9.3: MacConkey Media, MAC
- Discuss the purpose of MAC media for selective and differential colony growth.
- Inoculate MAC agar plates using aseptic technique.
- Determine rates of lactose fermentation in gram negative bacteria.
MacConkey Media
Mac Conkey Media, or MAC , is a differential and selective agar used to isolate bacteria in the Enterobacteriaceae family.
The Enterobacteriaceae family includes " enteric " or intestinal bacteria typically found in the human intestines. These bacterias are gram-negative facultative anaerobes who have adapted to the presence of intestinal bile salts and are capable of migrating to the urinary tract to cause infections. Enterobacteriaceae also have different rates of lactose fermentation, making the bacteria either a coliform or non-coliform class. Coliform bacteria, like E. coli, can rapidly and completely ferment lactose. This rapid fermentation produces acid as a by-product, which can alter the coliform's colony morphology and surrounding environment. Non-coliform bacteria either slowly and incompletely ferment lactose or they are unable to ferment lactose at all. Non-coliform bacteria, like Yersinia pestis, are more likely to produce very low, negligible levels of acid or none at all. Differences in lactose fermentation of Enterobacteriaceae contribute to colony morphology and media appearance when using MAC agar plates.
Figure 1. Examples of coliform and non-coliform bacteria.
MAC is a selective media as it has the ability to inhibit gram positive growth. MAC media is made with crystal violet dye and bile salts which are capable of inhibiting most gram-positive bacterial growth. Gram-negative enteric bacteria are not affected as these species normally grow in the presence of bile salts in the human gut.
MAC is a differential media based on the ability of the bacteria to ferment lactose present in the agar. MAC media is also made using a pH indicator called Neutral Red, and this indicator can change the color of the bacterial colonies based on their fermentation rate. Rapid lactose fermenters, enteric coliforms, will produce high levels of acid during lactose fermentation. High acid production will lower the pH of the MAC media and, once the pH drops below 6.8, the neutral red indicator will activate and turn the media a bright red/pink color. The Neutral Red will also be absorbed by the bacteria, changing colony color to the same bright red/pink color of the media. Bacteria that can not ferment lactose, non-coliforms, must use other nutrients for energy production. Instead of lactose, non-coliform bacteria can use nitrogen sources like peptone. Peptone fermentation will produce ammonia as a by-product instead of acid. Ammonia will raise the pH of the MAC agar, Neutral Red will not activated, and bacterial colonies will be white or colorless. Under these conditions, the MAC agar will also turn yellow in color.
Figure 2. Gram negative bacteria that can grow on MAC agar will have different colony colors depending on their ability to ferment lactose. Rapid lactose fermenters will produce high quantities of acid, turning the media red. Bacteria that can not ferment lactose will be forced to find other energy sources and will not produce acid. These colonies will remain colorless and the agar may turn yellow in color.
Pseudomonas aeruginosa
The genus Pseudomonas contains a diverse group of gram-negative rod-shaped bacteria. Many Pseudomonas species are pathogenic for humans including Pseudomonas aeruginosa. P. aeruginosa is an opportunistic pathogen that targets patients with cystic fibrosis and can cause urinary tract infections (UTIs). (Openstax, 2024) The CDC estimates that P. aeruginosa causes approximately 30,000 infections and 2,700 deaths annually. (CDC, 2024) P. aeruginosa infections are difficult to treat as the bacteria is highly resistant to antibiotics. One diagnostic tool to determine if an infection is caused by P. aeruginosa is MacConkey Agar.
MacConkey agar is made with the sugar lactose and pH indicator Neutral Red. This agar will promote the growth of gram-negative species like P. aeruginosa while differentiating species based on lactose fermentation and pH. Patients suffering from a UTI suspected to be caused by P. aeruginosa will first need submit a clean urine sample. The patient must first wash their urethral opening prior to providing the urine sample to prevent contamination by their normal microbiota. The uncontaminated urine sample will then be grown on MacConkey agar.
P. aeruginosa can not ferment lactose. This means the bacteria will not alter the color of the agar plate. Thus, if a patients urine sample on MacConkey comes back positive for growth but has no change to the color of the media, the UTI is likely the result of a P. aeruginosa infection. P. aeruginosa cells may produce a blue-green pigment called pyocyanin in response to environmental stress when grown on MacConkey, further helping identify the microbe.
Figure 3. Pseudomonas aeruginosa can grow on MAC as it is a gram-negative bacterium. The bacteria can not ferment lactose,and will not alter the color of the media (black arrows). P. aeruginosa may become stressed from growth on MAC, however, and start to produce a blue-green pigment called pyocyanin (white arrows). Pyocyanin helps the bacteria consume more iron in low-iron environments like MAC agar.
Attributions
"Microbiology Labs II: Results, 12.6.1 MacConkey Agar" by Dr. Gary Kaiser , LibreTexts: Biology , Community College of Baltimore County, Catonsville Campus is licensed under CC BY 4.0
"Microbiology Labs II: Pure Cultures, 3.4 Use of Specialized Media" by Dr. Gary Kaiser , LibreTexts: Biology , Community College of Baltimore County, Catonsville Campus is licensed under CC BY 4.0
"Bio 221 Lab: Physiological Tests, 22.2 Selective and Differential Media-MacConkey, EMB, MSA" by Kelly Burke , LibreTexts: Biology , College of The Canyons is licensed under CC BY
"Microbiology Textbook: Chapter 9, Microbial Growth" by Openstax , Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0
"Microbiology Textbook: Chapter 4, Prokaryotic Diversity" by Openstax , Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0
"About Pseudomonas aeruginosa" by Centers for Diseases Control and Prevention , Use of CDC Materials Statement is in the Public Domain
"Microbiology Textbook: Chapter 12, Modern Applications of Microbial Genetics" by Openstax , Digital ISBN: 13: 978-1-947172-23-4 is licensed under CC BY 4.0
"Microbiology Textbook: Chapter 21, Skin and Eye Infections" by Openstax , Digital ISBN: 13: 978-1-947172-23-4 is licensed under CC BY 4.0
Image Citations
Figure 1, Modified From:
- "E. coli Bacteria (7316101966).jpg" by National Institute of Allergy and Infectious Diseases , Wikimedia Commons is in the Public Domain
- "Yersinia pestis scanned with electron micrograph.jpg" by National Institute of Allergy and Infectious Diseases , Wikimedia Commons is in the Public Domain
Figure 2, Modified From:
- "MacConkey agar with LF and LF colonies.jpg" by Medimicro, Wikimedia Commons is in the Public Domain
Figure 3, Modified From:
- "Microbiology Labs II: Results, 12.6.1 MacConkey Agar" by Dr. Gary Kaiser , LibreTexts: Biology , Community College of Baltimore County, Catonsville Campus is licensed under CC BY 4.0