Preparation of a holidic medium for Drosophila melanogaster

• Protocol
• Discussion

Authors: Matt Piper & Linda Partridge

Abstract

We describe a novel holidic medium for Drosophila melanogaster that has been optimised for adult fitness traits. Development is also possible on this medium, albeit at a reduced rate.

On this medium, adult egg laying, lifespan and activity patterns are no different from those of flies on a sugar yeast diet.

Introduction

Drosophila melanogaster feeds on fermenting fruit.

Here we describe a synthetic, fully chemically defined (holidic) medium that it is sufficient for fruitfly development, albeit at a reduced rate. When adult flies are maintained on the diet, they are phenotypically similar to those kept on a natural yeast based (oligidic) diet. The holidic medium can produce more stable experimental outcomes than oligidic media, thus potentially improving inter-laboratory comparability. This medium also offers the opportunity to investigate the effects of subtle nutrient manipulations that could not be achieved otherwise.

Materials

Table 1: Ordering list for holidic diet components

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A list of holidic medium components with order numbers. These are supplied in an attempt to standardise how people make the media and enhance replication of data between laboratories and trials.

UPDATE FROM AUTHORS AUGUST 2014: we have become aware that catalogue number p2250 (D-Pantothenic acid hemicalcium salt) has been discontinued by Sigma. We have replaced this item with Sigma’s recommended substitute, order number: 21210. After one month of use, we have not observed any obvious differences in the flies indicating this is OK.

Equipment

1. silicone tubing
2. autoclave
3. autoclave-resistant bottles
4. magnetic stirrer
5. sterile syringes
6. 0.22 micron syringe-mounted filters

Procedure

IN ADVANCE:

1. purchase iungredients according to order sheet (Table 1)
2. prepare each of the stock solutions using the qauntities and according to the directions shown in Table 2, Table 3 and Table 4

MEDIA

Preparation of the medium is performed in two stages. (also see Table 5: example working recipes)

I. before autoclaving

1. in a 1l, autoclave-proof, glass bottle, mark on the side the level of the target pre-autoclave volume (=1 litre minus the sum of the ingredients to be added after autoclaving)
2. add magnetic stirrer flea to bottle
3. to milliQ water, add sucrose and low solubility amino acids (L, I, Y)
4. add stock solutions for buffer base and metal ions
5. stir well (may not fully dissolve at this stage)
6. add cholesterol stock solution (solution will turn cloudy)
7. add agar

II. autoclave at 120 °C for 15 min

(at the same time, autoclave dispensing tubing and any glass vials or bottles to house the medium)

III.

(all steps with constant stirring)

1. allow solution to cool at room temperature with stirring to ~65 °C
2. add sterile stock solutions in order: amino acids, nucleosides/choline/inositol mix, vitamins and preservatives
3. using sterile tubing, dispense the solution into sterile vials
4. cover the vials and allow to cool for 90 min at room temperature
5. stored at 4 °C until use

Timing

• Stock solution preparations: ~30 – 60min each
• medium preparation before autoclaving ~30 min
• autoclaving (variable depending on model) ~90 min
• medium mixing after autoclaving ~15 min
• dispensing medium ~30 min
• time to cool ~90 min

Troubleshooting

• MEDIUM DOESN’T SET:
  • Try sterilising the buffer base separately from the medium and adding it after autoclaving.

Acknowledgements

In addition to the authors, the following people contributed significantly to the development of the medium:

Eric Blanc, Ricardo Leitão-Gonçalves, Mingyao Yang, Xiaoli He, Nancy Linford, Matthew Hoddinott, Corinna Hopfen, George Soultoukis, Christine Niemeyer, Fiona Kerr, Scott Pletcher, Carlos Ribeiro

Figures

Table 2: Stock solutions part 1 (those added before autoclaving)

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These stock solutions are to be made in advance of medium preparation. This batch are added to the medium before autoclaving.

Table 3: Stock solutions part 2 (added after autoclaving)

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These tables contain the stock solutions to be added after autoclaving. In most cases they are filter sterilised using a syringe-mounted filter. They are added after autoclaving to avoid nutritional loss upon heating.

Table 4: Stock solutions part 3 (added after autoclaving)

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These are amino acid stock solutions. Note, there are two tables here, each containing all 20 amino acids, but in differet ratios. The Yaa ratio is that most similar to what is found in yeast.

Table 5: example working recipe

Download Table 5

These recipes describe the ingredient list and quantities required to make up holidic media for HUNTaa and Yaa amino acid mixtures. Note, there are three recipes for each corresponding to different final amino acid levels in the media. Modifying these levels will alter adult fitness measures.

Associated Publications

A holidic medium for Drosophila melanogaster Matthew D W Piper, Eric Blanc, Ricardo Leitão-Gonçalves, Mingyao Yang, Xiaoli He, Nancy J Linford, Matthew P Hoddinott, Corinna Hopfen, George A Soultoukis, Christine Niemeyer, Fiona Kerr, Scott D Pletcher, Carlos Ribeiro, and Linda Partridge Nature Methods doi:10.1038/nmeth.2731

Author Information

Matt Piper, Piper Lab

Linda Partridge, University College London

Correspondence to: Matt Piper ([email protected])

Source: Protocol Exchange (2013) doi:10.1038/protex.2013.082. Originally published online 18 November 2013.