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میکروبیولوژی دانشگاه کرج
 Materials and Methods
This three-part experiment has three objectives: the Isolation of an antibiotic-producing organism from soil, determining the number of colony forming units (CFU's) per gram of soil sample by using the following techniques:
• Aseptic technique
• Dilution series and plating
• Plate counting
• Streak plating
• Two assay methods
• Simple staining (an possibly complex staining)
• Oil immersion microscope

Part 1: Determine the number of colony forming units (CFU's) of the genus Streptomyces per gram of soil

Materials
A - DRY SOIL SAMPLES
Three sandwich bags of soild was allowed to dry at room temperature for about a week (or for less time in a drying oven) to kill off some of the many bacteria in soil that compete with those of the genus Streptomyces. Streptomyces will form spores in the dry soil and will grow on media.
B - DILUTION PLATING
Four dilution blanks were labled 1-4. One (1) gram of soil was placed into test tube #1 and mixed well. (The volume of soil solution should be 10 ml., if it wasn't more sterile water was added to a line that marks 10 ml on the test tube). A sterile pipette was used to transfer 1 ml of soil solution from tube #1 to blank tube #2 and mixed well. A new sterile pipette was then used to transfer 1 ml of soil solution from tube #2 to blank tube #3 and mixed well. A new sterile pipette was used for each transfer between test tubes, and for the last tube 1 ml of soil solution from tube #3 to blank tube #4 and again mixed well.
A marking pen was used to lable 3 separate Agar plates with the numbers 1,2,3 and 4 to correspond to the test tubes numbered #1-4. Starting with tube 4, 0.1 ml (100 microliters) of soil solution was placed onto the half-plate labeled 4. The sample was immediately spread with flamed loop or glass spreader accross the entire agar plate.
The same pipette was used to continue transfers from tube #3 to half-plate #3, and then continued with tubes #2 and #1 to half-plates #2 and #1, respectively. The plates were incubated at room temperature for 2 days at 30 degrees celcius in the dark to mimic natural conditions of the soil.

Part 2: Identification and pure culture of Streptomyces
After two days of growth, the agar plates were observed. Streptomyces are round, chalky colonies with different species appearing white, greenish brown, gray, pink or other colors.
Using a flamed loop, well-isolated colonies of Streptomyces were carefully scraped off the plates and streaked onto a plate of transfer media to create a pure culture. The plates were incubated at room temperature for 2 days at 30 degrees celcius in the dark to mimic natural conditions of the soil.

Staining
A simple stain of a selected colony was performed to observe the microscopic characteristics of Streptomyces. Using a flamed loop, part of a colony was scraped onto a drop of water on a slide and allowed to dry. The slide washeat fixed by gently passing the slide thru the flame 1 or 2 times until it is warm to the touch. The slide was then flooded with crystal violet for 1 min, then gently rinsed and blotted dry. The resulting slide was viewed under an oil immersion lens and characteristics noted.

Part 3: Assay
Materials
Pure cultures of test organisms (on solid or liquid media) were obtained for testing the antibiotic properties of Streptomyces that had been isolated. These were other organisms from the soil, any other culture such as those of the genus Bacillus, Micrococcus, etc. Procedure
A single streak of Streptomyces was made (from your pure culture) down the middle of a plate of assay media (same as transfer media and incubated for 2 days to allow for any antibiotics to be produced. A single streak of each test organism was then placed perpendicular to the Streptomyces streak. It was possible to place 4 or five test organisms perpendicular to one Streptomyces streak. The plates were then incubated for 24 hr at 30 degrees C. The inhibition by Streptomyces of the bacteria was then observed and recorded.


Media Recipes
Isolation Medium
0.4 g Casein
1.0 g Starch
0.5 g potassium nitrate
0.2 g potassium monohydrogen phophate
0.1g magnesium phosphate
0.1 g calcium carbonate
15 g agar
Sterilized distilled water to final volume of 1 liter (Sterilize 15 psi for 15 minutes. Just before pouring plates, add 1 ml cycloheximide, if available, to inhibit fungal growth)
NOTE: Pour plates fairly thick so they don't dry out . (about 45 plates per liter)
Transfer (growth) medium
10 g glucose
1 g yeast extract
1 g potassium nitrate
0.1 g potassium monohydrogen phosphate
15 g Agar
Sterilized distilled water to final volume of 1 liter (Sterilize 15 psi for 15 minutes. Just before pouring plates, add 1 ml cycloheximide, if available, to inhibit fungal growth)
Assay agar
Transfer medium may be used, or use the following recipe.
10 g peptone
1 g glucose
15 g agar
Sterilized distilled water to final volume of 1 liter (Sterilize 15 psi for 15 minutes. Just before pouring plates, add 1 ml cycloheximide, if available, to inhibit fungal growth)

Credits
This particular protocol was developed by Dr. Jerry Ensign, Emeritus Professor, Bacteriology Dept., University of Wisconsin-Madison.
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 The isolation of the antibiotic Streptomyces bacteria from the soil
The isolation of the antibiotic Streptomyces bacteria from the soil
Colleen Dunford and Willow Malick
Microbiology 342 Lab
Abstract
Streptomyces sp. are gram positive bacteria of the order Actinobacteria known for their production of bioactive metabilites that often have antibiotic properties. Strepomyces sp. were cultured from soil samples collected during the winter months in Anchorage Alaska. The bacteria were isolated through the use of specialized media and isolation streak procedures. The resulting pure cultures were tested for antibiotic properties through the use of streak plating with Bacillus cereus and Micrococcus luteus. A Streptomyces sp. Was isolated that produced a zone of inhibition when tested against M. luteus, while B. cereus was not inhibited by the Streptomyces cultures.


Introduction
Streptomyces is a genus of Actinobacteria, a group of Gram-positive bacteria and are found predominantly in soil and in decaying vegetation1,3. Commonly producing spores, the bacterium is noted for their "earthy" odor. They are also unique amongst bacteria in their mycelial, sporulating life cycle, which involves complex regulation of gene expression in space and time3,5.
None of the Streptomycetes are pathogenic but many are medically significant because of the antibiotics that they produce. To date the majority of these antibiotics are applied in human and veterinary medicine and agriculture, as well as anti-parasitic agents, herbicides, pharmacologically active metabolites (e.g. immuno-suppressants) and several enzymes important in the food and other industries1,2.
The purpose of this investigation is to isolate antibiotic-producing bacteria (genus Streptomyces) from local soil samples. This experiment replicates the first step in the search and screen research that is currently underway in many labs worldwide4.





Materials

Two Alaskan soil samples were obtained (approximately ½ cup each), for the isolation of Streptomyces. Each was from a residential garden but of different locations. Frozen, wet samples were air dried and then kept in refrigeration until they were required for use.
Isolation media required to isolate the organism consisted of agar, dextrose, soybean meal, NaCL, yeast extract, CaCO3, glycerol, and cycloheximide5. Exact measurements for all media can be found at the end of the materials section. The dry ingredients measured, distilled water was added (to bring total volume to 500ml) and heated to a rapid boil5. Once cooled the mixture was autoclaved and then poured into petri plates. Transfer (growth) medium consisting of dextrose, yeast extract, potassium nitrate, potassium monohydrogen phosphate, agar and cycloheximide5, were prepared and poured into petri plates as previously outlined.
Both Serial dilutions and dilution plating each required 1gram (g) of sample soil and each set of serial dilutions required four standard test tubes labeled 1-4. Test tube blanks were prepared with 4.5ml of distilled water.
Gram staining of select colonies employed the use of standard gram stain chemicals and dyes3, 4. Test bacteria, Bacillus.cereus and Micrococcus luteus were pure broth cultures (non-pathogenic) used in the Assay.

Isolation Media
Agar 7.5g
Dextrose 15.0g
Soybean meal 7.5g
NaCl 2.5g
Yeast extract 0.5g
CaCO3 0.5g
Glycerol 1.25g
Cycloheximide 0.5ml

Transfer (Growth) Media
Agar 7.5g
Yeast extract 0.5g
Potassium nitrate 0.5g
Potassium monhydrogen phosphate 0.05g
Cycloheximide 0.5ml
Dextrose 5.0g


Methods

Soil samples were allowed to dry for a week at room temperature. By drying the soil, other competing bacteria were killed off, and the remaining spore -forming Streptomyces were enabled to grow on the provided media.
In the preparation of the isolation and growth media, the addition of cycloheximide was important to inhibit fungal growth. The medias were both adjusted to a pH of 6.8 +/- 0.2 @ 25oC before being autoclaved.
The medias prepared, two serial dilutions, (one for each soil sample) were performed as follows. Labeling four test tubes 1-4, 1 gram of soil was transferred into test tube #1 and enough distilled water added to bring the volume to 5ml. Test tubes 2-4 were established as blanks by delivering 4.5ml of distilled water to each. The contents of test tube #1 mixed, 0.5ml of the soil solution was transferred via pipette to test tube #2 making a dilution factor of 10-1. The procedure carried out to test tube #4 to a dilution factor 10-3. Individual aseptic 0.1ml transfers of diluted soil solutions from test tubes #2, #3 and #4 were plated to three petri plates of isolation media resulting in the respective dilution plates of 10-2, 10-3 and 10-4.
Immediately after the transfers, each soil solution was spread aseptically with a glass spreader to completely cover the plate. Labeled with the appropriate dilution factor, the plates were incubated at 35oC for 4-7 days. Plates were incubated in the dark to mimic natural conditions of the soil.
After the incubation period, plates were observed and colonies counted. Viable colonies of Streptomyces identified (30-300), colonies were then aseptically transferred with a loop and an isolation streak made onto a plate of transfer media. Incubated once again in the dark at 35oC for 3-5 days, plates were then qualitatively observed and noted.
A gram stain of selected colonies was made. The organism viewed under the oil immersion lens of a microscope, observations were noted and recorded.
To test for the production of the secondary metabolite, a single streak of Streptomyces (from the isolation plate) was made down the middle of a plate of transfer media. Two plates were prepared in this manner and incubated at 35oC for 3-5 days. After such time, a single streak of each test organism (Bacillus.cereus and Micrococcus luteus) were made perpendicular to the Streptomyces streak plates and incubated 2-3 days at 35oC . Observation of inhibition by Streptomyces was then made.
Results

’er 300) for either soil sample. However, isolation plates of df 10-4 had the respective counts; soil sample A = 36, and soil sample B = 32. See table one for calculated CFU’s of the original soil samples.

Table one: Calculated CFU’s
Plate count Final Dilution Viable no. in original culture (CFU/ml)
Soil sample A = 36 1: 104 36 x 104 = 3.6 x 105
Soil sample B = 32 1: 104 32 x 104 = 3.2 x 105

Distinct colonies were observed on both dilution plates of df 10-4. Different species were observed as white, orangish pink and brown, where as the Streptomyces were round, chalky cream- white, colored colonies with a “fuzzy” edge. Once pure colonies were grown (3 days) on streak isolation plates, staining revealed gram positive, branching, filamentous arrangement of cells (mycelium) from exhibits A and B. when examining the organism under the oil immersion lens, spores were observed from the organism stained from soil sample A, but were not observed from sample B. Important to note was the distinct “earthy” smell from both isolation plates of the soil bacteria.

The filamentous mycelium of Streptomyces.
From: Natural Resources Conservation Service

Isolation plates were returned to the incubator and observed daily for three days, at which time secondary metabolites were observed. What was noted as the antibiotic product, appeared as a “dew drop” formed on top of the Streptomyces colonies.
From soil sample plate A, the performed assay test with test organism Micrococcus luteus revealed a zone of inhibition after only 5 days for but not for Bacillus.cereus. The zone of inhibition appeared as a slight cleared ring where the organism was streaked.



Isolation plate of Streptomyces with
secondary metabolite production.



Summary of Observations



Soil Sample A
Dilution plate Chalky texture color elevation Surface appearance Soil odor filaments spores Antibiotic production
10-5 yes creamy convex dull yes yes yes yes

Zone of clearing
M luteus yes
B cereus no




Soil Sample B
Dilution plate Chalky texture color elevation Surface appearance Soil odor filaments spores Antibiotic production
10-5 yes creamy convex dull yes yes no no

Zone of clearing
M luteus no
B cereus no

Discussion

The medias used in this experiment did contain cycloheximide to inhibit fungal growth. However, it would have been especially advantageous to have used an antibiotic while making the isolation media. To do so could have encouraged a variety of isolated species of Streptomyces. Although, we are convinced that we did successfully isolate one species of this organism from soil sample A.
The chalky-white appearance of colonies, and the production of the liquid (secondary metabolite), helped to validate the characteristic colony morphology of the soil bacteria. Other observations of a filamentous mycelium, spores (conidia) and a gram positive reaction of the organism were also factors leading to our conclusion. The strong earthy odor from the isolated colonies and prevalent zone of inhibition from the Assay test helped to confirm that we had isolated the antibiotic producing bacteria.

Bibliography

1. Booser, D.J. and Hortobagyi, D.N. (1994). Anthracycline Antibiotics in Cancer Therapy. Focus on Drug Resistance. Drugs 47, 223-258
2. Brockmann, H. and Bauer. K. (1950). Rhodomycin, ein rotes Antibioticum aus Actinomyceten. Naturwiss. 37, 492-493
3. Fujiwara, A. and Hoshino, T. (1986). Anthracycline antibiotics. CRC Crit Rev Biotechnol 3, 133-157
4. Hutchinson, C.R. (1988). Prospects for the discovery of new (hybrid) antibiotics by genetic engineering of antibiotic-producing bacteria. Med Res Rev 8, 557-567
5. Motamedi, H., Wendt-Pienkowski, E. and Hutchinson, C.R. (1986). Isolation of tetracenomycin C-nonproducing Streptomyces glaucescens mutants. J Bacteriol 167, 575 - 580
|+| نوشته شده توسط مصطفی در جمعه بیست و ششم بهمن 1386  |
  کریستال یا شیشه
: کریستال یا شیشه

 

 

میزان ابتلاء به اعتیاد و مصرف مواد مخدر در ایران بالاست. با وجود اقدامات مختلف قانونی و نظامی برای کنترل مواد مخدر در ایران متاسفانه روند مصرف مواد مخدر در بین جوانان شیوع داشته بویژه افزایش مصرف مواد مخدر جدید از مشکلات فعلی می باشد که بهترین راه در مسیر مبارزه با این خطر، بالا بردن سطح آگاهی جامعه بویژه جوانان و حمایتهای صحیح خانوادگی است.

یکی از مواد مخدر خطرناک که اخیرا" بین جوانان شایع شده، شیشه است این ماده به عنوان محرک( نه مخدر) در مجالس رقص و مراکز فساد در کشورهای انگلیس و استرالیا بین جوانان رواج دارد.

ترکیب اصلی این ماده از آمفتامین(Amphetamine) گرفته شده که یک ماده محرک و اعتیاد آور است و مغز و سیستم عصبی را به شدت تحریک می کند و از نظر شیمیایی اثر آن از آمفتامین بیشتر است. این مواد در لابراتوارهای غیر قانونی ساخته می شود و اسامی خیابانی مثل: کریستال، یخ، Meth و شیشه دارند. این ماده توهم زایی و اثرات محرک را باهم ایجاد می کند.

 

 

 

خطرات بهداشتی:

Methamphetamine وقتی وارد سیستم عصبی مرکزی CNS می شود، باعث آزاد شدن ناگهانی واسطه شیمیایی دوپامین ((Dopamine از مغز می شود که باعث تحریک سلولهای مغزی و افزایش خلق و خو و حالت تهاجمی و غیر قابل کنترل و افزایش حرکات جسمی می شود. که در صورت ادامه مصرف بعد از مدت طولانی علایم بدخلقی و افسردگی و علایم اختلال حرکتی مثل پارکینسون در فرد ظاهر می شود.

این ماده به صورت داخل بینی و خوراکی و داخل رگ و کشیدنی مصرف می شود و بلافاصله بعد از مصرف حالتی به نام rush یا flash در فرد ایجاد می گردد و افراد به سرعت اعتیاد پیدا می کنند و مجبورند هر روز مقدار مصرف را زیاد کنند.

مصرف این ماده می تواند باعث کاهش اشتها برای روزها، افزایش تعداد تنفس، افزایش فعالیت فیزیکی، افزایش دمای بدن و تحریک پذیری و بی خوابی گیجی، لرزش و تشنج، اضطراب و بدبینی و خشونت را سبب شود. که تشنج و افزایش زیاد دمای بدن باعث مرگ افراد می گردد. همچنین با صدمه روی عروق باعث سکته های مغزی و قلبی می گردد.

یکی از عوارض روانی آن ایجاد بیماری روانی شبیه اسکیزوفرنی است شامل توهمات بینایی و شنوایی و بدبینی و پرخاشگری است.

مصرف این ماده به تدریج در بین حیوانات افزایش دارد طبق بررسی در سال 2004 میلادی در ایالات متحده آمریکا 6.2% از دانش آموزان دبیرستانی حداقل یکبار مصرف شیشه را تجربه کرده اند.

متاسفانه افرار شیاد تبلیغ این ماده را در ایران با عنوان نشئه آور ولی بدون اعتیاد عنوان می کنند ولی این طور نیست و علاوه بر اعتیاد می تواند مرگ آور باشد.

اطلاع رسانی و معرفی صدمات جسمانی و روانی جدی این ماده از طریق رسانه های عمومی می تواند از گسترش مصرف این ماده خطرناک جلوگیری نماید.

 

منابع:

·          دکتر آذرخش مُکری ( عضو هیات علمی دانشگاه علوم پزشکی) – نگاهی به سوء مصرف مواد مخدر در ایران.

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 اکستازی

اکستازی ( جادوی مرگ )

  • امروزه اعتیاد به مواد مخدر معضل بزرگی برای تمامی کشورهای جهان اعم از در حال توسعه و توسعه یافته می باشد که دولتها مجبور به صرف هزینه سنگین برای کنترل این بحران هستند.

  • مشکل بزرگی که امروزه با آن روبرو هستیم استفاده از مواد مخدر صنعتی (شیمیایی ) است که در حال حاضر بیشتر در جوانان شیوع پیدا کرده است و اخیراً در کشور ما شاهد استفاده از آن به جای تریاک و هروئین می باشیم.

اکستازی چیست ؟

  • واژه اکستیسی ( ECSTASY )  به معنای نشاط و خوشی زیاد می باشد. ترکیب شیمیایی " متیلن دی اکسی متیل آمفتامین و دی فنیل اتیل آمین " که از خانواده آمفتامین ها با مواد محرک ( در مقابل مخدر ) می باشند. این ترکیبات را روان گردان هم می نامند.

  • این ترکیبات با مصرف دارویی ، قبلاً به عنوان ضد اشتها ، و تنظیم کننده خلق و خو معرفی شده بودند اما در دهه اخیر مورد سوء مصرف قرار گرفته اند یعنی برای توان یافتن کاذب ، ساعت ها رقصیدن و فعالیت خارج از توان بدن به طور اجباری ، مصرف می شوند. در بین جوانان این ترکیبات به نام قرص نشاط آور - اکسی - قرص X ( اکس ) - قرص E پارتی ، باران - محبت و ... متداول شده است.

  • این ماده در کارخانجات صنعتی ساخته می شود و به صورت خوراکی مصرف می شود در سطح خارجی این قرص ها اغلب اشکال و حروفی وجود دارد که مدت زمان تاثیر و نوع ماده موثر آن را مشخص می کند. البته در ایران طی بررسی انجام شده تمامی ترکیبات دست ساز بوده که از خطرات مضاعف این قرص ها می باشد. در سال 2003 پژوهشگران آقار مغزی این ماده را در مغز مشاهده نمودند که روی ماده سرتونین اثر می کند.

  • در بررسی آماری در سالهای 1999 - 1998 میزان مرگ و میر این ماده 400 درصد افزایش یافته و در سال 2004 در جهان از 185 میلیون معتاد 8 میلیون نفر به اکستازی معتاد بودند و در یک بررسی در شهر تهران مشخص شده میزان مصرف این ماده بیشتر شده است. ( مردان 76 درصد و زنان 24 درصد ).

علائم مصرف اکستازی :

  • علائم با مقدار مصرف ، دفعات مصرف ، میزان خلوص ماده ، وضعیت جسمانی و روحی فرد ارتباط دارد. از عوارض جانبی آن می توان به تاری دید ، گشاد شدن مردمک ها و بی اشتهایی اشاره نمود. به علت گشاد شدن مردمک ها و دریافت نور زیاد باعث می شود در میهمانی ها با وجود نور کم افراد مجبور به استفاده از عینک های آفتابی در شب باشند.

  • عوارض دیگر شامل افزایش ضربان قلب و فشار خون و قفل شدن دندان ها است که برای جلوگیری از این حالت مجبور به استفاده از آدامس هستند و تا ساعت ها این فک زدن های غیر طبیعی وجود دارد.

  • اثر دارو حدود نیم ساعت پس از مصرف شروع شده و تا شش ساعت ادامه دارد که در آغاز با هجوم ناگهانی انرژی و نشاط و تهوع و سرگشتگی همراه است و بعد از 24 ساعت تمایل شدید برای مصرف مجدد وجود دارد در آغاز فرد مصرف کننده با احساس انرژی زیاد ( علیرغم غذا نخوردن ) به فعالیت طولانی و خسته کننده می پردازد و به علت از دست دادن مایعات بدن و غذا نخوردن و تهوع و استفراغ به ویژه در میهمانی های شلوغ می تواند به سکته قلبی و مغزی منجر شود.

  • از نظر روانی جون فرد احساس انرژی کاذب دارد پرحرف شده نیاز به جروبحث و درگیری با دیگران و محکوم کردن آنها دارد احساس از خود بزرگ بینی و انرژی زیاد دارد و بدون این که کار مهمی انجام دهد احساس غرور و پرانرژی بودن و محبوب بودن می کند و از شنیدن حرف دیگران گریزان است و خود را مطرح معرفی می کند و نیاز به ارتباط کلامی و پرحرفی در او وجود دارد.

  • بزرگترین مشکل این افراد ، پایان تاثیر دارو است زیرا نئشگی حاصله یا به اصطلاح " اکتسازی فیلم " به افسردگی شدید و عمیق و احساس گناه می انجامد و باعث اضطراب ، خستگی و کوفتگی عضلات و بی انگیزگی و بی تفاوتی شدید می شود و میل به مصرف مجدد دارد ( Dependency ) .

عوارض دراز مدت شامل :

  • اختلال در حافظه بویژه حافظه کوتاه مدت - صدمات به کبد و کلیه و سکته مغزی و قلبی و مرگ می باشد.

  • از عوارض روحی دراز مدت می توان به ضعف اراده و عدم فعالیت و انفعال ، گریز از جمع ، ضعف ایفای نقش اجتماعی و عزت نفس اشاره نمود.

اقدامات و راه های پیشگیری :

  • ترک اعتیاد این ماده بسیار دشوار است و متاسفانه خطر مرگ در انتها وجود دارد و تا امروز هیچ پادزهری برای مسمومیت با اکس شناسایی نشده است درمان اعتیاد به این ماده نیز همانند سایر مواد بایستی در پیشگیری خلاصه شود یعنی بایستی آموزش و آگاهی دادن به افراد جزء رئوس پیشگیری باشد.

تهیه و تنظیم : دکتر علیرضا شکرگزار

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امیدوارم که مو فق باشیم 

|+| نوشته شده توسط مصطفی در پنجشنبه پنجم مهر 1386  |
 
 
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