SOP: Preparation of Bacteriological Media

GIFSQ — Global Institute for Food Safety & Quality

Principle

A culture medium must supply the nutrients the organisms need, at the correct acidity, in a sterile container. The ingredients are therefore dissolved in a fixed order (agar last, since it needs heat to dissolve), the pH is adjusted to the required value and checked, the medium is filtered and dispensed into plugged tubes, and it is sterilized by a heat treatment that kills vegetative cells and spores without destroying heat-sensitive ingredients such as sugars.

Apparatus required

• Hot water bath (for dissolving agar)
• pH meter, or the indicator set-up below (test tubes of uniform wall thickness and bore, six-hole comparator block, colour standards — sealed coloured-liquid tubes or coloured glass discs — test tubes of distilled water and of plain broth)
• Burette (for adding NaOH)
• Filter paper or Buchner suction funnel (for liquid media)
• Cotton wrapped in gauze (for filtering melted agar)
• Glass funnel on a stand, fitted with rubber tubing, a pinch cock and a tapered delivery tube (for tubing of media)
• Lid of a large sterile Petri dish (to cover the funnel)
• Arnold sterilizer (“steamer”) (for intermittent heating of sugar-containing media)
• Autoclave
• Bottles with screw caps, flasks, or culture bottles and screw-capped jars (for storage)
• Petri dishes (for plating)

Reagents

a. Medium ingredients: salts, peptone, beef extract and similar soluble ingredients; agar (added last).

b. Indicator solution, 0.02 to 0.04% — for pH adjustment; the book’s indicator table lists the suitable indicators for each range (that table is not part of this chapter).

c. 0.1 N sodium hydroxide — added dropwise from a burette for fine pH adjustment.

d. 1 N sodium hydroxide — added in calculated quantity for bulk pH adjustment.

Procedure — Preparing the medium

Add the soluble ingredients (salts, peptone, beef extract, etc.) first, and the agar last, to the required amount of cold water

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Dissolve the agar in a hot water bath, never over an open flame

Procedure — Adjusting the pH

Pipette 10 ml of the solution of unknown pH into a clean test tube

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Add 0.5 ml of the 0.02 to 0.04% indicator solution and mix carefully

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Place the tube in the central hole of the six-hole comparator block, with a test tube of distilled water behind it

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In the slot to the right of the unknown, insert the colour standard nearest in colour to the unknown, with a tube of broth (no indicator) behind it

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In the slot to the left of the unknown, insert a colour standard 0.2 pH units higher, with a tube of broth behind it

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Look through the block at a good light source (preferably daylight) and match the colours; read the pH from the standard that most closely matches the unknown

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If the medium is acid, add 0.1 N NaOH dropwise from a burette until the colour for the desired pH is obtained, and note the quantity used

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From the 0.1 N NaOH used for the known volume, calculate the amount of 1 N NaOH required for the whole batch, add it, and recheck the pH

Note: a pH meter may be used instead of the indicator and comparator block.

Procedure — Filtering the medium

Filter liquid media through filter paper or a Buchner suction funnel

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Filter melted agar (80–90°C) through cotton wrapped in gauze

Procedure — Tubing of media

Fill the test tubes using the glass funnel on a stand, fitted with rubber tubing, pinch cock and tapered delivery tube

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Cover the funnel with the lid of a large sterile Petri dish to keep out aerial contamination

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Plug the filled tubes with cotton wool or fit them with aluminium caps (screw-capped culture bottles or jars may be used instead)

Procedure — Sterilizing the medium

For media containing sugars, use discontinuous (intermittent) heating: heat at 100°C or less for 20–30 min on each of 2 or 3 successive days — the intervals let spores germinate, so the next heating kills the cells that grew from them

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Use the Arnold sterilizer (“steamer”) for this; an autoclave with the steam escape valve left wide open (so no steam pressure develops) may be used instead

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For media without sugars, use steam under pressure in the autoclave: usually 20 min at 15 psi is sufficient

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When autoclaving, expel all air before letting the pressure rise, and raise or lower the pressure slowly

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At the end of sterilization, release the pressure gradually

Procedure — Storing the medium

Store the medium in bulk in bottles or flasks, or distribute it into tubes

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Bottles of suitable size with screw caps may be used; sterilize the bottles with the caps screwed on tightly

Procedure — Plating the medium

Pour the melted agar medium into the Petri dish, lifting the lid just enough to let the medium in

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Let it solidify as a thin layer — about 10–15 ml of medium is enough for a 10 cm dish

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Allow the medium to set for a few hours

Precautions

• Do not heat media containing sugars longer than the required time at reduced pressure (10 lb): prolonged heating hydrolyzes many carbohydrates, especially in alkaline or very acidic media.
• Add the agar last and dissolve it in a hot water bath; an open flame can scorch it.
• Release autoclave pressure gradually at the end of sterilization, so the plugs are not blown out and the cotton is not moistened by boiling medium.
• Recheck the pH after the bulk NaOH addition; do not rely on the calculation alone.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.