A Chocolate - Not For Humans

 Introduction

Chocolate agar, also known as chocolate blood agar, is a nonselective, enriched growth medium used for the isolation of fastidious pathogenic bacteria. It is a variant of blood agar that contains lysed red blood cells. The red blood cells have been lysed by slow heating to 80°C; on heating, the colour of the medium becomes chocolate-brown. Hence, the name of the agar is derived from its colour, and it does not contain any chocolate products. The lysed red blood cells release nutrients that help fastidious bacteria grow, most notably Haemophilus and Neisseria species. With the addition of bacitracin, chocolate agar becomes selective for the genus Haemophilus. Thayer-Martin agar is a chocolate agar variant that contains an assortment of antibiotics that select for Neisseria species.

Principle

Microorganisms such as Haemophilus influenzae require growth factors such as nicotinamide adenine dinucleotide (factor V or NAD) and hemin (factor X), which are found inside red blood cells; thus, the presence of red blood cell lysates is required for these bacteria to grow. Heating the blood agar inhibits enzymes that can degrade NAD.

Chocolate agar, prepared in a laboratory or on a small scale, is a modified blood agar that has been heated to lyse the RBC. The lysis of RBC releases intracellular nutrients into the agar, such as haemoglobin, hemin (the X factor), and the coenzyme nicotinamide adenine dinucleotide (NAD or the V factor), for use by fastidious bacteria. Peptone provides nitrogen, amino acids, and other growth-promoting elements to the organism; sodium chloride maintains the osmotic balance; and agar acts as a solidifying agent.



In commercial preparation, casein and animal tissue digests provide nitrogen, amino acids, and other elements to the organism. Corn starch is added to neutralise potentially toxic metabolites because Neisseria species are highly sensitive to toxic substances such as fatty acids. Potassium phosphate aids in the maintenance of a constant pH during growth. Isovitox enrichment provides the V-factor (nicotinamide dinucleotide) and other complex compounds that enhance the growth of Neisseria species, while haemoglobin solution provides the X-factor (hemin) required by Haemophilus species.

Composition Of Chocolate Agar

Ingredients

Gm/L

Casein/animal tissue digest

15.0

Corn starch

1.0

Potassium phosphate, dibasic

4.0

Potassium phosphate, monobasic

1.0

Sodium chloride

5.0

Agar

10.0

Hemoglobin solution (2%)

500.0 ml

Isovitox enrichment

10.0 ml

 How To Prepare A Chocolate Agar Plate

  • First, make the blood agar base.

  • Sterilize it by autoclaving at 121°C for 15 minutes.

  • Add 5-7% v/v defibrinated blood (horse or sheep blood) and place the media in a 75-80°C water bath, gently swirling until the colour changes to dark brown.

  • After the medium has cooled to 50-55°C, pour it into sterile Petri plates under aseptic conditions.

A Ready-To-Use Solution

TM Media product portfolio includes Ready-to-Use Chocolate Agar Plates. The Ready-to-Use Chocolate Agar Plates are an easy-to-use, time-efficient solution. There is no need to weigh, autoclave, and cool. TM Media culture plates are gamma irradiated sterilized, which means there is no chance of contamination if opened and used properly.

Open the TM Media Chocolate Agar Plate under aseptic conditions and inoculate with the desired sample. Incubate at 37o C for 24-48 hours and interpret the results on the basis of colony characteristics.

For more products, check https://www.tmmedia.in/

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