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Dehydrated Culture Media

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HOYLE MEDIUM BASE

Code: CM0083

A modification of Neill’s medium for the isolation and differentiation of Corynebacterium diphtheriae types.

Typical Formula*

gm/litre

`Lab-Lemco’ powder

10.0

Peptone

10.0

Sodium chloride

5.0

Agar

15.0

pH 7.8 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Suspend 40g in 1 litre of distilled water and bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 55°C and add 50ml of Laked Horse Blood SR0048 and 10ml of 3.5% Potassium Tellurite solution SR0030, mix, and pour plates.

Description
Hoyle medium is the well known modification1 of Neill’s medium for the cultural isolation and differentiation of Corynebacterium diphtheriae types. Hoyle medium does not exert the inhibitory effect manifested by Neill’s on some mitis types, but gives very rapid growth with all types of Corynebacterium diphtheriae, so that diagnosis is possible after 18 hours’ incubation.

Technique
This is a highly selective medium which is used in parallel with non-selective media such as blood agar (e.g. Blood Agar Base CM0055 with 10% of horse blood). Unlike non-selective media, Hoyle medium should be inoculated by rubbing the throat swab (or other material) over the entire surface - spreading with a platinum loop is not necessary.

Normally incubation for 18 hours at 35°C is sufficient but, when a negative result is obtained, incubation for up to 72 hours may be advisable. Gentian violet stained films made from colonies picked straight off the medium, are satisfactory for Corynebacterium diphtheriae morphology. For the demonstration of the characteristic morphology and staining reactions of Corynebacterium diphtheriae by Neisser’s or Albert’s method, it is preferable to utilise colonies from Loeffler medium. The toxigenicity of Corynebacterium diphtheriae strains may be determined by the Elek2 method.

Colonial characteristics
It is best to examine with a low-power microscope, the colonies being illuminated from above by daylight.

Type differentiation is good, and typical colonial appearances after 18 hours’ incubation are as follows:
Corynebacterium diphtheriae type gravis - grey colonies, 1.5-2.5mm diameter dull, matt surface. May be slightly umbonate and show indented margins. Colonies can be pushed along the surface of the medium.
Corynebacterium diphtheriae type mitis - grey colonies, 1.5-2.0mm diameter with regular margins and shining surface. Variation in size common.
Corynebacterium diphtheriae type intermedius - grey colonies, 0.5-0.75mm diameter with shining surface. Colonies are very uniform in size with darker centres.
Corynebacterium hofmannii - usually rounded, white or greyish, 0.5-0.75mm diameter. Colonies may be up to 1mm diameter, when they are black in more heavily inoculated areas and white when well isolated.
Corynebacterium xerosis - black shining colonies of variable size.
Streptococci - minute black or brownish-black colonies.
Other organisms may occasionally grow which resemble Corynebacterium diphtheriae type intermedius but are larger, while sporing anaerobes may produce brownish mucoid colonies.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

Positive controls:

Expected results

Corynebacterium diphtheriae biotype gravis
ATCC® 19409

Good growth; dull grey/black colonies

Corynebacterium diphtheriae biotype intermedius ATCC® 14779

Good growth; shiny grey/black colonies

Negative control:

 

Uninoculated medium

No change

* This organism is available as a Culti-Loop®

Precautions
It should be noted that not all corynebacteria produce the typical colonies described above - so in all cases it is advisable to use Hoyle medium in conjunction with the additional media and tests mentioned above.

References
1. Hoyle L. (1941) Lancet. i. 175-176.
2. Elek S. D. (1948) Brit. Med. J. 1. 493-496.

 
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