Асептики лаборатории: Покрытие методы

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Sanders, E. R. Aseptic Laboratory Techniques: Plating Methods. J. Vis. Exp. (63), e3064, doi:10.3791/3064 (2012).

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Abstract

Materials

1. Yeast Tryptone Agar (YTA)

Yeast extract 2.0 g
Tryptone 10.0 g
Agar 15.0 g
Distilled water up to 1000.0 ml
pH 7.0

Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C.

If preparing tubes for the pour-plate procedure, allow the agar to cool to ~55°C then add 2.0 ml of 50 mg/ml cycloheximide. Aseptically dispense 18.0 ml of the melted agar per 18 mm tube then store at 4°C. The agar will solidify and will need to be melted in a steamer or microwave prior to use.

2. Minimal Salts Agar (MSA) + 0.1% (w/v) carbon source

NH4Cl 1.0 g
NH2HPO4•2H2O 2.14 g
KH2PO4 1.09 g
MgSO4•7H2O 0.2 g
Carbon source* 1.0 g
Trace salts solution** 10.0 ml
Agar 15.0 g
Distilled water up to 1000.0 ml
pH 7.0

Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C.

* Carbon sources used for experiments presented in Figure 13 include acetamide, lactose, and glycine.

** Trace salts solution is prepared in 0.1 N HCl as follows. It is added to the base before sterilization (autoclave at 121°C for 20 minutes).

FeSO4•7H2O 300.0 mg
MnCl2•4H2O 180.0 mg
Co(NO3)2•6H2O 130.0 mg
ZnSO4.7H2O 40.0 mg
H2MoO4 20.0 mg
CuSO4•5H2O 1.0 mg
CaCl2 1000.0 mg
HCl (0.1 N) up to 1000.0 ml

3. EHA soft agar (0.65 % w/v)

Agar 6.5 g
Tryptone 13.0 g
NaCl 8.0 g
Na Citrate•2H2O 2.0 g
Glucose 3.0 g
Distilled water up to 1000.0 ml

Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C.

4. EHA hard agar (1.2% w/v)

Agar 12.0 g
Tryptone 13.0 g
NaCl 8.0 g
Na Citrate•2H2O 2.0 g
Glucose 3.0 g
Distilled water up to 1000.0 ml

Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C.

5. 1X Middlebrook Top Agar (MBTA soft agar, 0.5% w/v)

100 mM CaCl2 stock* 1 ml
7H9 liquid medium: Neat ** 50 ml
2XTA *** 50 ml

Melt 50 ml of 2XTA and allow it to cool to ~55°C. Using aseptic technique, add the CaCl2 and 7H9 broth to the melted agar. Aseptically dispense 4.5 ml of the mixture per 13 mm tube and store in a 55°C incubator ≤7 days. Cooling MBTA to room temperature or 4°C will cause the CaCl2 to precipitate out of solution.

* 100 mM CaCl2. stock must be stored at room temperature to prevent CaCl2 from precipitating out of solution.

** 7H9 liquid medium: Neat

7H9 broth base 4.7 g
40% glycerol stock 5 ml
Distilled water up to 900.0 ml

Mix the base with water then add the glycerol while stirring. Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C.

*** 2X Middlebrook Top Agar (2XTA, 1.0% w/v)

7H9 broth base 4.7 g
Agar 1.0 g
Distilled water up to 1000.0 ml

Autoclave at 121°C for 20 minutes to sterilize. Dispense 50 ml aliquots into 100 ml bottles and store at 4°C.

6. Middlebrook 7H10 Agar Plates (MHA hard agar, 1.9% w/v)

7H10 agar base 19.0 g
40% glycerol stock 12.5 ml
Distilled water 887.5 ml

Mix the agar base with water then add the glycerol while stirring. Heat the solution to boiling then stir for one minute to completely dissolve the base powder. Autoclave at 121°C for 20 minutes to sterilize. Allow the agar to cool to ~55°C then aseptically add the following reagents:

AD supplement (pre-warmed to 37°C)* 100 ml
50 mg/ml Carbenicillin ** 1.0 ml
10 mg/ml Cycloheximide ** 1.0 ml

* AD supplement

NaCl > 17 g >
Albumin (Fraction V)> 100 g>
Dextrose (D-Glucose)> 40 g>
Distilled water> up to 2000.0 ml>

Filter-sterilize this solution; do not autoclave. Store at 4°C.

** Filter-sterilize and store these solutions at 4°C for ≤60 days.

7. LB agar (1.5% w/v) + X-Gal (60 μg/ml)

Tryptone 10.0 g
Yeast extract 5.0 g
NaCl 10.0 g
Agar 15.0 g
Distilled water up to 1000.0 ml
pH 7.5 at 25°C

Autoclave at 121°C for 20 minutes to sterilize. Allow the agar to cool to ~55°C then aseptically add 3.0 ml of 20 mg/ml X-Gal solution. Freshly prepare X-gal stock by dissolving 400 mg X-Gal in 20 ml dimethylformamide (DMF).

Table of specific reagents:

Name of the reagent Company Catalogue number
Yeast extract Becton Dickenson 212750
Tryptone Becton Dickenson 211705
Agar Becton Dickenson 214030
NH4Cl Acros Organics 123340010
NH2HPO4•2H2O Sigma-Aldrich 30435
KH2PO4 Fisher Scientific BP 303-500
MgSO4•7H2O Sigma-Aldrich 230391
Acetamide Sigma-Aldrich A-0500
Lactose Fisher Scientific L6-500
Glycine Sigma-Aldrich G-7126
FeSO4•7H2O Sigma-Aldrich F8048
MnCl2•4H2O Sigma-Aldrich M-3634
Co(NO3)2•6H2O Sigma-Aldrich 230375
ZnSO4.7H2O Sigma-Aldrich Z4750
H2MoO4 Acros Organics 213621000
CuSO4•5H2O Sigma-Aldrich 209198
CaCl2 Sigma-Aldrich C1016
HCl Fisher Scientific A144-212
Cycloheximide Sigma 038K1561
Carbenicillin Cellgro 46-100-RG
NaCl Fisher S271-1
Na Citrate•2H2O Fisher S279-500
Glucose (Dextrose) BD 215530
7H9 broth base BD 271310
7H10 agar base BD 262710
Glycerol Shelton IB15760
Albumin (Fraction V) Fisher S71907
X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) Teknova X1205
Dimethylformamide (DMF) Sigma D4551
Ethanol Fisher CDA19
Chlorine Bleach Chlorox 02490/06644884
CiDecon (disinfectant) Decon Laboratories, Inc. 8504

Table of specific equipment:

Name of equipment Company Catalogue number Experiment
Metal loops American Educational Products S17352 Streak plating
Disposable plastic loops Fisher 22-363-602 Streak plating
Wooden sticks Fisher 23-400-104 Streak plating
Flat Toothpicks American Educational Products S67859 Streak plating
Turn tables Fisher 08-758Q Spread plating
Glass rods Bellco Glass NC9004380 Spread plating
4 mm glass beads Fisher 11-312B Spread plating
Velveteen cloth Bel-Art Products 09-718-2 Replica plating
Cylindrical block Bel-Art Products 09-718-1 Replica plating

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References

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Comments

2 Comments

  1. I am not sure why you keep lots of care flaming your clean loop starting at the base and then up to the loop, but in contrast, flame directly the loop when it is full of bacteria.
    My suggestion is to flame always starting from the bottom of the wire and then up to the loop, which in theory may reduce the amount of aerosols produced.

    Reply
    Posted by: Ed K.
    June 8, 2012 - 11:02 PM
  2. Shouldn't the scientist be using gloves?

    Reply
    Posted by: Josh F.
    June 30, 2012 - 3:15 PM

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