium into a 45+1°C water bath to cool, and adding 20 ml of (1%) potassium tellurite; dividing a prepared medium into Erlenmeyer flasks, where one part is used as a VJ agar plate for S. aureus screening (without antibiotics), and the other part is immersed with antibiotics at the minimum inhibitory concentration as a VJ-R agar plate for antibiotic resistance screening;
[12] FE. coli: accurately weighing 36.5 g of an MI medium (BectonDickinson, New
Jersey), adding 1 L of sterile water, heating to dissolve the MI medium under constant stirring, boiling for 1 min and sterilizing (103 Kpa, 121°C for 15 min); put a sterilized MI medium into a 45+1°C water bath to cool, preparing and adding cefsulodine sodium to a final concentration of 5 pg/ml; dividing a prepared medium into Erlenmeyer flasks, where one part is used as an MI agar plate for £. coli screening (without antibiotics), and the other part is immersed with antibiotics at the minimum inhibitory concentration as an MI-
R agar plate for antibiotic resistance screening;
[13] (3) antibiotic resistance test:
[14] 3.1, antibiotic resistance test of water samples:
[15] 3.1.1, selecting appropriate an appropriate volume of samples according to an estimation of a sample collection area and a contamination status, and complementing using a 0.1 M phosphate-buffered saline (PBS) with a pH of 6 when an initial volume of a sample is not more than 50 ml, to prepare a 10-fold gradient dilution homogenate, where it takes not more than 15 min from preparation of sample homogenate to completion of sample inoculation;
[16] 3.1.2, selecting 3 serial dilutions of the sample homogenate, filtering a homogenized test solution through a sterile 0.45 um filter membrane, removing the filter membrane and moving the test solution to solidified MI and VJ control petri dishes and solidified MI-R and VJ-R resistant petri dishes, where three parallel groups are made for each dilution;
[17] 3.1.3, diluting a standard E. coli solution without antibiotic resistances and a standard S. aureus solution without antibiotic resistances to a 0.5 McFarland turbidity standard, operating following steps in 3.1.1 and 3.1.2 and filtering through a sterile filter membrane, and moving the above two bacterial solutions to MI and VJ (as a positive control) culture plates and MI-R and VJ-R (as a negative control) culture plates as a quality control group;
[18] 3.1.4, after the sample is filtered each time, weighing 100 ml of ultrapure water or physiological saline with a graduated cylinder, pouring into a funnel to rinse the funnel twice, scrubbing a top of the funnel with alcohol and burning with a flame to eliminate interferences between the samples;
[19] 3.1.5, holding sterile forceps on an edge of the filter membrane, and lifting the filter membrane slightly to move the filter membrane onto an agar plate, to slide the filter membrane onto the agar plate, wherein a rolling action is conducted to avoid bubbles between the filter membrane and the agar at a bottom; if bubbles appear in non-wet areas, the filter membrane is reset; and
[20] 3.1.6, turning over the petri dish and placing in an incubator, and conducting incubation at a constant temperature of 35+0.5°C for 24-36 h; after the incubation, counting blue colonies on the membrane in the MI petri dish under natural light, and counting gray-black round colonies on the membrane in the VJ petri dish under the natural light, and repeating the counting under long-wave ultraviolet (366 nm) to determine results;
[21] 3.2, antibiotic resistance test of sediment samples:
[22] 3.2.1, selecting appropriate weight of samples according to an estimation of a sample collection area and a contamination status, putting weighed sediment samples into a sterile centrifuge tube containing 50 mL of sterile seawater, vortex-shaking for 3 min and standing for 1 min;
[23] 3.2.2, filtering a test solution after standing for 1 min through a sterile 0.45 um filter membrane, removing the filter membrane and moving the test solution to solidified
MI and VJ control petri dishes and solidified MI-R and VJ-R resistant petri dishes, wherein three parallel groups are made for each sample;
[24] 3.2.3, diluting a standard E. coli solution without antibiotic resistances and a standard S. aureus solution without antibiotic resistances to a 0.5 McFarland turbidity standard, operating following steps in 3.1.1 and 3.1.2 and filtering through a sterile filter membrane, and moving the above two bacterial solutions to MI and VJ (as a positive control) culture plates and MI-R and VJ-R (as a negative control) culture plates as a quality control group;
[25] 3.2.4, after the sample is filtered each time, weighing 100 ml of ultrapure water or physiological saline with a graduated cylinder, pouring into a funnel to rinse the funnel twice, scrubbing a top of the funnel with alcohol and burning with a flame to eliminate interferences between the samples;
[26] 3.2.5, holding sterile forceps on an edge of the filter membrane, and lifting the filter membrane slightly to move the filter membrane onto an agar plate, to slide the filter membrane onto the agar plate, wherein a rolling action is conducted to avoid bubbles between the filter membrane and the agar at a bottom; if bubbles appear in non-wet areas, the filter membrane is reset; and
[27] 3.2.6, turning over the petri dish and placing in an incubator, and conducting incubation at a constant temperature of 35+0.5°C for 24-36 h; after the incubation, counting blue colonies on the membrane in the MI petri dish under natural light, and counting gray-black round colonies on the membrane in the VJ petri dish under the natural light, and repeating the counting under long-wave ultraviolet (366 nm) to determine results; and
[28] (4) data analysis and calculation:
[29] calculating abundance and resistance level of the £. coli and the S. aureus in the sample, where the following basic conditions are needed:
[30] 4.1, a total number of the colonies on the plate 1s <200/plate;
[31] 4.2, atarget number of the colonies on the plate is <100/plate (20-80 is the best);
[32] 4.3, a calculation formula for the abundance of the £. coli and the S. aureus is as follows:
[33] E.coli (S.aureus)/ 100ml =
Number of blue colonies (black colonies)
Ere 100
Number of blue colonies (black colonies) , on a culture plate
[34] E.coli (S.aureus)/g = ee E
[35] 4.4, a calculation formula for the antibiotic resistance rate of the £. coli and the
S. aureus is as follows:
Number of blue colonies (black colonies)
[36] Antibiotic resistance rate % = a oF Solan Ey x 100%. on a control agar plate 5 [37] When collecting samples, sampling tools and related reagent materials should be prepared and sterilized in advance. The water and the sediment samples are collected in a sterile leak-proof polypropylene sample container, refrigerated at 1-4°C and sent back to the laboratory for analysis as soon as possible. The samples should be analyzed within 12 h after collection, and the analysis of a single sample should not exceed 15 min.
[38] Instep 3.1.1, the initial volume selection is as follows: the estuary and the sewage outlet into the sea: 10 ml; the coastal ocean: 300-500 ml.
[39] In step 3.2.1, the initial sample mass selection is as follows: the estuary and the sewage outlet into the sea: 0.1-0.3 g; the coastal ocean: 0.3-0.5 g.
[40] In the MI medium, the E. coli in water is detected using a specific enzyme reaction. The medium includes two enzyme substrates: MUGal (fluorescence) and IBDG (color). Coliform bacteria can produce B-galactosidase to react with the MUGal, to generate a compound that emits fluorescence under the long-wave ultraviolet (366 nm); the E. coli can produce B-glucosidase to react with the IBDG, to produce a blue compound, such that the colony is blue under natural light. A certain concentration of the cefsulodine sodium is added to the medium to inhibit the growth of gram-positive bacteria and other gram-negative bacteria that belong to non-coliform bacteria. In addition, antibiotic- resistant microorganisms are selected by immersing antibiotics at the minimum inhibitory concentration in the medium.
[41] Inthe Vogel-Johnson (VJ) medium, due to the reducing capacity to tellurium and the adaptability to potassium chloride of the S. aureus, the growth of gram-negative bacteria and other bacteria are inhibited by the potassium chloride and the potassium tellurite in the agar medium. In addition, sodium pyruvate is added to repair damaged cells to stimulate the growth of S. aureus, to further increase the detection rate. The 5. aureus can reduce the tellurium to make the colony gray-black.
[42] The present disclosure has the following beneficial effects.
[43] In the present disclosure, the method for isolation and screening and evaluation of antibiotic resistance of £. coli and S. aureus in an estuary and a nearshore marine environment has simple and easy operations, high accuracy, safety and environmental protection. The method is suitable for rapid analysis and detection of antibiotic resistance levels of the £. coli and the S. aureus in water and sediment samples of coastal oceans, estuaries and sewage outlets into the sea.
[44] Although the embodiments of the present disclosure have been illustrated and described, it should be understood that those of ordinary skill in the art may make various changes, modifications, replacements, and variations to the above embodiments without departing from the principle and spirit of the present disclosure, and the scope of the present disclosure is limited by the appended claims and their legal equivalents.

Claims (4)

ConclusiesConclusions 1. Werkwijze voor isolatie en screening en evaluatie van antibioticaresistentie van1. Method for isolation and screening and evaluation of antibiotic resistance of L. coli en S. aureus in een riviermond en een zeeomgeving nabij de kust, die specifiek de volgende stappen omvat: (1) sterilisatie; (2) bereiding van kweekmedia; (3) antibioticaresistentietest:L. coli and S. aureus in an estuary and near-coastal marine environment, which specifically includes the following steps: (1) sterilization; (2) preparation of culture media; (3) antibiotic resistance test: 3.1 antibioticaresistentietest van watermonsters:3.1 Antibiotic Resistance Test of Water Samples: 3.1.1 het selecteren van een geschikt volume monsters, en het aanvullen met behulp van een 0,1M fosfaatgebufferde zoutoplossing (PBS) met een pH van 6 wanneer een initieel volume van een monster niet meer dan 50 mL is, om een verdunningshomogenaat met 10-voudige gradiënt te bereiden, waarbij het niet meer dan min duurt vanaf bereiding van een monsterhomogenaat tot voltooiing van een 15 monsterinoculatie;3.1.1 Selecting an appropriate volume of samples, and supplementing with 0.1M Phosphate Buffered Saline (PBS) with a pH of 6 when an initial volume of a sample is not more than 50 mL, to obtain a dilution homogenate with 10 -fold gradient, taking no more than 10 minutes from preparation of a sample homogenate to completion of a sample inoculation; 3.1.2 het selecteren van 3 seriële verdunning van het monsterhomogenaat, het filteren van een gehomogeniseerde testoplossing door een 0,45 um steriel filtermembraan, het verwijderen van het membraan en het overbrengen van de testoplossing naar uitgeharde MI- en VJ-controlepetrischalen en uitgeharde MI-R- en VJ-R-resistente petrischalen, waarbij voor elke verdunning drie parallelle groepen gemaakt worden;3.1.2 selecting 3 serial dilution of the sample homogenate, filtering a homogenized test solution through a 0.45 µm sterile filter membrane, removing the membrane and transferring the test solution to cured MI and VJ control petri dishes and cured MI -R and VJ-R resistant petri dishes, making three parallel groups for each dilution; 3.1.3 het verdunnen van een standaard £. coli-oplossing met antibioticaresistenties en een standaard S. aureus-oplossing zonder antibioticaresistenties tot een 0,5 McFarland- troebelheidsstandaard, het handelen volgens stappen in 3.1.1 en 3.1.2 en het filteren door een steriel filtermembraan, en het overbrengen van de bovenstaande twee bacteriële oplossingen naar MI- en VJ-kweekplaten (als positieve controle) en MI-R- en VJ-R- kweekplaten (als een negatieve controle) als een kwaliteitscontrolegroep;3.1.3 diluting a standard £. coli solution with antibiotic resistances and a standard S. aureus solution without antibiotic resistances to a 0.5 McFarland turbidity standard, proceeding according to steps in 3.1.1 and 3.1.2 and filtering through a sterile filter membrane, and transferring the above two bacterial solutions to MI and VJ culture plates (as a positive control) and MI-R and VJ-R culture plates (as a negative control) as a quality control group; 3.1.4 nadat het monster elke keer gefilterd is, het meten van 100 ml ultrazuiver water of fysiologische zoutoplossing met een maatcilinder, het gieten in een trechter om de trechter tweemaal te spoelen, het wassen van een bovenkant van de trechter met alcohol en het met een vlam branden om verstoringen tussen de monsters te elimineren;3.1.4 after filtering the sample each time, measuring 100ml of ultrapure water or physiological saline with a graduated cylinder, pouring into a funnel to rinse the funnel twice, washing a top of the funnel with alcohol, and wiping with burn a flame to eliminate disturbances between the monsters; 3.1.5 het houden van een steriele pincet op een rand van het filtermembraan, en het optillen van het filtermembraan om het filtermembraan naar een agarplaat te verplaatsen, om het filtermembraan op de agarplaat te schuiven, waarbij een rolhandeling uitgevoerd wordt om bubbels tussen het filtermembraan en de agar op een bodem te vermijden; waarbij het filter opnieuw geplaatst wordt als er bubbels verschijnen in niet-natte gebieden; en3.1.5 Holding sterile tweezers on an edge of the filter membrane, and lifting the filter membrane to move the filter membrane to an agar plate, slide the filter membrane onto the agar plate, performing a rolling action to create bubbles between the filter membrane and avoid the agar on a bottom; repositioning the filter if bubbles appear in non-wet areas; and 3.1.6 het omkeren van de petrischaal en het plaatsen ervan in een incubator, en het uitvoeren van incubatie bij een constante temperatuur van 35 + 0,5 °C gedurende 24 — 36 uur; na de incubatie, het tellen van blauwe kolonies op het membraan in de MI- petrischaal onder natuurlijk licht, en het tellen van grijszwarte ronde kolonies op het membraan in de VJ-petrischaal onder het natuurlijke licht, en het herhalen van de telling onder langegolflengte-ultraviolet licht (366 nm) om resultaten te bepalen;3.1.6 inverting the petri dish and placing it in an incubator, and incubating at a constant temperature of 35 + 0.5 °C for 24 — 36 hours; after the incubation, count blue colonies on the membrane in the MI Petri dish under natural light, and count gray-black round colonies on the membrane in the VJ Petri dish under the natural light, and repeat the count under long-wavelength ultraviolet light (366 nm) to determine results; 3.2 antibioticaresistentietest van sedimentmonsters:3.2 Antibiotic Resistance Test of Sediment Samples: 3.1.2 het selecteren van een geschikt gewicht van monsters volgens een schatting van een monsterverzamelingsgebied en een contaminatiestatus, het plaatsen van gewogen sedimentmonsters in een steriele centrifugebuis die 50 mL steriel zeewater bevat, het vortexschudden gedurende 3 min en het staan gedurende 1 min;3.1.2 selecting an appropriate weight of samples according to an estimate of a sample collection area and a contamination status, placing weighed sediment samples in a sterile centrifuge tube containing 50 mL of sterile sea water, vortexing for 3 minutes and standing for 1 minute; 3.2.2 het filteren van een testoplossing na het staan gedurende 1 min door een steriel 0,45 um filtermembraan, het verwijderen van het membraan en het overbrengen van de testoplossing naar uitgeharde MI- en VJ-controlepetrischalen en uitgeharde MI-R- en VJ-R-resistente petrischalen, waarbij voor elke monster drie parallelle groepen gemaakt worden;3.2.2 Filtering a test solution after standing for 1 min through a sterile 0.45 µm filter membrane, removing the membrane and transferring the test solution to cured MI and VJ control Petri dishes and cured MI-R and VJ R-resistant petri dishes, making three parallel groups for each sample; 3.2.3 het verdunnen van een standaard E. coli-oplossing met antibioticaresistenties en een standaard S. aureus-oplossing zonder antibioticaresistenties tot een 0,5 McFarland- troebelheidsstandaard, het handelen volgens stappen in 3.1.1 en 3.1.2 en het filteren door een steriel filtermembraan, en het overbrengen van de bovenstaande twee bacteriële oplossingen naar MI- en VJ-kweekplaten (als positieve controle) en MI-R- en VJ-R- kweekplaten (als een negatieve controle) als een kwaliteitscontrolegroep;3.2.3 diluting a standard E. coli solution with antibiotic resistances and a standard S. aureus solution without antibiotic resistances to a 0.5 McFarland turbidity standard, proceeding according to steps in 3.1.1 and 3.1.2 and filtering through a sterile filter membrane, and transferring the above two bacterial solutions to MI and VJ culture plates (as a positive control) and MI-R and VJ-R culture plates (as a negative control) as a quality control group; 3.2.4 nadat het monster elke keer gefilterd is, het meten van 100 ml ultrazuiver water of fysiologische zoutoplossing met een maatcilinder, het gieten in een trechter om de trechter tweemaal te spoelen, het wassen van een bovenkant van de trechter met alcohol en het met een vlam branden om verstoringen tussen de monsters te elimineren;3.2.4 after filtering the sample each time, measuring 100ml of ultrapure water or physiological saline with a graduated cylinder, pouring into a funnel to rinse the funnel twice, washing a top of the funnel with alcohol, and burn a flame to eliminate disturbances between the monsters; 3.2.5 het houden van een steriele pincet op een rand van het filtermembraan, en het optillen van het filtermembraan om het filtermembraan naar een agarplaat te verplaatsen, om het filtermembraan op de agarplaat te schuiven, waarbij een rolhandeling uitgevoerd wordt om bubbels tussen het filtermembraan en de agar op een bodem te vermijden;3.2.5 holding sterile tweezers on one edge of the filter membrane, and lifting the filter membrane to transfer the filter membrane to an agar plate, slide the filter membrane onto the agar plate, performing a rolling action to create bubbles between the filter membrane and avoid the agar on a bottom; waarbij het filter opnieuw geplaatst wordt als er bubbels verschijnen in niet-natte gebieden; enrepositioning the filter if bubbles appear in non-wet areas; and 3.2.6 het omkeren van de petrischaal en het plaatsen ervan in een incubator, en het uitvoeren van incubatie bij een constante temperatuur van 35 + 0,5 °C gedurende 24 — 36 uur; na de incubatie, het tellen van blauwe kolonies op het membraan in de MI- petrischaal onder natuurlijk licht, en het tellen van grijszwarte ronde kolonies op het membraan in de VJ-petrischaal onder het natuurlijke licht, en het herhalen van de telling onder langegolflengte-ultraviolet licht (366 nm) om resultaten te bepalen; en (4) data-analyse en berekening: het berekenen van een abondantie en resistentieniveau van de £. coli en de S. aureus in het monster, waarbij de volgende basisomstandigheden nodig zijn:3.2.6 inverting the petri dish and placing it in an incubator, and incubating at a constant temperature of 35 + 0.5 °C for 24 — 36 hours; after the incubation, count blue colonies on the membrane in the MI Petri dish under natural light, and count gray-black round colonies on the membrane in the VJ Petri dish under the natural light, and repeat the count under long-wavelength ultraviolet light (366 nm) to determine results; and (4) data analysis and calculation: calculating an abundance and resistance level of the £. coli and the S. aureus in the sample, requiring the following basic conditions: 4.1 een totaal aantal van de kolonies op de plaat is < 200/plaat; 42 een doelaantal van de kolonies op de plaat is < 100/plaat (20 — 80 is het beste);4.1 a total number of colonies on the plate is < 200/plate; 42 a target number of colonies on the plate is < 100/plate (20 - 80 is best); 4.3 een berekeningsformule voor de abondantie van de £. coli en de S. aureus is als volgt: Aantal blauwe kolonies (zwarte kolonies)4.3 a calculation formula for the abundance of the £. coli and the S. aureus is as follows: Number of blue colonies (black colonies) E. coli (S. aureus) / 100ml = ee x 100; Aantal blauwe kolonies (zwarte kolonies)E. coli (S. aureus) / 100ml = ee x 100; Number of blue colonies (black colonies) E. coli (S. aureus) / g= I x 100; 44 een berekeningsformule voor de antibioticaresistentiemate van de £. coli en deE. coli (S. aureus) / g = I x 100; 44 a calculation formula for the antibiotic resistance rate of the £. coli and the S. aureus als volgt 1s: Aantal blauwe kolonies (zwarte kolonies) Antibioticaresistentiemate % = een screenig ela x 100; op een controleagarplaatS. aureus as follows 1s: Number of blue colonies (black colonies) Antibiotic resistance rate % = a screenig ela x 100; on a control agar plate
NL2030349A 2021-12-29 2021-12-29 Method for isolation and screening and evaluation of antibiotic resistance of e. coli and s. aureus in estuary and nearshore marine environment NL2030349B1 (en)

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