Download Microbiology Lab Manual

yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Dr Janet Fulks
Bakersfield College
August 2010
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
MSA plate © Jackie Reynolds, author.
Licensed for use, ASM MicrobeLibrary
represents an environment created to provide all the
(linked to
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
Lab Exercise 5 - Selective and Differential Media
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..
Stock cultures:
 Staph. epidermidis
 E.coli
 Staph. Aureus
 Other stocks
EMB plates (purple)
TSA plates
MSA plates (pink)
mixed broth
MacConkey agar
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 pieshaped pieces. Label and date the bottom
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 and
look at the Levine EMB, MSA agar, MacConkey and TSA II with 5% sheep blood.
Dr Janet Fulks
Bakersfield College
August 2010
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
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
mixed broth
Type of media
Enriched, selective,
differential or a
1. What can you deduce about your unknown mixed broth by looking at the growth on the
media, before making a microscopic slide? Why?
Dr Janet Fulks
Bakersfield College
August 2010
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
g. endotoxins
h. virulence
i. presumptive identification
j. final identification
k. oxygen optimums
l. temperature optimums
Check box if you can conclude
this about an unknown by
observing growth on selective
and differential media
a. gram stain reaction
b. metabolic activity
c. sensitivity to drugs
d. pathogenicity
e. colony morphology
f. toxic activity
Dr Janet Fulks
Bakersfield College
August 2010
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 -)
Neisseria sicca
Bacillus cereus
Gram Negative
Rod (GNR)
Gram Negative
cocci (GNC)
Gram positive
coccus GPC)
Gram positive
Rod (GPR)
Appearance on
Look at the following websites
Note any important medical aspects related to identifying the bacteria above.
Bacterial species
Medical Significance
Staphylococcus aureus
Dr Janet Fulks
Bakersfield College
August 2010