CbMM:

(Modified from the laboratory manual for axenic cultivation of Caenorhabditis elegans, by Nancy C. Lu, Ph.D., R.D., Department of nutrition and food science, San Jose State University, San Jose, CA 95192-0058)* some modifications to methods are present)

 

HySoy-Yeast Extract (HS-YE)

Component

mass needed per liter final volume

Hy-Soy Powder (Quest International)

40

Yeast Extract (Difco)

10

* Autoclave and keep in refrigerator or in sterile bottles on shelf

 

HySoy-Yeast Extract- Heated Liver Extract (HS-YE-HLE)

When needed remove HLE from freezer and thaw.

Use 1ml of HLE for every 9ml HS-YE.

 Small cultures

 Large cultures of (HS-YE-HLE)

500ml Wheaton bottles containing 40ml of HS-YE and 4 ml HLE have been successfully maintained for ~3 months. Bottles do not need to be shaken. Stand them upright on a shelf.

 Setup of a 500ml of culture

Heated Liver Extract (HLE):

 Method for continual subculture of Axenic Caenorhabditis elegans nematodes:

Stock cultures are kept as follows:

 

Method for determination of inoculation volume:

 

Method for determination of inoculation volumes for CbMM assays

 

Nutrient Broth-Yeast Glucose extract (NYG):

component

mass of component with final volume = 1 liter

Glucose

5.0 grams

Nutrient Broth (DIFCO)

8.0 grams

Yeast Extract (DIFCO)

5.0 grams

Place `2ml into a quantity of small capped vials and autoclave at 110C-122C, 15 psi for 15 min..

Store in refrigerator or shelf.

Salts:

Compound three salt solutions separately

Salts solution A

CHEMICAL

grams 1 batch

grams 10 batches

CaCl2*2H2O

0.2205

2.2050

CuCl2*2H2O

0.0065

0.0650

MnCl2*4H2O

0.0222

0.2220

or CaCl2 anhydrous

0.1665

1.6650

ZnCl2

0.0102

0.1020

To compound salt solution A

Salts Solution B

CHEMICAL

grams 1 batch

grams 10 batches

Fe(NH4)2(SO4)2*6H2O

0.0588

0.5880

K3Citrate*H2O

0.486

4.8600

KH2PO4

1.2255

12.2550

To compound Salt Solution B

 

Salts Solution C

CHEMICAL

grams 1 batch

grams 10 batches

Citric Acid*H2O

0.6303

6.3030

Mg(OH)2

0.1740

1.7400

To Compound Salt Solution C

 

Complete Salt Solutions

Water Soluble Components frozen stock (WSC)

CHEMICAL

Grams/ 25ml

Grams/200ml

canocobalamine (vitamin b-12)

0.015

0.1200

N-Acetylglucosamine

0.06

0.4800

niacinamide

0.03

0.2400

pantethine

0.015

0.1200

pantothenate (Ca) pantothenic acid hemicalcium salt

0.03

0.2400

Pyridoxal*PO4

0.015

0.1200

pyridoxamine*2HCl

0.015

0.1200

pyridoxine*HCl

0.03

0.2400

riboflavin-5'-PO4(Na)*2H2O Flavin mono-nucleotide

0.03

0.2400

Thiamine

0.03

0.2400

To compound media WSC

 

TEA Soluble Components (TSC):

CHEMICAL

grams 25ml

grams 250ml

biotin

0.0150

0.1500

dl-thioctic acid (lipoic acid)

0.0150

0.1500

niacin (nicotinic acid)

0.0300

0.3000

p-aminobenzoic acid

0.0300

0.3000

pteroylglutamic acid (folic acid)

0.0300

0.3000

To compound above media TSC:

TEA STOCK

CHEMICAL

10%TEA(w/v) TEA(Triethanolamine)(2,2;,2;;-Nitrilotriethanol)

To compile stock solution:

Stock Solution KOH-10%

CHEMICAL

KOH- 10%

To compile stock solution:

Beta-sitosterol stock

CHEMICAL

AMOUNT (grams)/(50ml)

beta-sitosterol stock solution

0.2500

To construct above stock solution follow instructions below:

Essential Amino Acids (EA)

CHEMICAL

AMOUNT (grams)

l-argenine

0.9750

l-histidine

0.2830

l-isoleucine

0.8610

l-leucine

1.4390

l-lysine*HCl

1.2830

l-methionine

0.3890

l-phenylalanine

0.6230

l-threonine

0.7170

l-tryptophan

0.1840

l-valine

1.0200

To combine above media:

Nucleic Acid Substitutes

CHEMICAL

AMOUNT (grams)

adenosine-3'(2')-phosphoric acid*H2O

0.3652

cytidine-3'(2')-phosphoric acid

0.3232

guanosine-3'(2')-PO4(Na)2*H2O

0.3632

uridine-3'(2')-phosphoric acid

0.3242

or uridine-3'(2')-PO4Na

0.3681

thyamine

0.1261

To combine above media:

Other Growth Factors (GF)

CHEMICAL

AMOUNT (grams)

glutathione(reduced)

0.204

Weigh and place into a small dry covered beaker.

CHEMICAL

AMOUNT (grams)

choline dihydrogen citrate

0.0885

Weigh and place in a small dry covered beaker with myo-inositol.

CHEMICAL

AMOUNT (grams)

myo-inositol

0.0645

Weigh and place in a small dry covered beaker with choline dihydrogen citrate.

CHEMICAL

AMOUNT (grams)

cytochrome c

0.05

Weigh and place in a small dry covered beaker.

 

Energy Source (use either A. or B.)

CHEMICAL

AMOUNT (grams)

d-glucose(Source A)

32.5000

K-acetate(Source B)

5.0000

 

Non-Essential Amino Acids (NEA)

CHEMICAL

AMOUNT (grams)

glycine

0.7220

l-alanine

1.3950

l-aspartic acid

1.6200

l-cysteine*HCl*H2O

0.0280

l-glutamate(Na)*H2O

0.5500

l-glutamine

1.4630

l-phenyalanine

0.1800

l-proline

0.6530

l-serine

0.7880

l-tyrosine

0.2720

To compile above media:

Compounding CbMM Media for use:

To compound CbMM: (makes 500ml 2X solution)

 

Rules for Experimental setups:

Standard Rules to follow concerning volumes

 

Procedure for experimental tube setup:

 

Counting Experimental Cultures

RULES:

 

©Copyright by James Randolph Hocker 1997

All Rights Reserved.