Lab Exercise 5 - Selective and Differential Media

 

MSA plate © Jackie Reynolds, author. Licensed for use, ASM MicrobeLibrary (linked to http://www.microbelibrary.org)

Exercise 5 - Objectives

1.      Compare and contrast enriched, differential, and selective media.

2.      Explain the strategy behind the use of selective and differential media.

3.      Interpret the results of bacterial growth on EMB, MacConkey and MSA agar.

4.      Predict the appearance of a bacterial species on MSA or EMB given data regarding fermentation & Gram stain reaction.

Bacteria are primitive single-celled organisms that have very specific growth requirements. Artificial media represents an environment created to provide all the optimum requirements for their growth but also a means to make the invisible microscopic organisms, visible either through sheer numbers or through chemical reactions. Three categories of media enable macroscopic study of bacteria: enriched, selective, and differential. Enriched media is formulated with all the necessary ingredients for a wide variety of organisms and grows a multitude of bacterial species. Tryptic soy agar (TSA), Nutrient agar (NA) and Blood agar are commonly used enriched media. Selective media are formulated with ingredients that inhibit the growth of some bacteria, such as an antibiotic, but enhance growth of the target organism. Differential media includes ingredients, such as chemical indicators, that produce observable differences between species of bacteria. This allows the microbiologist to macroscopically distinguish between species. Often a medium is formulated so that it is both selective and differential functions. This is particularly useful when growing clinical or environmental cultures where richly diverse communities of organisms.

As seen in exercise 2 clinical and environmental cultures display incredible variety on enriched media. While interesting this makes the recovery and identification of a particular pathogen far more difficult. In practice clinical specimens are generally inoculated on enriched, differential, and selective media to facilitate rapid identification, diagnosis, and treatment. However, identification of bacterial species and testing for antibiotic sensitivity require PURE, ISOLATED bacterial colonies. Growing two species together in a biochemical test would give confusing test results and the wrong identification. In addition commensal organisms, such as the normal flora on our body, often inhibit and out-grow the pathogens on artificial media. Culturing a wound on the hand, would require selective media to inhibit the growth of normal flora such as Staph. epidermidis and diphtheroids, and give the suspected pathogen an opportunity to grow. The scope of selection may be large group - such as Gram Negative Rods, or narrow for an individual species, such as Neisseria gonorrhea.

The use of media that is both selective and differential not only selects for a particular group or species of organisms, it also reveals specific metabolic information that can point to a particular species. For instance, EMB agar is both differential and selective. Eosin and Methylene-blue stains are added to a nutrient agar base containing lactose. The nutrient agar provides a smorgasbord of nutrients to nourish the bacteria, but the stains embedded in the agar inhibit the ability for most Gram positive bacteria to grow, thus selecting for Gram negative bacteria. In addition, as the bacteria use the lactose they produce acid by products which alter the color of the stains and change the appearance of the medium. This indicates metabolic activity that can quickly lead to a presumptive identification of the bacteria species present. On EMB Gram negative rods that can ferment lactose appear pink, and those that cannot ferment lactose remain colorless. Due to the large quantity of acid E.coli produce the dyes precipitate out on the surface of the colonies causing a green metallic sheen. MacConkey agar is also differential and selective. Bile salts and crystal violet dye in the agar inhibit most Gram-positive bacteria, a red dye changes color when microbes ferment  lactose. Another agar used in this exercise is MSA agar which is selective for Gram positive organisms from the family Micrococcaceae and differential for Staphylococci..

Materials:

Stock cultures:

·         Staph. epidermidis                                                      EMB plates  (purple)

·         E.coli                                                                        TSA plates

·         Staph. aureus                                                          MSA plates (dark pink)

·         Other Stock                                                         MacConkey agar (light pink)

.        unknown broth                                                        loops  

        PPG

Methods:

1. Work as groups. Use the sharpie to write on the bottom of the TSA, MSA, MacConkey and EMB plates to divide them into four pie-shaped pieces. Label and date the bottom plate.

2. Streak section 1 with a very light inoculum of Staph. epidermidis then incinerate the loop. Inoculate section 2 with E.coli then incinerate the loop. Inoculate section 3 with Proteus vulgaris, then incinerate the loop. Finally, inoculate the fourth section with a sample of your unknown mixed broth.

3. Incubate the media upside down at 37C.

4. For homework go to http://www.troybio.com/images/Product_Images_BBL/BBL.htm and look at the Levine EMB, MSA agar, MacConkey and TSA II with 5% sheep blood.

 Lab Day 2

Observe the results of the media inoculated. Collect your data and answer the following questions. If you would like to practice your gram stain technique you may use these bacteria.  Discard all of your media in the biohazard container when you are done. Refer to the Atlas and the Internet for information and pictures of growth media.

Lab Exercise 5 Selective and Differential Media

Name___________________

On the table below indicate growth by writing G and describing any color changes in the media. Use NG for no growth.

Differential and Selective Media Data Table 

Specimen

 

Media

Staph. epidermidis

Staph. aureus

E.coli

Other stock

mixed broth

Type of media

Enriched, selective, differential or a combination

 

TSA

 

 

 

 

 

 

 

 

 

MSA

 

 

 

 

 

 

 

 

 

EMB

 

 

 

 

 

 

 

 

 

 

MacConkey Agar

 

 

 

 

 

 

 

11.      What can you deduce about your unknown mixed broth by looking at the growth on the media, before making a microscopic slide? Why?

 

2.2. A. What ingredients cause MSA to be selective?

 B.      What ingredients allow MSA to be differential?

 C.      What ingredients cause MacConkey to be selective?

 D.     What ingredients allow MacConkey to be differential?

3.  Gram stains indicate microscopic information concerning the cell membrane, shape, and arrangement. Culturing on media displays macroscopic information such as colony morphology, but it also indicates chemical reactions occurring. Circle the type of information the selective and differential media used in this lab exercise provided about the organisms in your unknown broth? (circle none, one or many)

 

Check box if you can conclude this about an unknown by observing growth on selective and differential media

check

Check box if you can conclude this about an unknown by observing growth on selective and differential media

check

a. gram stain reaction

 

g. endotoxins

 

b. metabolic activity

 

h. virulence

 

c. sensitivity to drugs

 

i. presumptive identification

 

d. pathogenicity

 

j. final identification

 

e. colony morphology

 

k. oxygen optimums

 

f. toxic activity

 

l.  temperature optimums

 

 

4. Using the information in the first two columns predict the growth and appearance of the bacterial species listed on EMB and MSA agar.

Use G for growth and NG for no growth. Predict any color changes in the media.

You may double check your predictions by looking in the atlas, text or online – but make the hypothesis about the results before you look for the answer.

Bacterial species

Gram Stain

fermentation (+ or -)

Appearance on

 

 

Lactose

Mannitol

EMB

MSA

MacConkey

Pseudomonas aeruginosa

Gram Negative

Rod (GNR)

 

-

 

+

 

 

 

Neisseria sicca

 

Gram Negative cocci (GNC)

 

-

 

-

 

 

 

Staphylococcus aureus

Gram positive coccus GPC)

 

+

 

+

 

 

 

Bacillus cereus

Gram positive

Rod (GPR)

+

weak

 

-

 

 

 

Look at the following websites  

http://www.cdc.gov/ncidod/diseases/submenus/sub_staphylococcus.htm

http://textbookofbacteriology.net/pseudomonas.html

http://www.cdc.gov/healthyswimming/derm.htm

Note any important medical aspects related to identifying the bacteria above.

Bacterial species

Medical Significance

 Pseudomonas aeruginosa

 

 Staphylococcus aureus

 

 

 

 Bakersfield College | Kern Community College District | Dr. Janet Fulks
1801 Panorama Dr. - Bakersfield, CA 93305 - (661)395-4381
Date last updated 09/12/2011
©Janet Fulks