Download Lab Day 2 - Bakersfield College

Lab Exercise 5- Selective and Differential Media
1
Lab Exercise 5 - Selective and Differential Media
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
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
author.
Licensed for use, ASM MicrobeLibrary
(linked to
http://www.microbelibrary.org)
MSA plate © Jackie Reynolds,
2
Lab Exercise 5- Selective and Differential Media
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. Another agar used in this
exercise is MSA agar which is selective for Gram positive organisms from the family
Micrococcaceae and differential for Staphylococci. The Atlas describes the composition
and characteristic results of various selective and differential media.
Materials:
Stock cultures:
 Staph. epidermidis
 E.coli
 Proteus vulgaris
 mixed broth
PPG
EMB plates (purple)
TSA plates
MSA plates (pink)
loops
Methods:
1. Work as groups. Use the sharpie to
write on the bottom of the TSA, MSA,
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, and TSA II with 5% sheep blood.
3
Lab Exercise 5- Selective and Differential Media
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
Staph.
epidermidis
E.coli
Proteus
vulgaris
mixed broth
Media
Type of media
Enriched, selective,
differential or a
combination
TSA
MSA
EMB
1. What can you deduce about your unknown mixed broth by looking at the growth
on the media, before making a microscopic slide? Why?
2. Refer to the atlas.
A. What ingredients cause MSA to be selective?
B. What ingredients allow MSA to be differential?
4
Lab Exercise 5- Selective and Differential Media
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)
a. gram stain reaction
b. metabolic activity
c. sensitivity to drugs
d. pathogenicity
e. colony morphology
f. toxic activity
g. final identification
h. virulence
i. presumptive identification
1. 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 -)
Lactose
Pseudomonas
aeruginosa
Neisseria sicca
Staphylococcus
aureus
Bacillus cereus
Gram Negative
Rod (GNR)
Gram Negative
cocci (GNC)
Gram positive
coccus GPC)
Gram positive
Rod (GPR)
Mannitol
-
+
-
-
+
+
weak
+
Appearance on
EMB
-
Look at the following websites
http://www.cdc.gov/ncidod/diseases/submenus/sub_staphylococcus.htm
http://vm.cfsan.fda.gov/~mow/chap3.html
http://textbookofbacteriology.net/pseudomonas.html
http://www.cdc.gov/healthyswimming/derm.htm
1.
Note any important medical aspects related to identifying the bacteria above.
Bacterial species
Medical Significance
Pseudomonas
aeruginosa
Staphylococcus aureus
Date last updated 6/19/2017
©Janet Fulks
MSA