Updated 28 December 04
Review appropriate sections of chapter 3 in the text.
Microbiologists may inoculate several kinds of media with clinical specimens for microbial analysis. The types we will examine differentiate species by certain characteristic responses to ingredients in the culture medium and/or selectively encourage growth of those species of interest while suppressing the normal flora.
Differential media contain a component that can be used by some microorganisms but not by others. An indicator that demonstrates any change in this component may be added, along with the usual nutrients.
Selective media contain one or more components that suppress the growth of some microorganisms without seriously affecting the ability of others to grow. They may also contain ingredients for differentiating among the species that do grow.
For each of these special media and trests you need to find outside photos or descriptions of the expected results. Use these to provide expected results in your lab book.
EMB solid medium is recommended for the selective isolation and differentiation of gram-negative enteric bacilli. See lab manuals for photos.
1. Make a short streak of known (from the list below) or unknown bacteria (suspected of being on this list) on a designated sector of the surface. Dip a sterile loop in inoculum and then shake off the excess before streaking.
2. Incubate for 24 hours at 37 degrees C.
3. Results expected:
| Organism | Colonial appearance |
| Escherichia | black, metallic sheen |
| Enterobacter | brown or pink, mucoid |
| Klebsiella | brown or pink, mucoid |
| Salmonella | colorless to amber, transparent |
| Proteus | opaque, colorless, swarming |
| Staphylococcus | inhibited |
Mac solid media is used to isolate and separate non-fermenting enteric pathogens from lactose fermenting coliforms. See Identibacter Interactus or lab manuals for photos and more information.
1. Use the same procedure as above but focus on this species list.
2. Results expected:
| Organism | Colonial appearance |
| Salmonella | colorless |
| Proteus | colorless |
| Escherichia | brick red to dark pink |
| Klebsiella | dark pink, mucoid |
| Streptococcus | inhibited |
V-J solid media is selective for coagulase positive, mannitol positive Staphylococcus aureus in clinical specimens and foods. Also see the Coagulase test in Identibacter Interactus.
1. Use the same procedure as above with attention to this species list.
2. Results expected:
| Organism | Colonial appearance |
| Staphylococcus aureus | black, yellow zones |
| Staphylococcus epidermidis | transparent |
| Escherichia | inhibited |
| Streptococcus | inhibited |
TSI agar is used to differentiate enteric bacteria (see lab manuals).
1. Obtain and label two tubes of TSI agar. Choose one lactose-fermenting organism and a nonlactose fermentor.
2. Streak a sample from a colony onto the surface of the slant and then stab the needle into the butt of the agar. Check that the cap is loose and incubate for 24 hours at 37 degrees C.
3. Observe the slant and butt of the tube within 18 to 24 hours to determine whether alkaline(red) or acid(yellow) conditions exist. Note whether gas was evolved by the presence of cracks of fissures in the medium and whether hydrogen sulfide was produced by noting any blackening of the medium.
The IMViC series of biochemical tests for indole production(I), the methyl red test(M), the Voges-Proskauer test(Vi), and the citrate test(C) are primarily used to distinguish between Escherichia coli and Enterobacter aerogenes. See Identibacter Interactus for Citrate, Indole, Methyl Red, and Voges-Proskauer tests or available lab manuals for photos and more details.
1. Inoculate a set of labeled tubes (a peptone broth, 2 MR-VP broths, and a citrate) with an organism and incubate for 24 to 48 hours.
2. Slowly add 0.5 ml of Kovac's reagent to the peptone broth culture. The appearance of a dark red color is a positive reaction.
3. Add several drops of methyl red indicator to one MR-VP broth culture. A orange-red color is positive reaction indicating a pH of 4.5 or below. A yellow color is negative.
4. Add 0.5 ml of VP reagent #1 and 0.5 ml of VP reagent #2 to the second MR-VP broth culture. Development of a red color on standing for several minutes indicates the production of acetylmethylcarbinol and a positive test.
5. Observe the citrate broth for cloudiness of turbidity. Bacterial growth indicates a positive reaction. Broth remaining clear without growth is a negative reaction.
How did your actual results compare with what you expected? If different, how do you account for the differences?
A group database containing characteristics of our microbial cultures will be essential for the unknowns project. Class results from the tests and observations on our cultures will aid you in developing a key for identification of your unknowns.