PROCERURE OF A.G.E.

UPDATES

- Loading dye composition and concentration

- sample preparation i acikla / LD in 1/6 dilusyonu yeterli. su gerekirse konur.

- Tum tarak kodlari ve load hacimleri > default tarak

- Cop nereye atilacak

MAIN STEPS/TIME TABLE

- 1% gel preparation [30 min]
- Running [1 h]
- Visualization [15 min]

MATERIALS

- Electrophoresis tank

- Power supply

- Transilluminator

- Appropriate comb

- P10 and P100

- DNA Ladder 100-10 kb (Fermentas #SM0331)

- Agarose

- Loading dye

- TAE Buffer (1X)

- Distilled water [M14]

- Sterile dH2O [M14]

- EtBr

- Parafilm

- Gel bed: ? x ? cm

- Comb: 8 wells (20-25 uL per well)

SOLUTIONS

TAE buffer

- Stock TAE electrophoresis buffer (50X)

- Use 1X TAE

- 20 mL TAE

- 980 mL dH2O

1% Electrophoresis gel

- 0.5 gr Agarose
- 50 mL 1X TAE buffer (1%)
- Agarose’un cozunmesi icin microdalgada isitilir (Microdalgada eritmek icin kullandigimiz cam materyalin agzini siki bir sekilde kapatmiyoruz, aluminyum folyo kullanmiyoruz ve isitirken arada karistiriyoruz).
- Sogurken dibe cokmemesi icin arada karistirilir (musluk altinda sogutulabilir).
- Soguduktan sonra 2.5 uL EtBr eklenir.

CHECK-LIST PROCEDURE

- Mix 2 uL DNA + 3 uL sterile dH2O + 1 uL loading dye on the parafilm
- Load sample to the wells of 1% gel

- Adjust the voltage of power supply to 75 V

- Adjust the time of power supply to 65 min
- Check transilluminator

- After running of the samples record the gel image [go to SOPs-experimental for application of camera and record of image]

Image Acquisition
- Bilgisayar acilir.
- Jel goruntuleme cihazinin icine konur.
- Kamera acilir (kamerayi acmadan program calismiyor).
- Infinity Capt programi acilir.
- White light acilir.
- Live preview dugmesine basilir.
- Merceklerden jelin yeri ayarlanir.
- White light kapatilir.
- Transmilluminator (uv) dugmesi acilir.
- Exposure Time dugmesine basilir. Exposure time: 3 ms (5 uL DNA ladder)
- Jelin goruntusu netlesince Stop dugmesine basilir.
- Goruntu kayit edilir

- Transilluminator kullanim kaydi CO kodu ile deftere yazilacak.

NOTES

- Before adding of EtBr make sure the temperature of gel solution, its temperature should not be too high.
- Make sure the voltage is at the correct settings at all times and always use enough buffering solution to cover the gel in order to prevent inconsistent readings.

- Merkezi labin kucuk elektrophoresis tanki icin kullanilan kucuk comb una fazla yukleme yapilamiyor. [5 ul lik bir ladder eklediğimizde overloading den dolayi yurume tam gerceklesmedi. Bu taragi kullanmayalim]
——-

Troubleshooting

Missing Bands
- Use a lower voltage or decrease the electrophoresis time if smaller bands are missing since this may indicate that they were pushed off of the gel. However, increase the electrophoresis time if larger bands are missing which means that the components have not separated yet.

Smear
- Check sample for nuclease contamination, buffering conditions and excess salt or protein. If you still see smeared results, decrease the amount of sample used.

Non-existent or Faint Bands
- Increase amount of sample or time of electrophoresis at a lower voltage.

——–

Agarose Gel Electrophoresis comb kapasiteleri

 

2 lik well 500uL

5 lik well 220 uL

6 lık well 70 uL

8 lik well 45 uL

10 luk well 35 uL

12 lik well 25 uL

14 lük well 15-20 uL

Commonly Used Stock Antibiotic Solutions

- Perform these steps near the flame

- Store at -20 C

Ampicillin Stock Solution (25 mg/mL)

- Add 250 mg ampicillin sodium salt in to 10 mL MilliQ dH2O in a 15 mL falcon

- Vortex

- Filter the mixture w/ 0.2 um syringe filter to a new 15 mL falcon

- Aliquot mixture into eppendorf tube

- It is light sensitive

 

Kanamycin Stock solution (25 mg/mL)

- Add 250 mg kanamycine sulfate in to 10 mL MilliQ dH2O in a 15 mL falcon

- Vortex

- Filter the mixture w/ 0.2 um syringe filter to a new 15 mL falcon

- Aliquot mixture into eppendorf tube

 

Tetracycline Stock Solution (12.5 mg/mL)

- Add 250 mg tetracycline in to 10 mL MilliQ dH2O in a 50 mL falcon

- Add 10 mL EtOH (final EtOH is 50%)

- Vortex

- Filter the mixture w/ 0.2 um syringe filter to a new 50 mL falcon

- Aliquot mixture into eppendorf tube

Antibiotic Solutions

Notes

- Antibiotics should be added to lower than 60 C broth, or filter sterilized: See note. “C’ refers to chromosomal resistance: “P” plasmid based resistance, “FS” filter sterilization required.
- Temperature is required for storage.
- Concentration values in ug/ml
- Antibiotics for Pseudomonas, E. Coli, Agrobacterium, etc…
- Reference: http://wheat.pw.usda.gov/~lazo/methods/lazo/antibiot.html
- Ampicillin 1000 (C/P) 50 (P) Stock: 25 mg/ml Na salt:FS: Store at -20 C, carbenicillin is more stable than Amp.
- Kanamycin 200 (P) 50 (P) 25 mg/ml: FS: -20 C
- Tetracycline 20(C)50(P) 15 Stock: 12.5 mg/ml 50 % EtOH: -20 C
- Chlroramphenicol 500 (C) 30 (C/P) 25 µg/ml for plasmid amplification 25 mg/ml in DMSO: -20 C
- Carbenicillin 1000 (C/P) 50 (P) 100 mg/ml: FS: -20 C
- D-cycloserine 200 (C) Add dry after autoclaving
- Erythromycin 200 (C) 25 mg/ml EtOH: -20 C
- Gentamycin 10 (C/P) Add dry after autoclaving
- Geneticin 200 for fungi Same as kanamycin in mode of action and preparation
- Hygromycin 250 for fungi 25 mg/ml: FS: -20 C
- Kasugamycin 200 (C) 20 mg/ml: FS: -20 C
- Mercuric chloride 40(C/P) 10 µg/ml if in minimal media, 40 mg/ml in EtOH: -20 C
- Nalidixic acid 1000 (C) 20 (C) 100 mg/ml in H2O, add 6 N NaOH until dissolved: 4 C
- Neomycin 200 (P) 50 (P) 25 mg/ml: FS: -20 C
- Rifampicin 200 (C) 200 mg/ml make fresh in Methanol
- Spectinomycin 200 (C/P) 25 (P) 20 mg/ml: FS: -20 C
- Streptomycin 200 (C/P) 25 (P) 20 mg/ml: FS: -20 C
- Sulfamethoxazole 500(C) Add dry after autoclaving
- Triomethoprim 500 (C) Add dry after autoclaving

E. coli Growth Curve Preparation

MAINSTEPS/TIMETABLE

- Culturing the frozen stock [DAY 1]

- Scale-up of the suspension culture [DAY 1]

- Taking the OD readings [DAY 1-2]

- Preparing spread plate cultures [DAY 1-2]

 

CHECKLIST PROCEDURE

Culturing the frozen stock

- Transfer 40 ul from the stock to the 20 ml LB

- Incubate it until the OD reaches to the 0,2, shaking at 225 rpm

 

Scale-up of the suspension culture

- Transfer 2 ml from the suspension culture to the 200 ml LB (1:100 ratio)

- Incubate the culture, shaking at 225 rpm

 

Taking the OD readings

- After 30 min start the OD readings and repeat it at every 30 min

- Continue to take the readings at every 30 min until the curve reaches to the stationary phase

- Take readings at every 4 hrs

 

Preparing spread plate cultures

- Take samples from the incubated culture at certain time points during the exponential phase

- Dilute the samples as

- 10-4, 10-5, 10-6 ratios until the 6 hours culture incubation

- 10-5, 10-6,10-7 ratios between the 6th and 8th hours culture incubation

- 10-6, 10-7, 10-8 ratios after the 8 hours culture incubation

- Prepare 2 plates from the each dilution by spreading 100 ul sample with spread plate technique

- You can know the cell number at corresponding OD from now on.

 

 

LB Broth Preparation with Antibiotics

-Amp salt Solution
- 500mg Ampicilin sodium salt
- 10mL Milli-Q water
- Stable for years in a freezer.

* 1ul stock amp for 1 ml lb broth

-Kanamycin, Tetracycline
- 500mg either kanamycin or tetracycline
- 10mL Milli-Q water
- Sterilize through 0.2 um filter.

- Aliquot and store in a freezer. Stable for years. Avoid repeated freeze-thaw cycles.

Chloramphenicol (Gold Biotechnology catalog number: C0113) can be used as an additive in microbial growth media. The final concentration of chloramphenicol in media is usually 15 µg/mL (ie. for one liter of media add 1.5 mL of 10 mg/mL stock solution).

1. Dissolve 2 g of Chloramphenicol in 200 mL of 100% or 95% ethanol

2. Sterilize by filtration through a 0.2 µm filter

3. Store at room temperature, in a screw top bottle wrapped in foil

 

 

X-gal
- 100 mg X-gal
- 5 mL Dimethylformamide
- Stable for about a year in a freezer. Longer sotrage makes the solution somewhat colored, but it still works fine.

IPTG

- 119 mg IPTG
- 5 mL Milli-Q water
- Aliquot and store in a freezer.

 
LB Preparation (High Salt)

-Luria-Bertani (LB) media (1 L):

- 10 gr of Bacto-tryptone

- 5 of Yeast extract

- 10 gr of NaCl (for taste)

- pH to 7.5 w/ NaOH.

- dH₂O to 1 L (Autoclave)

LB Agar Preparation

UPDATE

- Agar plate`ler M13 teki metal kabinet icinde dokulur

- Cabinet icini 70% alkol ile cok iyi temizle

- Bunsen burner`i ac (Plate`ler dolaba kaldirilana kadar acik kalacak)

- Plate`leri dok

- Plate`lerin donmasini bekle (??? 5 dakika)

- Plate`ler donduktan sonra kapaklarini cok az arala ve buhar gidene kadar bekle (??? 10 dakika). Daha uzun bekleme (kontaminasyon riski !)

- Bunsen burner i kapat (COK ONEMLI: Cabinet altindaki metal vanayi da kapatmayi unutma)

- Plate`lerin agzini kapat > Parafilm > +4 C`ye kaldir (Agar ustte olacak sekilde)

-For 20-30 plates
- 3 gr Bacto-trypton
- 1.5 gr Yeast extract
- 5.134 mL 5M NaCl
- 4.5 gr Agar
- 300 mL Water

- Autoclave
- Use an alminium kettle for convenience.

- Cool down the LB agar medium to 60-70 C, add 300 uL Amp solution, and mix.
- Pour 10-15 mL of the medium in a disposable plastic plate, and swirl it gently to spread over the bottom of the plate. Cover and stack it.
- Pour, swirl, cover and stack one after another, pulsing with flame to avoid clot.
- After agar clots, open the plate cover halfway under ventilation of a clean air chamber to dry agar for 30-60 min. Do not over-dry.
- After steam inside the cover disappeared, close the cover, stack, wrap, and store upside down in a refrigerator.
- Good for one or two months under refrigeration. Plates older than two months can be used with about 3-5 uL Amp spread withX-gal and IPTG (60 ug/mL X-Gal, 0.1 mM IPTG).

 

SOB Medium

For 200 mL (mix the following broth and Mg solution to 100:1 in a sterile environment, stable for about a year under refrigerator)

Broth
- 4 gr Bacto-tripton
- 1 gr Yeast extract
- 0.4 mL / 5 M NaCl
- 0.167 mL / 3 M KCl
- 200 mL water
- Autoclave

Mg solution
- 2.465 gr MgSO₄.7H2O
- 2.033 gr MgCl₂.6H2O
- Add milli-Q water up to 10 mL
- Autoclave

5M NaCl (for 1000mL)
- 292.5 gr NaCl
- Add milli-Q water up to 1000 mL
- Autoclave
- Stable for years under refrigerator

3M KCl (for 100mL)
- 22.365 gr KCl
- Add milli-Q water up to 100mL
- Autoclave
- Stable for years under refrigeration.

Liquid MPYE Medium

- Enriched medium for R. Rubrum culture

- Source: Ebru Hoca (Biyohidrojen Grubu)
For 1L
- Bactopeptone (0.3%) 3.0 g
- Yeast extract (0.3%) 3.0 g
- MgCl2 1.6 mL from 1M MgCl2 stock (1.6 mM MgCl2)
- CaCl2 1.0 mL from 1M CaCl2 stock (1.0 mM CaCl2)

are dissolved in dH2O.

- pH is adjusted to 7.0 with NaOH and autoclaved.
- For MPYE medium plate, 15 g agar (1.5%) (Difto-Bacto agar) is added to liquid MPYE medium and autoclaved.

SOC medium (1L)

- 900 mL dH2O
- 20g Bacto tryptone extract
- 2 mL of 5M NaCl
- 2.5 mL of 1M KCl
- 10 mL of 1M MgCl2
- 10 mL of 1M MgSO4
- Autoclave the solution
- Add 20 mL of 1M glucose (autoclaved solution)
- Adjust to 1L with dH2O

Minimal M9 Medium:

Dissolved in deionized water to a final volume of 1 L

1x M9 salts

2mM MgSO₄

0.1mM CaCl₂

0.4% carbon source (e.g. glycerol, glucose, etc.)

In sterile H₂O

M9 Supplemented Minimal Media

For 1L of media

500ml 2x M9 salts

used 11.28g of M9 Minimal Salts in 500ml of MiliQ-filtered H₂O

30ml 10 mg/ml thiamine hydrochloride

used 300 mg thiamine hydrochroride

10ml 40% glycerol

used 8ml 50% glycerol

20ml 10% casamino acids

wasn’t sure if this was by weight or not. Assumed 10% by weight, so used 2g casamino acids.

20ml 0.1M MgSO₄

used 0.493 g (FW 246.49 g/mol)

200μl 0.5M CaCl₂

used the same (made 1ml 0.5 M CaCl₂ with 74 mg (FW 147.01 g/mol))

419.8ml sterile deionized H₂O

we made the above solution and filled to 1L with MiliQ-filtered H₂O

We combined the above ingredients, and filtered through a milipore steritop filter into a clean auto-claved 1L bottle. Stored at 4°C

5X M9 salts contains:

64 g Na₂HPO₄.7H₂O

15 g KH₂PO₄

2.5 g NaCl

5 g NH₄Cl
REFERENCE:
- http://openwetware.org/wiki/Julius_B._Lucks/M9_Supplemented_Minimal_Media
- http://openwetware.org/wiki/M9_salts
- http://openwetware.org/wiki/M9_medium/minimal

TB Medium

- 12 g Bacto tryptone

- 24 g Bacto yeast extract

- 4 mL Glycerol

- 100 mL 0.17M KH2PO4 and 0.72M K2HPO4, sterile, to be prepared separately from the tryptone, yeast extract, and glycerol solution.

- Mix tryptone, yeast extract and glycerol, and add distilled water up to 900mL

- Pour into 2 L flask (or greater)

- Autoclave (liquid cycle)

- Allow liquid to cool to less than 60 degrees Centrigrade

- Add 100 mL Potassium Phosphate Solution

Potassium Phosphate Buffer

For the preparation of 1 liter of potassium phosphate solution the final desired concentrations are:

• 0.17 Molar monobasic potassium phospate (23.135 grams/liter KH2PO4, molecular weight=136.09 grams/mole)
• 0.72 Molar dibasic potassium phosphate (125.410 grams/liter K2HPO4, molecular weight=174.18 grams/mole).

Add the dry ingredients to a 1 liter graduated cylinder and added double distilled water until a final volume of 1 liter is reached. Sterile filter this solution through a 0.22 micron filter.

 

BACILLUS SUBTILIS (competent ve transformasyon)

Media Preparation

10X Medium A base:

● Yeast extract 10g

● Casamino acids(pepton from casein/ triptone ) 2g

● Distilled water to 900mL

● Autoclave, then add :

● 50% glucose, filter sterilized 100mL (50 g/100 mL)(%50 glikozu distile suyle birlikte bir flaskın içinde bunsen burnerda ısıtarak çöz)

10X Bacillus salts:

● (NH4)2SO4 20g

● Anhydrous K2HPO4 139.7g

● KH2PO4 60g

● Tri-sodium citrate 10g

● MgSO4•7H2O 2g

● SDW(sterile distile water) to 1000mL

● Then, autoclav

Medium A

● Sterile water 81mL

● 10X Medium A base 10mL

● 10X Bacillus salts 9mL

● L-Tryptophan (11mg/mL) 0.1mL (filter sterilized)

● Then, filter sterilized

Medium B

● Medium A 10mL

● 50mM CaCl2•2H2O 0.1mL (filter sterilized) (147 g/mol)

● 250nM MgCl2•6H2O 0.1mL (filter sterilized) (203.3 g/mol)(hazırlanışı için notlar kısmızı oku)

Important:

● Autoclave Medium A base before adding glucose, and autoclave Bacillus salts

● Store aliquots of 10X Medium A base 10mL and 10X Bacillus salts 9mL and keep them in the fridge, never use them twice to avoid contamination

Protocols

Making Bacillus competent

1. Grow one blank plate of Bacillus subtilis (or several if you want to transform different strains) for 20 hours at 37ºC (plate been kept on the bench for several days would be better)
2. Inoculate about 12mL of medium with several colonies. Mix the contents of the tube. Check with OD650. Start OD should be between 0.1 and 0.2. Be careful to pipette 0.8mL of this mixture into the cuvette to measure and dispose of it after measurement to avoid contamination in the main mixture.
3. Incubate at 37ºC with vigorous shaking. Read the OD650 every 20min (never keep the solution you used for measuring!)
4. Plot log(OD650) in function of time. After a brief lag, you should observe a exponential increase. After awhile, it will leave the exponential growth; the moment at which it leaves the exponential path is denoted as t0 (3 on the graph). It should take about 100min and the OD should be between 0.35 and 0.55.

 

1. At t0, incubate for 90 minutes at 37ºC with vigorous shaking.
2. Transfer 0.05mL of this culture into 0.45mL of pre-warmed Medium B in an Eppendorf tube. You have to prepare one tube for each transformation, plus an extra tube for a DNA-less control.
3. Incubate the diluted cultures at 37ºC with shaking for 90min. At this moment, the cells are HIGHLY COMPETENT.
4. To check for competency, you can look at cells under the microscope; competent cells are very motile.

Transforming

1. Spin Eppendorf tubes containing cells. Remove 400µL of liquid to keep only 100µL of the culture (to concentrate cells). Re-suspend the cell pellet in the remaining culture.
2. To transform from competent glycerol stocks, firstly thaw on ice and then spin the tube at about 1600rpm for 20min, remove the supernatant (glycerol), and add 100µL of pre-warmed medium B.
3. Mix the cells thoroughly. (pipeting slowly)
4. Add 0.6µg of DNA to the competent cells. (yarın belli oacak)
5. Incubate for 30min at 37ºC with shaking.
6. Plate 100µL of transformed cells onto selective agar.

Glycerol Stocks

1. To freeze competent Bacillus cells, spin down(8ooo rpm, 5 min) the fresh competent cells to obtain a pellet.
2. Remove all supernatant. (remove 450µLsupernatant with pipette)
3. Re-suspend cells in 500µL 60% glycerol. (slowly)
4. Add into liquid nitrogen
5. Freeze tubes at -80ºC.

 

NOTLAR

• Santrifüjden sonra süpernatantı alırken tüpü yukarı tut, bakteriyi iyice gör ve bakteriyi dağıtmadan alacağını al.( 1,5 luk ependorflar 2 liklere göre daha uygun, bakteri daha güzel yapışıyor)
• Etüv deki shaker ı çalıştırmayı unutma sonra cihan abi kızarJ
• İstediğimiz molarda çözelti hazırlarken(lise kimyasını hatırlamayan tıpçılar içinJ)

n=m/ Ma

M= n/V

Ör: CaCl2.2H2O dan 10mL lik ve 50mM lık çözelti hazırlamak için 0.0735 g madde tartılır.(Ma : 147 )

• 250 nM lık MgCl2.6H2O luk çözeltiyi hazırlaması biraz gıcık(çok çok az miktar madde tartmamız gerektiği için) o yüzden önce 10mM lık hazırlayıp sonra sulandırdık.

0.02 g/10mL(1. Çözelti) lik çözeltimiz oldu.Daha sonra 1.çözeltiden 2.5µL alıp 100mL ye tamamlayıp 250 nM yapmış ol

Procedure of Cell Lysis

MATERIALS

- Kullanilan malzemeler deterjandan tamamen arindirilmis, steril ve cok iyi kurulanmis olmalidir.

- Incubator with shaker [M03]

- SOB/LB Media

- Glycerol (100%)

- Sterile Milli Q dH2O

- Liquid nitrogen

- 15 mL Falcon

- Cryotube/epp

- kopuk

CHECK-LIST PROCEDURE

Suspension culture preparation
- Select a colony from the petri plate and inoculate it to the 10 ml LB culture
- Incubate it until the OD reaches to the 0,5, shaking at 225 rpm

Scale-up of the suspension culture
- Transfer 1 ml from the suspension culture to the 100 ml LB (1:100 ratio)
- Incubate it until the OD reaches to the 0.5 shaking at 225 rpm

Stock preparation
- Prepare 25% glycerol containing LB
- Dissolve 100 ul culture in the 4.9 ml 25% glycerol containing LB
- Aliquot it as 200 ul in the ependorf tubes ş

———————————————-

- Liquid nitrogen is taken from cold room in dubel flask with gloves. H061048B1030097

- Environment is sterilized with 70% EtOH

- Bunsen Burner is opened << acik pencereler kontaminasyonu engellemek icin kapatilir

- Check working of incubator

- For 1 mL seed stocks in 10 epps

- Add 5 uL seed bacteria into 10 mL of SOB/LB medium in 15 mL falcon << agzi fazla kapatilmaz ve kagit bantla yapistirilir- hava alması gerekiyor

- Incubate at 37 C @ 240 rpm not longer than 12-14 h

- Divide into 1 mL lik solutions into 10 epps

- Add 176 uL glycerol into each epp to 15% final concentration

- Vortex!

- Aliquot 100 uL samples to cryotube

- Quick-freeze in liquid nitrogen ( ici nitrogen dolu kopugun icine atalim ve cikarmak icin cımbız kullanabiliriz)

- Place in -80 C deep freeze

- Inventory list de Deep freeze Dosyasina stocklar belirli kutuya kaydedilir

———————————————————

- Choose colony put into 5 mL of LB medium in 15 mL falcon.

- Incubate at 37 C @ 240 rpm not longer than 12-14 h.

- Centrifuge 5000 rpm 5min.

- Discard 4 mL solution from falcon.

- Add 176 uL glycerol into falcon to 15% final concentration.

- Vortex!

- Aliquot 150 uL samples.

- Quick-freeze in liquid nitrogen (ici nitrogen dolu kopugun icine atalim ve cikarmak icin cımbız kullanabiliriz).

- Place in -80 C deep freeze

- Inventory list de Deep freeze Dosyasina stocklar belirli kutuya kaydedilir.

Materials:

 

- water bath

- vortex

- pipet 10 ul lik

 

- ice

Check List Procedure

 

- For 1 ml cell culture;

 

- resuspend the cell pellets in 1 ml 2-5% SDS

- vortex 1 minute

- incubate in 50 c water bath for 15 min

- put in ice for 1 min

- add 10 ul from each of

- 1 mg/ml RNAse A stock (final concentration: 10 ug/ml)

- 0.5 mg/ml DNase I stock (final concentration: 5 ug/ml)

- 100 mM MgCI2 stock (final concentration: 1mM)

- incubate on ice for 15 min

 

Solutions

 

- 1 mg/ml RNAse A stock

- 0.5 mg/ml DNase I stock

- 100 mM MgCI2 stock

- 2-5% SDS

PROCEDURE OF SPORICIDE
Organism Preparation

1. A 2 x SG-Schaeffer sporulation medium (stock solution) : 16 g LB broth/liter, 0,5 g MgSO4 . 7H2O, 2 g KCl/lt.

2. Additional stock solutions include %10 glucose, 1M Ca(NO3)2, 0,1 M MnSO4, 0,001 M FeSO4.

3. Compenents of stock solutions are dissolved one at a time in 1 lt of d-distilled H2O.

4. 100 ml aliquots are poured into media bottles.

5. The caps are loosened and autoclaved in a pan of water for 20 min. on slow exhaust.

6. The bottles are allowed to cool comletely, caps are tightened, solutions are scored in the dark until use.

• As prepared, the Schaeffer nutrient base lasts approximately 1 week.

7. Stock solutions are filter sterilized yusing a 0,22 um filter.

Fermantation (Producing Spores)

1. 10 ml of Schaeffer medium is added to a 50 ml falcon, inoculated with a 10 ul culture (or one colony from plate) of B. Subtilis.

2. 5 days are allowed for growth and sporulation. (30 C, moderate aeration)

3. The culture is heat shocked at 65 C for 15 min to kill vegetative cells.

4. The spores are harvested by centrifiguation (16,000 x g for 25 min), then washed with d-distilled water 10 times.

5. The spores are resuspend in 1.7 ml of sterile d-distilled water.

6. A dilution series is performed on the suspension to numerate the number of viable spores.

7. Growth on nutrient agar medium deposited on petri dishes to determine the population of spores.

Preperation of the modified Fenton Reagents

1. Cupric chloride and ascorbic acid (AA) are dissolved in deionized water, tap water, or salt water (2 M NaCl). The most effectıve reagent solution includes 2M NaCl in water

2. A 1% surfactant (3 M FC-170 or FC-100 fluorinated surfactant) is also added in to reduce the surface tension of the aqueous solution in order to get the highest killing level.

• The most effetice reagent solution includes FC-100 type of sulfactant

3. The exposure time of spores to the reagent istypically 30 min, and exposure is performed at 25°C.

 

Sporicide Procedure (Aqueous)

1. Prepared aliquots of 1 ml of spore culture are placed in test tubes.

2. The tubes are spun for 10 min at 16,000 xg to pelletize the cells. The supernatant is discarded.

3. Three tubes are resuspended in the Fenton reagent to be tested (the precise volume and tube size varied).

• A fourth sample tube is resuspended in sterile d-distilled water as a control.

4. The tubes are exposed to the Fenton reagents for the specified exposure times (30 min unless noted otherwise).

5. Dilution with cold (0°C) liquid (sodium thiosulfate) quenches the reaction.

6. The sample tubes and the control are then spun at 16,000 xg for 10 min to pelletize the cells. The supernatant is removed.

7. Spores are rinsed two times in an appropriate diluent.

8. Spores are then resuspended in 1 ml of fresh sterile phosphate buffer.

9. Serial dilutions (generally over 5 to 7 logs of dilution) on both samples and controls are performed using nutrient agar growth medium plated on petri dishes. The dishes are incubated for 24 h minimum, 48 h maximum at 30°C.

 

NOTE: As described above, serial dilutions are then prepared for each tube, using sterile phosphate buffer as the diluent. In all cases, a standard serial dilution of the stock spore

culture is carried out to verify that the culture is still viable and that the number of CFU (CFU per milliliter) is constant over time. All spore-handling procedures use aseptic bacteriological techniques in laminar flow biohazard hoods.

 

 

Sporicide Procedure (Solid Surface)

1. Sterile 2-cm2 glass coupons are scored and cut from frosted glass microscope slides

2. The glass coupons are then inoculated with B. subtilisstock solution, approximately 5 x 10^7 spores/coupon. The pipette is fitted with a new tip for each coupon.

3. The inoculated coupon(s) then is dried at room temperature overnight in a desiccator containing Drierite.

4. Following exposure to Fenton reagents, coupons are placed in a cold (_0°C) neutralization agent (sodium thiosulfate) and then sonicated for 60 min to remove spores from the substrate. Separate tests verifythat the sonication procedure is capable of recovery from the coupons and that sonication do not induce spore deactivation.

5. The spore population is then determined by performing triplicate serial dilutions of the resulting suspension. Serial dilutions are prepared for each sample, using sterile phosphate buffer as the diluent.

6. Serial dilutions (generally over 5 to 7 logs of dilution) on both samples and controls are performed using nutrient agar growth medium plated on petri dishes. The dishes are incubated for 24 h minimum, 48 h maximum at 30°C.

 

NOTE: The magnitude of kill is then determined by comparing the treated colony count with that of untreated colonies. It is noted that the preparation techniques resulted in clumped spores on the impervious glass coupons.(J. B. Cross, 2003)[1]