Exercise 12: Selective and Differential Plates

Introduction

Most clinical and environmental specimens begin as a mixed culture containing many different species. Each species must be isolated and cultivated as pure cultures before they can be properly identified. The isolation streak is the most common means of isolating an organism from a mixed culture. Selective media are designed to enhance the isolation procedure by inhibiting (discouraging) growth of some organisms while promoting the growth of others. Some types of media also contain indicators to reveal metabolic differences between organisms. In this case we refer to them as differential media.

Clinical microbiologists generally look for specific pathogens that are associated with human infections. They will start by choosing selective and differential media that will inhibit the normal microflora that are also likely to be in the patient sample. Environmental microbiologists use selective and differential media to detect coliform bacteria. Coliforms are a subgroup of Enterobacteriaceae that produce acid and gas from lactose. Most coliforms are normal microflora of the human intestinal tract. When found in water samples their presence indicates fecal contamination.

The physiological characteristics of bacteria are the result of enzymatic reactions that are used in metabolic pathways. Certain divisions, genera and species of bacteria use only specific metabolic pathways, and therefore, specific enzymes. Differential tests are really looking for the ability of an organism to make a particular enzyme or group of enzymes. Indicators added to the media will cause a recognizable color change in or around the organism that is a result of the action of the enzyme. The most common indicators used change colors based on the pH of the medium. Phenol red is an example: it is orange at a neutral pH, turns yellow in acid and pink when conditions are alkaline.

In this lab you will use four different types of selective and differential media: mannitol salt agar (MSA), MacConkey Agar (MAC), Eosin Methylene Blue Agar (EMB) and xylose lysine desoxycholate agar (XLD). Each has a slightly different composition of indicators and selective agents.

• Manitol Salt Agar (MSA): This medium contains 7.5% NaCl and is selective for members of the genus Staphylococcus due to their salt tolerance. It further allows differentiation based on the ability to ferment the carbohydrate mannitol. Organisms that produce acid from mannitol will turn yellow.
• MacConkey Agar (MAC): This medium is commonly used as selective and differential media for Enterobacteriaceae. It contains lactose, bile salts, neutral red, and crystal violet. The bile salts and the crystal violet inhibit the growth of Gram-positive bacteria. Organisms that ferment lactose to acid end products lower the pH and their colonies turn pink to red color.
• Eosin Methylene Blue Agar (EMB): This medium is a complex, selective, and differential medium. It contains digest of gelatin, lactose, and the dyes eosin Y and methylene blue. Lactose is fermented to acid end-products by coliforms such as Escherichia coli and Enterobacter aerogenes, whereas it is not by pathogens such as Proteus, Shigella, and Salmonella species. The dyes inhibit the growth of Gram-positive bacteria.
• Xylose Lysine Deoxycholate Agar (XLD): Fermentation of xylose, lactose and sucrose generates acid products, which causes a color change in the medium from red to yellow. Non-fermenters will be red. Xylose is incorporated into this medium because it is fermented by practically all enterics (yellow) except for the Shigella species (red). Lysine is included to enable detection of Salmonella. Salmonellae rapidly ferment xylose but then lysine is attacked reverting to an alkaline pH. This mimics the Shigella reaction.

Course Intended Outcomes

Describe different types of media according to their physical state, chemical composition and their function and list some of their applications; interpret microbial growth patterns.

Describe and perform at least 3 biochemical tests and describe their roles in microbial identification. Interpret results obtained and draw conclusions from biochemical tests performed.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Bunsen burner
• Inoculation loop
• Test tube rack
• Additional materials and cultures will be assigned by your instructor

Procedure, Day 1

Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Students will be inoculating multiple organisms on the same plate. Follow the specific directions from your instructor.
3. Label clearly and inoculate carefully – preferably with only one organism at a time to avoid mistakes and cross contamination.
4. Refer to illustration in figure 12.1 for possible inoculation schemes

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for inoculation by your group.
2. Collect the required materials for the lab.
   • Place all tubes in a tube rack.
   • Never hold a culture tube by its cap.
   • Bacterial culture tubes are always to be shared with your table group.
3. Clearly label plates (on the bottom side) with your group name or initials, organism name(s), and section day/time.
4. Place inoculated plates in the rack labeled “to be incubated” on the back bench. Always place the plates with lids down and bottoms up.

Procedure, Day 2

1. Collect your group’s plates and tubes from the back bench area labeled “last week’s lab”.
2. Examine your plates to determine results for each organism inoculated. Did the organism grow or was it inhibited?
3. If the organism grew, what was its reaction with the indicator in the media?
4. Record your results in your lab notebook.
5. Examine plates from other groups so that you can see results for all the organisms and representative positive and negative results for each media type.
6. Students can also observe prepared plates at the front bench for representative positive and negative results.

Lab Clean-up

Return bacteria cultures to the front bench after inoculations are complete.

Remove labels from tubes and place in the tube rack ABOVE the biohazard container.

Plates are disposable and should be thrown away in the biohazard container when everyone in the group has completed the exercise.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

Complete table for each of the four S&D media used in your lab notebook. You can use the tables below as an example.

Medium Used: Eosin Methylene Blue Agar (EMB)

Medium Used: MacConkey Agar (MAC)

Medium Used: Xylose Lysine Desoxycholate Agar (XLD)

Medium Used: Mannitol Salt Agar (MSA)

Critical Thinking Questions

Explain the difference between a negative result/reaction and a result of no growth or inhibited.

What color does the indicator phenol red turn in the presence of acid?

Which S&D media inhibit Staphylococcus epidermidis?

Which S&D media inhibit E.coli?