1.17: MSA, MacConkey, EMB Plates
The student will:
- Use aseptic techniques in the safe inoculation of various forms of media.
- Follow oral and written instructions and manage time in the lab efficiently.
- Apply correct terminology regarding microbiological techniques, instruments, microbial growth, biochemical testing, and media types when making observations.
- Correctly perform various inoculation techniques and describe each technique’s purpose.
- Make accurate observations and appropriate interpretations of biochemical test results and use them in the identification of potentially disease causing microbes.
In this course you will encounter many types of growth media. Most of the types, TSA/TSB (Tryptic Soy Agar/Broth) and NA/NB (Nutrient Agar/Broth) for example, are all purpose or general media . They contain a wide variety of complex carbon, nitrogen and sugar compounds. You will often see terms like beef extract, peptone, tryptone, soytone in the list of ingredients. These components are slurries of animal and plant tissue that have been partially hydrolyzed (broken down) so that the components are more readily available. These media contain many different complex molecules, however the exact amount and types of each is unknown. These media will support a wide range of nonfastidious microbes with differing nutritional requirements.
Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition are called selective media . (OpenStax CNX, 2018) Selective media are formulated with inhibitors such as antibiotics or high NaCl concentration. When studying a mixed sample, selective media can be helpful. For example, if you suspect a patient is carrying Salmonella (a pathogenic Gram negative bacillus), you may plate a stool sample on a selective medium containing an antibiotic effective against Gram positive bacteria. By eliminating the Gram positive organisms, the range of organisms growing on the plate will be narrowed to Gram negatives. Thus, the variety of bacteria you will need to study is reduced.
The fact that a medium does not grow every microbe, does not make it selective. For example, TSA is an all-purpose medium and a wide range of organisms grow on it. Certain fastidious organisms, however, will fail to grow or grow poorly on TSA because it lacks the specific nutrients required by those bacteria. Even so, TSA is not classified as selective. To be selective, a medium must contain a specific substance intentionally added to inhibit certain microbes and not others.
Differential media contain substrates and indicators (often pH indicators) that make a certain biochemical process visible. Differential media allow one to differentiate between types of organisms growing on the plate because each has a distinct appearance based on whether or not it is carrying out a particular biochemical reaction. “Color changes are the result of end products created by interaction of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium” (OpenStax CNX, 2018). Differential media can be used to distinguish between bacteria that can ferment a specific type of sugar and those that cannot or between bacteria that utilize a certain electron acceptor and those that do not.
Some media are selective, some are differential and some are both. We will study Mannitol Salt Agar, MacConkey agar, and EMB as examples.
Mannitol Salt Agar (MSA) can be used to presumptively isolate and identify Staphylococci from human samples. Refer to the compositions of MSA and MacConkey agar below. MSA contains 75 g/L NaCl (7.5%) compared to the 5 g/L found in TSA and other all-purpose media. MSA favors the growth of salt tolerant microbes, namely Staphylococci , because other bacteria from a human sample, are inhibited by the high NaCl component. In addition, to distinguish pathogenic Staphylococci, namely S. aureus from other common Staphylococci, the substrate mannitol (a sugar) and the pH indicator phenol red are added. If the organism ferments mannitol, acids will be produced as byproducts. These acids will lower the pH changing the indicator from pink to yellow. S. aureus can ferment mannitol, while other common Staphylococci found in humans cannot.
| MS Agar | ||
| Selectivity | Interpretation | Identification |
| Growth | Organism not inhibited by NaCl | E.g., Staphylococcus , Micrococcus |
| No growth | Organism inhibited by NaCl | Not Staphylococcus |
| Differentiation | ||
| Yellow halo | Organism ferments mannitol | Probable S. aureus |
| No yellow halo |
Organism does not ferment
mannitol |
Staphylococcus species (other than S. em>aureus); Micrococcus (yellow colonies) |
MacConkey agar contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria, particularly the Enterobacteriaceae . These species, commonly named enterics, reside in the intestine, and are adapted to the presence of bile salts. Enterics can be further characterized by their ability to ferment lactose. In MacConkey agar, the lactose fermenters ( coliforms ) utilize lactose in the medium producing acid, lowering the pH. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. (OpenStax CNX, 2018) Thus, lactose fermenters are observed as bright pink colonies or with a bright pink halo surrounding the growth. Non-lactose fermenters (noncoliforms) include some notable human pathogens, such as Salmonella spp., Shigella spp., and Yersinia pestis . (OpenStax CNX, 2018)
Eosin-methylene blue agar (EMB) contains peptone, lactose, sucrose and the dyes eosin Y and methylene blue. Gram positive organisms are inhibited by the dyes, so this medium is selective for Gram negative bacteria. The medium differentiates based on the ability to ferment lactose (and/or sucrose.) Organisms that cannot ferment either of the sugars produce colorless colonies. Organisms that ferment the sugars with some acid production produce pink or purple colonies; organisms that ferment the sugars and produce large amounts of acid form colonies with a green metallic sheen. This medium is commonly used to detect the presence of fecal coliforms (like E. coli )—bacteria that grow in the intestines of warm-blooded animals. Fecal coliforms produce large amounts of acid when fermenting lactose and/or sucrose; non-fecal coliforms will produce less acid and appear as pink or purple colonies.
| EMB Agar | ||
| Result | Interpretation | Identification |
| No or poor growth | Organism inhibited by dyes | Organism is Gram-positive |
| Good growth |
Organism not inhibited by
dyes |
Organism is Gram-negative |
| Colorless growth |
Organism does not ferment
sucrose or lactose |
Non-coliform |
| Growth is pink and mucoid |
Organism ferments lactose
and/or sucrose with some acid production |
Coliform bacteria |
|
Growth is dark (purple to black with or without
green metallic sheen) |
Organism ferments lactose and/or sucrose, with large
amounts of acid production |
Possible fecal coliform ( E. coli ) |
Coliform bacteria are microbes found in the digestive systems of warm-blooded animals, in soil, on plants, and in surface water. (Note their ability to assist mammals in the digestion of milk sugar, lactose.) These microbes typically do not make you sick; however, because microbes that do cause disease are hard to test for in the water, “total coliforms” are tested instead. If the total coliform count is high, then it is very possible that harmful germs like viruses, bacteria, and parasites might also be found in the water. Thus, they are considered one of several a water quality indicators. (U.S. Centers for Disease Control and Prevention, 2019)
MacConkey Agar (MacC) Pancreatic digest of Gelatin 17g/L Peptones (meat and Casein) 3g/L Lactose 10g/L
Bile Salts 1.5g/L Sodium Chloride 5g/L Agar 13.5g/L
Neutral red 0.03g/L Crystal Violet 1mg/L
Mannitol Salts Agar (MSA) Pancreatic digest of Casein 5g/L Peptic digest of Animal Tissue 5g/L Beef extract 1g/L
Sodium Chloride 75g/L D-Mannitol 10g/L Phenol red 25mg/L Agar 15g/L
Tryptic Soy Agar (TSA)
Tryptone 17g/L Soytone 3g/L
Sodium Chloride 5g/L Dipotassium Phosphate 2.5g/L Agar 15g/L
Materials per student
1 EMB plate*
1 MSA plate*
1 MacConkey Agar plate*
* Be sure to label each as you take it. They look very similar!
Cultures
Fresh overnight broth cultures
E. coli
Staphylococcus epidermidis
Staphylococcus aureus
Pseudomonas aeruginosa
Procedure Lab 1
- Obtain one EMB plate, one MSA plate and one MacConkey agar plate. On the plate base write the medium abbreviation when you remove it..
- Add the following to the bottom of each plate around the edge: your name, date.
- Divide each plate into 4 sections. Label each section with an abbreviation for each organism. Write small but legibly! Each plate will be inoculated with each of the 4 organisms.
- Aseptically spot inoculate each sector with the corresponding microbe. A spot inoculation is a short (1 cm) streak line as shown. (DO NOT STAB the agar.)
- Keep the inoculation lines short and away from the other inoculations on the plate.
- Be sure to make each inoculation separately and refrain from “double dipping.”
- Be sure to hold the lid of the plate above the plate surface to protect it from airborne contaminants.
- It may help to set the plate on a piece of white scratch paper so that you can see the sector lines.
- Place the plates upside-down in the location designated for cultures to be incubated.
- They will be incubated for 24-48 hours at 37 o C.
Procedure Lab 2
- Obtain your plates and make observations in the data table.
- Use + for growth and – for no growth. If growth is poor, simply write “poor growth” in the table.
- In the appearance column, describe any color change in the growth and/or the surrounding medium. If the organism did not grow, you will not be able to describe an appearance. In this case write “N/A” in the table.
- Be sure to use the organism’s full scientific name written correctly.
- After making observations, dispose of your plates in the hazardous waste.
References:
OpenStax CNX. (2018, Mar 19). OpenStax Microbiology. Retrieved from http://cnx.org/contents/e42bd376-624b-4c0f-972f-e0c57998e765@4.24
U.S. Centers for Disease Control and Prevention. (2019, June 30). Well Testing . Retrieved from Healthy Water: https:// www.cdc.gov/healthywater/drinking/private/wells/testing.html