The first ever developed solid differential medium for the growth of bacteria on the globe was MacConkey Agar Medium (MAC medium), was formulated by and is named after a British bacteriologist, Alfred Theodore MacConkey in the 20th century.
Besides being a Differential solid medium, MacConkey Agar Medium is a selective and Indicator medium too and primarily used for the isolation of Gram-negative and enteric bacilli, the rod-shaped bacteria. Moreover, MAC is the most commonly used media for the differentiation of Lactose fermentors and Non-Lactose fermentors and particularly for the members of Enterobacteriaceae family.
MacConkey Agar Medium is a selective medium for the gram-negative bacterium. The presence of Bile salts and Crystal violet inhibits the growth of gram-positive bacteria especially the Staphylococcus species. MAC is also commonly used for the isolation of Pseudomonas species which appears as the green-brown colonies (Pseudomonas aeruginosa) on MacConkey agar medium.
The most widely used differential medium in the laboratories for the differentiation of Bacteria is MacConkey Agar Medium (MAC) medium which differentiates the bacteria in two categories as Lactose & Non-Lactose fermentors.
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.
In this article, I’m gonna explain the procedure of preparing MacConkey Agar Medium in Laboratory…..
PRINCIPLE OF MACCONKEY AGAR MEDIUM
MacConkey Agar Medium (MAC), 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 colonies as per the pH – the acidic one as Pink colored and Neutral one as colorless routinely used in Microbiology laboratory consists of various components and each component plays an important role which is described below…..
Peptone – It is a semi-digested protein which is soluble in water and easily metabolized by the bacterial cell, provides the rich source of protein to the bacterial cell of the rapid growth.
Pancreatic digest of gelatin – Similar to Peptone, the Pancreatic Digest of Gelatin is a rich source of Nutrients, Vitamins, and Nitrogenous substances that support the rapid growth of the bacterial cell.
Lactose – The Lactose present in the MacConkey Agar Medium (MAC) provides a rich source of carbohydrate for the rapid growth of the Bacterial cell and is the basis of making the MAC a differential medium, categorizing the bacteria into Lactose and Non-Lactose fermentors.
Bile salt – The Bile salts present in MacConkey Agar medium (MAC) is the basis for making MAC a Selective media. 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.
Crystal Violet – Similar in action to the Bile salts, the Crystal violet present in MacConkey Agar Medium (MAC) makes the medium selective for Gram-Negative Bacteria by inhibiting the growth of Gram-Positive Bacteria.
Neutral Red – Here comes the main ingredient that makes the MacConkey Agar Medium an Indicator medium. The Neutral Red is a Dye present in the MAC 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 MAC, the pH of the medium changes to Acidic and hence the colonies appear as Pink colored.
Agar-Agar – It is often called as Agar, is a complex polysaccharide, a carbohydrate consisting of 3, 6-anhydro-L-galactose and D-galactopyranose, free of nitrogen, produced from various red-purple algae belonging to Gelidium, Gracilaria, Gigastina etc. It liquefies on heating to 96 °C and hardens into a jelly on cooling 40-45 °C. It basically solidifies the medium and commonly used in the microbiology laboratory.
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 Agar (MAC) Medium which you can check here
Also, MacConkey Agar 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 AGAR MEDIUM
The MacConkey Agar 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. However, the composition may vary from lab to lab as per their Standard Operating Procedure (SOPs) and the results required.
The liquid form of this medium i.e. MacConkey Broth Medium consists of only some of these contents as Pancreatic Digest of gelatin (source of vitamin & nitrogenous substances), Lactose (Source of Carbohydrate), dehydrated bile (selective action) & Bromo cresol purple (Indicator). The final pH of the content is 7.3 ± 0.2 at 25ºC.
MacConkey Agar Medium without Crystal Violet is commonly used in laboratories, a good differential medium but lack of crystal violet make it less selective and do not inhibit the growth of Staphylococcus species and Enterococcus species. The final pH of the content is same as 7.1 ± 0.2 at 25ºC.
MacConkey Agar Medium lacking Crystal violet and Bile Salt is another preparation of MAC medium which is used whenever there is need to prevent the swarming of Proteus species.
The Sorbitol MacConkey Agar Medium, an important modification to Traditional MacConkey agar medium, contains Sorbitol as a source of carbohydrate instead of lactose, specifically used to differentiate Pathogenic strains of E.coli from non-pathogenic strains.
MATERIALS REQUIRED FOR PREPARATION OF MACCONKEY AGAR MEDIUM
- Sterile Conical Flask / Erlenmeyer Flask
- Peptone (Meat & Casein)
- Pancreatic Digest of Gelatin
- Lactose Monohydrate
- Bile Salts
- Sodium Chloride
- Crystal violet powder
- Neutral Red powder
- Agar-Agar powder
- Measuring Cylinder
- 1N HCl
- 1N NaOH
- pH Strip
- Weighing Scale
- Distilled Water
- Butter Paper
PROCEDURE FOR THE PREPARATION OF MACCONKEY AGAR MEDIUM
⇒ Weigh the quantity of Peptone, Lactose, the Pancreatic digest of gelatin, Bile salts, Sodium chloride, Crystal violet, Neutral red and Agar using the weighing scale for 1000 ml of MacConkey Agar Medium (MAC) as follows:
COMPONENTS QUANTITY (in grams)
Peptones (meat and casein) 3.0
Pancreatic digest of gelatin 17.0
Lactose monohydrate 10.0
Bile salts 1.5
Sodium chloride 5.0
Crystal violet 0.001
Neutral red 0.030
⇒ Put the butter paper on the weighing scale and transfer the required quantity of Peptone on to the paper using a spatula. Repeat the step to obtain the required quantity of Lactose, a Pancreatic digest of gelatin, Bile salts, Sodium chloride, Crystal violet, Neutral red and Agar-Agar powder.
Note: Use the separate piece of butter paper to avoid the errors in measurements.
Alternatively, You can use the commercially available MacConkey Agar media powders and simply weigh the mixture of content as prescribed on the carton or box of the manufacturer. As per HiMedia, its 49.53 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, Pancreatic digest of gelatin, Bile salts, Sodium chloride, Crystal violet and Neutral red. Mix well the content by swirling the flask.
⇒ Now add the weighed quantity of Agar-Agar to the above solution.
⇒ Mix well the content 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 7.1 ± 0.2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
⇒ Mix well the content and apply the Non-absorbent cotton plug to the flask.
⇒ Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
⇒ Allow the content to cool down to 40-45 °C and pour in the empty media plates under a strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature.
⇒ Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.
PRECAUTIONS TO BE TAKEN WHILE PREPARING MACCONKEY AGAR MEDIUM
⇒ Carefully measure the quantity of Peptone, Lactose, a Pancreatic digest of gelatin, Bile salts, Sodium chloride, Crystal violet, Neutral red and Agar-Agar 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.