Preparation of MacConkey Broth medium in Laboratory
   

PREPARATION OF MACCONKEY BROTH MEDIUM IN LABORATORY

The MacConkey Broth medium, which is similar to the first ever developed solid differential medium for the growth of bacteria i.e. the MacConkey Agar Medium, formulated by and is named after a British bacteriologist, Alfred Theodore MacConkey in the 20th century, contains almost same components as MAC medium except for Solidifying agent that is Agar-Agar. 

Besides being a Differential solid medium, MacConkey Broth Medium is a selective and differential medium too same as that of MacConkey Agar medium and primarily used for the isolation of Gram-negative and enteric bacilli, the rod-shaped bacteria. Moreover, MacConkey Broth Medium is not commonly used in laboratories but is an excellent medium for the differentiation of Lactose fermentors and Non-Lactose fermentors in the liquid medium and particularly for the members of Enterobacteriaceae family.



Due to the presence of a pH indicator in the medium, the colonies appear in two colors as those which ferment the lactose present in Medium produces pink color colonies due to the change in pH from neutral to Acidic whereas those which do not ferment the Lactose appears as colorless, transparent colonies.

MacConkey Broth Medium is a selective medium for the gram-negative bacterium. The presence of Bile salts inhibits the growth of gram-positive bacteria especially the Staphylococcus species.

In this article, I’m gonna explain the procedure of preparing MacConkey Broth Medium in Laboratory…..

Check out the Preparation of Sabouraud Dextrose Broth (SDB) medium in Laboratory

PRINCIPLE OF MACCONKEY BROTH MEDIUM

MacConkey Broth Medium, the selective medium for Gram-negative bacteria, the differential media that differentiate bacteria as Lactose and Non-Lactose fermentors and the Indicator medium by showing two colors of the broth medium as per the pH – in the acidic one the broth medium appears as Pink colored and in Neutral one as colorless, not routinely used in Microbiology laboratory however is an excellent medium for gram-negative bacteria consists of various components and each component plays an important role which is described below…..

Peptone A – It is an enzymatic digest of the animal tissues which is soluble in water and easily metabolized by the bacterial cell, provides the rich source of Proteins, Nutrients, Vitamins and other Nitrogenous substances to the bacterial cell for the rapid growth.

Lactose – The Lactose present in the MacConkey Broth Medium provides a rich source of carbohydrate for the rapid growth of the bacterial cell and is the basis of making the MacConkey Broth medium a differential medium, categorizing the bacteria into Lactose and Non-Lactose fermentors.

Bile salt – The Bile salts present in MacConkey Broth medium is the basis of making MacConkey Broth medium a Selective medium for Gram-negative bacteria. It inhibits the growth of most of the Gram-Positive bacteria and the Gram-Negative remains unaffected.

Sodium Chloride – It maintains the osmotic pressure in the broth medium so that the movement of molecules takes place in and out of the bacterial cell. It must be present in right proportion otherwise it will lead to the lysis of the bacterial cell.

Neutral Red – Here comes the main ingredient that makes the MacConkey Broth Medium an Indicator medium. The Neutral Red is a Dye present in the MacConkey Broth medium is the pH-dependent Dye that changes its color as Pink to Red in the Acidic medium. If the Bacterial cell is capable of fermenting the Lactose present in the broth, the pH of the medium changes to Acidic and hence the color of the broth changes to pink colored.

The final pH of the solutions is adjusted to 6.9 – 7.3 preferably the 7.1 at 25 °C. The above-mentioned components can be modified by adding the various substances in a variety of ways as per the results required so that the rapid and satisfactory growth of the bacterial cell takes place.

The methodology explained in this article is based on HiMedia Labs MacConkey Broth Medium which you can check here

Also, MacConkey Broth medium is often modified by adding various substances to suppress the growth of one type of bacteria and to promote the growth of other types of bacterial cells. Some of the modifications are described as follows:

MODIFICATIONS IN MACCONKEY BROTH MEDIUM

The MacConkey Broth Medium can be modified in no. of ways as per the requirements of the organisms and to prevent the growth of contaminants and to produce a controlled growth of certain microbes etc. The modifications described below are just for consideration and cannot be taken as the only correct methodology because the results of the final broth medium may vary as per the constituents added and the quality of the contents used. Also, the composition may vary from lab to lab as per their Standard Operating Procedure (SOPs) and the results required.

The Solid form of this medium i.e. MacConkey Agar Medium consists of many other components and is an excellent solid differential medium which is routinely used in the microbiology laboratory which you can check here…..

Check out the Preparation of MacConkey Agar Medium (MAC medium) in Laboratory

Another modification to the MacConkey Broth medium is the addition of Bromo cresol purple dye instead of Neutral red and the medium is commonly known as MacConkey Broth Purple Medium. The Bromo cresol purple dye is also an excellent indicator which turns Yellow in the Acidic pH, indicating the production of acid and ultimately the fermentation of sugar in the medium by the test organism.



MacConkey Broth Medium with Crystal Violet is an excellent modification which is used in laboratories, is a good differential medium due to the presence of crystal violet make it more selective and inhibits the growth of Staphylococcus species and Enterococcus species in the medium. The final pH of the content is same as 7.1 ± 0.2 at 25ºC.

MacConkey Broth Medium lacking Bile Salt is another preparation of MacConkey Broth medium which is used whenever there is need to prevent the swarming of Proteus species.

The Sorbitol MacConkey Broth Medium, an important modification to Traditional MacConkey Broth medium, contains Sorbitol as a source of carbohydrate instead of lactose, specifically used to differentiate Pathogenic strains of E.coli from non-pathogenic strains but commonly the Sorbitol MacConkey Agar medium is used for this purpose.

Check out the Preparation of Mueller Hinton Broth (MHA) medium for Antibiotic Sensitivity Test (AST)

MATERIALS REQUIRED FOR PREPARATION OF MACCONKEY BROTH MEDIUM

  • Sterile Conical Flask / Erlenmeyer Flask
  • Spatula
  • Peptone (Meat & Casein)
  • Lactose Monohydrate
  • Bile Salts
  • Sodium Chloride
  • Neutral Red powder
  • Measuring Cylinder
  • 1N HCl
  • 1N NaOH
  • pH Strip
  • Weighing Scale
  • Distilled Water
  • Butter Paper

PROCEDURE FOR THE PREPARATION OF MACCONKEY BROTH MEDIUM

⇒ Weigh the quantity of Peptone, Lactose, Bile salts, Sodium chloride, and Neutral red using the weighing scale for 1000 ml of MacConkey Broth Medium  as follows:

COMPONENTSQUANTITY (in grams)
Peptone A20.0
Lactose monohydrate10.0
Bile salts5.0
Sodium chloride5.0
Neutral red0.070

⇒ Put the butter paper on the weighing scale and transfer the required quantity of Peptone A on to the paper using a spatula. Repeat the step to obtain the required quantity of Lactose, Bile salts, Sodium chloride, and Neutral red powder.

Note: Use the separate piece of butter paper to avoid the errors in measurements.

AlternativelyYou can use the commercially available MacConkey Broth media powders and simply weigh the mixture of content as prescribed on the carton or box of the manufacturer. As per HiMedia, its 40.07 grams for 1000 ml medium.

⇒ Take a clean and dry Conical Flask/ Erlenmeyer flask.

Note: The size of the flask should be at least 1.5 times larger than the quantity of media you are preparing, for e.g. use 1500 or 2000 ml flask to prepare 1000ml of broth.

⇒ Pour 500 ml of distilled water to the flask and add the weighed quantity of Peptone, Lactose, Bile salts, Sodium chloride, and Neutral red. Mix well the content by swirling the flask and Heat it with continuous agitation to dissolve the constituents.

⇒ Now add more distilled water to the medium and make the volume 1000 ml.

⇒ Check the pH of the solution using pH strip, it should be 7.5 ± 0.2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.

⇒ Now Pour about 5-10 ml of the broth medium in the Test tubes and apply the Non-absorbent cotton plug to all the tubes.

⇒ Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.

⇒ Allow the content to cool down and then use the prepared broth tubes to inoculate the specimen to be cultured under a strict aseptic atmosphere (preferably in Laminar Air Flow) and then place in the incubator at optimum temperature.

Check out the Preparation of Nutrient Broth Medium (NAM) in Laboratory

PRECAUTIONS TO BE TAKEN WHILE PREPARING MACCONKEY BROTH MEDIUM

⇒ Carefully measure the quantity of Peptone, Lactose, Bile salts, Sodium chloride, and Neutral red as per the quantity of medium to be prepared.

⇒ Maintain the pH of the medium carefully. A single extra drop can change the pH of the entire solution.

⇒ Store the Medium at low temperature in dust and contamination free environment for later use.

⇒ Acid and Alkali are corrosive to the skin, handle with care.



SAHIL BATRA

Hi, I'm the Founder and Developer of Paramedics World, a blog truly devoted to Paramedics. I am a Medical Lab Tech, a Web Developer and Bibliophiliac. My greatest hobby is to teach and motivate other peoples to do whatever they wanna do in life.

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