• Title, Summary, Keyword: Microtiter plate assay

Search Result 21, Processing Time 0.039 seconds

Biofilm Formation of Food-borne Pathogens under Stresses of Food Preservation (식품 보존 스트레스에서의 식중독세균의 생체막 생성)

  • Lee, No-A;Noh, Bong-Soo;Park, Jong-Hyun
    • Korean Journal of Food Science and Technology
    • /
    • v.38 no.1
    • /
    • pp.135-139
    • /
    • 2006
  • Most bacteria form biofilm as self-defence system, making efficient food sanitization, preservation, and instrument washing more difficult. Biofilm formation of Salmonella, E. coli, B. cereus, and S. aureus was observed during 24 hr food preservations by performing microtiter plate and glass wool assays. Most cells formed biofilm and attached onto glass wool. When biofilm formation and injury were analyzed on the microtiter plate, 10 and 20% acid-injured E. coli and S. aureus, respectively, 30-50% cold temperature $(4^{\circ}C)-injured$ B. cereus and E. coli, and 30-55% 6% sodium chloride solution-injured Salmonella showed significant biofilm formation. Results indicate biofilm formation level differed within species depending on type of stress.

Detection of the cell wall-affecting antibiotics at sublethal concentrations using a reporter Staphylococcus aureus harboring drp35 promoter - lacZ transcriptional fusion

  • Mondal, Rajkrishna;Chanda, Palas K.;Bandhu, Amitava;Jana, Biswanath;Lee, Chia-Y.;Sau, Subrata
    • BMB Reports
    • /
    • v.43 no.7
    • /
    • pp.468-473
    • /
    • 2010
  • Previously, various inhibitors of cell wall synthesis induced the drp35 gene of Staphylococcus aureus efficiently. To determine whether drp35 could be exploited in antistaphylococcal drug discovery, we cloned the promoter of drp35 ($P_d$) and developed different biological assay systems using an engineered S. aureus strain that harbors a chromosomally-integrated $P_d$ - lacZ transcriptional fusion. An agarose-based assay showed that $P_d$ is induced not only by the cell wall-affecting antibiotics but also by rifampicin and ciprofloxacin. In contrast, a liquid medium-based assay revealed the induction of $P_d$ specifically by the cell wall-affecting antibiotics. Induction of $P_d$ by sublethal concentrations of cell wall-affecting antibiotics was even assessable in a microtiter plate assay format, indicating that this assay system could be potentially used for high-throughput screening of new cell wall-inhibiting compounds.

Development of assay method for the activities of new compounds, and the effect of several fungicides against spore germination, adhesion, and myceial growth of Colletotrichum sp. causing red pepper anthracnose (고추 탄저병균의 포자 발아와 부착, 균사 생장에 미치는 화합물의 활성 검정법 확립 및 살균제의 효과)

  • Kim, Jae-Jeung;Kim, Joon-Tae;Park, Sung-Woo;Park, Eun-Suk;Kim, Heung-Tae
    • The Korean Journal of Pesticide Science
    • /
    • v.7 no.3
    • /
    • pp.159-168
    • /
    • 2003
  • With microtiter plate, the assay method was developed for detecting the fungicidal activity of new compounds against spore germination, spore adhesion and mycelial growth of Colletotrichum sp. JC24 cal1Sing red pepper anthracnose. Also, the effects of some commercialized fungicides on fungal development like above mentioned were investigated by measuring the optical density of mycelia grown into wells of microtiter plate. For the standardization of assay method, some factors, such as the treatment of MTT and/or propanol, inodulum density and incubation period, affecting on mycelial optical density were investigated. For obtaining precise and consistent mycelial optical density, it was necessary the treatment of MTT for 12 hrs and propanol for 1 hr. inoculum density adjusted to $1\times10^5$ spores/mL and incubation period for 36 hrs at $25^{\circ}C$. For fungicidal activities, 6 protective fungicides, 6 ones inhibiting sterol biosynthesis, and one inhibiting respiration were used in this study. While mancozeb, chlorothalonil and dithianon among 6 protective fungicides inhibited strongly spore germination, adhesion, and mycelial growth at $6.25{\mu}g/mL$, propineb, iminoctadine and fluazinam inhibited intermediately spore germination and mycelial growth at $100{\mu}g/mL$. Washing above 3 fungicides with new PD broth, their activity against spore adhesion decreased. With hexaconazole, tebuconazole and myclobutanil, the tendency of the activity against fungal differentiation of the early infection stage was similar to the latter group of protective fungicides, showing the decrease of the inhibitory activity against spore adhesion by washing 2 hrs after incubation. However, kresoxim-methyl inhibited spore adhesion distinctly, depending on the applied concentrations. Based on these results, it might be able to assess the fungicidal activity of many compounds against spore germination, adhesion and mycelial growth by the use of microtiter plate in vitro. Using the assay developed in this report, it was possible to investigate the inhibitory activity of some commercialized fungicides, too.

Enzyme-linked immunosorbent assay for detection of bovine antibody to Brucella abortus (축우 부루셀라병의 ELISA 진단법에 관한 연구)

  • Lim, Yoon-kyu;Lee, Doo-sick;Park, Jun-hong;Yang, Ki-chun;Kim, Seung-ho;Kim, Kong-sick;Hyun, Kwan-jong;Kim, Woo-tack;Lee, Yong-soon
    • Korean Journal of Veterinary Research
    • /
    • v.33 no.1
    • /
    • pp.131-135
    • /
    • 1993
  • Enzyme-linked Immuno sorbent Assay (ELISA) for the serological diagnosis of Brucella abortus was developed and compared with plate agglutination test. Cell wall antigen was extracted from Brucella abortus 1119-3 by sonication and with a sodium deoxychlate solution. Optimum protein concentration of coating antigen were $0.4{\mu}g/100{\mu}{\ell}$ protein on each microtiter plate well. Horse radish peroxidase (HRP) labeled protein-G was used as a tracer of reacted antibodies. ELISA confirmed the agreeable results of 40 cases out of 43 cases by plate aggulutination test. ELISA diagnosed positive cases(10 out of 12) and negative cases (1 out of 12) with dubious sera by plate agglutination test. From this results ELISA could be used for the early diagnostic tools of Brucellosis in cattle.

  • PDF

Selection of Fungicides for the Control of Soybean Black Root Rot Caused by Calonectria ilicicola (콩 검은뿌리썩음병 방제를 위한 살균제 선발)

  • Park, Seong-Woo;Kang, Beom-Kwan;Kim, Hong-Sik;Woo, Sun-Hee;Kim, Heung-Tae
    • The Korean Journal of Pesticide Science
    • /
    • v.11 no.1
    • /
    • pp.18-26
    • /
    • 2007
  • Fungicidal screening was performed to control soybean black root rot caused by Calonectria ilicicola through in vitro and greenhouse assays. In in vitro assay, 25 fungicides were assessed by an agar dilution method and a 96-well microtiter plate method. While protective fungicides including dithianon, dichlofluanid, mancozeb, and captan showed a very low activity against the mycelial growth C. ilicicola SC03-15 in the agar dilution method, they displayed potent inhibitory activity against spore germination in a 96-well microtiter plate method with $EC_{50}$ values of 4.65, 0.61, 4.64, and $0.29{\mu}g\;mL^{-1}$, respectively. Ergosterol biosynthesis-inhibiting (EBI) fungicides showed different antifungal activity against mycelial growth and spore germination according to molecules. Difenconazole displayed higher antifungal activity against spore germination rather than mycelial growth, and prochloraz inhibited potently both mycelial growth and spore germination with EC50 values less than $1.8{\mu}g\;ml^{-1}$. In contrast, the other EBI fungicides inhibited more highly mycelial growth than spore germination. Carbendazim+diethofencarb and dazomet also inhibited both mycelial growth and spore germination of C. ilicicola SC03-15 at very low concentrations. In greenhouse assay, carbendazim+diethofencarb effectively controlled a soybean black root rot by drenching 2 days before or after inoculation. In addition, tebuconazole showed potent curative activity against soybean black root rot.

Screening of Lactic Acid Bacteria with Potent Adhesive Property in Human Colon using Colonic Mucin-binding Assay (Colonic mucin-binding assay를 이용한 장내 우수 점착능 유산균주의 선별)

  • Kim, Seong-Yeong;Shin, Kwang-Soon;Lee, Ho
    • Korean Journal of Food Science and Technology
    • /
    • v.36 no.6
    • /
    • pp.959-967
    • /
    • 2004
  • To screen probiotic lactic acid bacteria with potent adhesive property on human colonic mucosa, colonic mucin-binding assay was introduced. This colonic mucin-binding assay actually measures the binding activity of surface lectin-like protein (SLP) on colonic mucin, and the optimal conditions were examined. The optimal pH for colonic mucin coating on plate wells was 4.8, and ${\times}24,000$ diluted solution of commercially available horseradish peroxidase (HRP) conjugated streptoavidin yielded good results, for rapid screening, $5.0\;{\mu}g/mL$ of biotinylated SLP from lactic acid bacteria was optimal, and optimal scintillation time of 3,3',5,5'-tetramethyl benzidine (TMB) was 10 min. These conditions were useful for both rapid selection and quantitative analysis of lactic acid bacteria that have high adhesion property to human intestinal tract. Among 50 strains of lactic acid bacteria, including 32 type culture strains and 18 isolated strains from infant feces, Lactobacillus species FSB-1 isolated from kimchi showed the highest binding activity to colonic mucin. From taxonomical viewpoints based on morphological study, physico-biochemical study, partial 16S rDNA seguencing, and phylogenetic analysis, L. species FSB-1 was identified as Lactobacillus brevis.

Studies on the Rapid and Competitive Enzyme-linked Immunosorbent Assay for the Detection of Thyroxine (T$_4$) in Human Sera (경쟁적 효소면역측정법을 이용한 환자 혈청 내 Thyroxine (T$_4$)의 검출)

  • Sang-Wook Park;Jong-Bae Kim
    • Biomedical Science Letters
    • /
    • v.5 no.1
    • /
    • pp.11-15
    • /
    • 1999
  • Thyroxine (3,5,3',5'-L-tetraiodothyronine; T$_4$) is the most commonly measured thyroid hermono for the diagnosis of various thyroid disorders. Although radioimmunoassay (RIA) is still considered as the reference technique for the measurement of T$_4$, it is generally regarded that RIA has its primary disadventages in handling the wastes and controling the human and material resources. Therefore, establishment of enzyme-linked immunosorbent assay (ELISA) has of great significance. To verify the usefulness of our enzyme immunoassay, we have obtained the standard dose response curve of T$_4$ in patient's sera which is inversely proportional to the amount of herseradish peroxidase (HRP) conjugated monoclonal antibody of T$_4$ bound to the wells. The correlation coefficient (r) between the ELISA and chemiluminescent assay was 0.444 (n=38). Thus we have investigated the establishment of rapid and sensitive competitive ELISA assay method for detection of T$_4$ in patient's sera.

  • PDF

Detection of Antistaphylococcal and Toxic Compounds by Biological Assay Systems Developed with a Reporter Staphylococcus aureus Strain Harboring a Heat Inducible Promoter - lacZ Transcriptional Fusion

  • Chanda, Palas Kumar;Ganguly, Tridib;Das, Malabika;Lee, Chia Yen;Luong, Thanh T.;Sau, Subrata
    • BMB Reports
    • /
    • v.40 no.6
    • /
    • pp.936-943
    • /
    • 2007
  • Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cellwall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region ($P_g$) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the $P_g$-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that $P_g$ in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced $P_g$ efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.

Measurement of the Affinity Constant of Monoclonal Antibody to Human Apolipoprotein A-I by ELISA (효소면역 분석법에 의한 아포지단백질 A-I 단일클론항체의 친화상수의 측정)

  • Mic Hung Yoon;Hyun Hee Lee
    • Biomedical Science Letters
    • /
    • v.1 no.1
    • /
    • pp.1-8
    • /
    • 1995
  • The present study was undertaken to determine the dissociation constant (Kd)of monoclonal antibody to human apolipoprotein A-I (apo A-I) using enzyme-linked immunosorbent assay (ELISA). First the monoclonal antibody was incubated in solution with the antigen until the equilibrium was reached; then the free antibody which remains unsaturated at equilibrium was captured by binding to antigen on the microtiter plate and be measured by a classical indirect ELISA. The value of Kd determined from Scatchard plot was 0.625$\times$10^{-9}$ for purified antibody and 0.720$\times$10$^{-9}$ for unpurified antibody. This method was valuable for the measurement of true dissociation constant and found to be simple, reproducible, and accurate.

  • PDF