Search box
Microbiology Concept Inventory
Please note, you must be an educator in higher ed or maybe high school to qualify to recieve the MCI
( 133459 Reads)
|Period 1
Materials
Saline suspension of two enterics (differing in abilities to ferment lactose and/or produce hydrogen sulfide) plus a non-enteric (Pseudomonas)
2 plates of Modified MacConkey Agar
Optionally, plates of Brilliant Green Agar and/or XLD Agar also may be available.
Record the number of your unknown.
- Streak the mixed unknown onto each plate. Be sure to streak for isolated colonies!
- Incubate the plates at 37°C. If the next period is two or more days away, bring the plates to the stage for incubation.)
Period 2
Materials
3 or more slants of Kligler Iron Agar (KIA)
(Optionally, slants of Lysine Iron Agar (LIA) may also be available.)
Demonstrations of various enterics on selective-differential plating media
Figure 14.1. Examples of various enterics. Some examples of typical enterics growing on various media. These various media are explained very well by John Lindquist.
Figure 14.2. A typical initial streak plate. The appearance of a streak plate (enlarged for easy viewing) after 24 hours of incubation. You should be able to discern three colony types.
- Observe the demonstrations of some enterics Figure 14-1 (including Salmonella and Shigella) on various plating media used in the isolation of enterics. The instructor will provide explanatory material.
- Examine your streak plates carefully, Figure 14-2. Note the characteristics and colors associated with only the well-isolated colonies. The three different organisms should be easy to differentiate on the Modified MacConkey Agar. Label each of the different chosen colonies by number or letter and record in your notes the appearance of each. (Also, indicate the specific medium of isolation if media in addition to MacConkey Agar were used.)
- Inoculate each chosen colony into the same-numbered tube of KIA by the use of the needle as follows: First, stab the "butt" of the medium all the way to the bottom and then streak along the top of the slanted portion from bottom to top. (If LIA is also used, the medium is inoculated in like manner.)
- If the next period is more than one day away, place the KIA tubes in the baskets on the stage.
(1)How is a non-lactose-fermenting organism able to grow on MacConkey Agar?
(2)At this point, would you expect to be able to differentiate between enterics and other organisms (such as Pseudomonas) on the plates? Why or why not?
The tubes will be refrigerated and then placed in the 37°C incubator a day before the next period. For the proper interpretation of KIA, it is very important that the reactions in the tubes be recorded for just one day of incubation.
Period 3
Materials
2 slants of Phenylalanine Agar
2 tubes of Lactose Fermentation Broth
2 tubes of MR-VP Broth
2 tubes of Motility Indole Ornithine (MIO) Medium
2 slants of Simmons Citrate Agar
2 tubes of Lysine Decarboxylase Broth
2 tubes of Decarboxylase Control Broth
4 tubes of sterile mineral oil
Demonstration of KIA reactions
Figure 14.3. Reactions in KIA.
Tube 4: Much gas is often seen for this tube, evidenced by cracks in the medium. Also, lactose fermenters which are methyl red-negative may show a "reversion" toward an alkaline reaction as neutral products are formed from some of the acid. This appears as shown in Tube 4A where a slight reddening of the slant occurs as the alkaline deamination reaction becomes no longer over-neutralized by acid from fermentation. How might such a tube appear after two or more days of incubation? (Recall the methyl red test.)
** Tube 5: Enough acid can be produced to cause the black iron sulfide precipitate to break down and not be seen. In this case, the tube will look like no. 4.
Photo and text courtesy of John Lindquist, University of Wisconsin-Madison
- Observe the demonstration of reactions in KIA, Figure 14-3. Note how Salmonella and Shigella differ from each other and how they are similar to certain other enterics in the medium.
- Observe your tubes of KIA. Discard the tube for any isolate which is negative for glucose fermentation (red slant and orange butt). This is the non-enteric (Pseudomonas) to which you need not pay any more attention. The remaining tubes should differ in the indication for lactose fermentation (yellow slant if positive) and/or H2S production (black color throughout the "butt" if positive). Retain two clearly-different tubes (discarding the rest, if any) and record the reactions carefully. These two slant cultures are your enteric isolates to identify and will provide the inocula for the next step.
- From each tube of KIA, inoculate each of the media listed under Materials, above. Carefully stab-inoculate (with the needle) the tube of MIO and do not mix! All of the other media should be inoculated in "normal" fashion; do not stab-inoculate the slants.
- Overlay each inoculated tube of Lysine Decarboxylase Broth and Decarboxylase Control Broth with sterile mineral oil as you did in Experiment 4-3 for Glucose O/F Medium.
- Incubate the tubes as follows:
- Unless you are coming into lab tomorrow, bring the MIO and Phenylalanine Agar to the front of the lab for one-day incubation (immediate refrigeration followed by incubation at 37°C for one day prior to the next period).
- Place all of the other tubes in the 37°C incubator. These media may be examined at any time after one day of incubation except for the MR-VP Broth which must be incubated for at least two days before any tests are made.
What is the principle behind the development of anaerobic conditions in media overlayed with mineral oil? Consider the oxygen relationship of these organisms. (Recall what was done in setting up the enrichment for photosynthetic bacteria)
Period 4
Materials
Dropper bottles of FeCl3, methyl red and Kovacs reagent
Be sure to consult with your neighbors such that positive and negative reactions can be seen for each of the media.
Figure 14.4. Reactions in phenylalanine agar. Positive and negative reaction in phenylalanine agar. Ec - E. coli showing a negative reaction on phenylalanine agar. Mm - Morganella morganii showing a positive reaction on phenylalanine agar.
Figure 14.5. Lactose fermentation broth. Positive and negative reactions in lactose fermentation broth. Pf - Pseudomonas fluorescens a negative reaction in lactose fermentation broth. Kp - Klebsiella pneumoniae a positive reaction in lactose fermentation broth.
Figure 14.6. Methyl red reactions. Negative, equivocal, and positive reactions in the methyl red test. Strains of Enterobacter will give a negative reaction in methyl red, while Klebsiella will give a negative to equivocal (orange) reaction. Salmonella, Escherichia, Citrobacter, Proteus, Morganella and Providencia will all give positive reactions.
Figure 14.8. MIO reactions. The various reactions found in MIO medium. This medium tests for three different properties at once. Motility, indole production and ornithine decarboxylation. Photo and table couresy of John Lindquist. For more information, see the web page written by John.
Figure 14.9. Simmon citrate medium. Positive and negative reactions in Simmons citrate medium. Strains of Enterobacter, Klebsiella, Salmonella, Citrobacter and Providencia are positive, while strains of Escherichia, Shigella and Morganella are negative.
Figure 14.10. Lysine Debarboxylation. Positive and negative reactions in the lysine decarboxylation test. This test involves two tubes. One containing lysine (Lysine Decarboxylase Broth - LDB) and the other (Decarboxylase Control Broth - DCB), a control, having the identical medium, but no lysine. DCB and LDB are both rich media supplemented with glucose. Enterics will ferment the glucose causing an acidic reaction. Enterics that can decarboxylate lysine will cause a net alkaline reaction, and turn the medium purple. 1 - a non-enteric, note the inability to ferment glucose in DCB. 2 - a positive reaction. DCB turns acidic due to the fermentation of glucose, while LDB turns purple due to carboxylation of lysine. 3 - a negative reaction. Both broths are yellow due to fermentation of glucose, but lysine is not decarboxylated.
- Phenylalanine Agar. Add about one-half dropperful of the FeCl3 solution. If deamination of phenylalanine has taken place, the reagent will react with the phenylpyruvic acid formed, and a dark green color will appear in the slant region. Figure 14-4 shows + and - reactions for phenylalanine agar.
- Lactose Fermentation Broth. Any degree of yellow color indicates fermentation. How does the result compare with what was seen in KIA and the original isolation medium? Figure 14-5 shows + and - reactions for Lactose Fermentation Broth
- A slight amount of yellow color with little or no gas should be recorded as a weakly-positive reaction. Such organisms probably would have appeared lactose-negative on MacConkey Agar and KIA.
- A whitish color in the bottom half of the tube is due to reduction of the pH indicator and is not an indication of acid.
- MR-VP Broth. (Be sure that this medium was incubated for at least two days.) Add a half-dropperful of the methyl red reagent to the tube. Do not mix; just let the reagent form a layer at the top of the medium. Figure 14-6 shows the + and - methyl red reactions
- Red color: Positive reaction, indicative of mixed-acid fermentation; pH has dropped and remained at or below 4.4.
- Yellow color: Negative reaction, indicative of butanediol fermentation; pH has dropped and then risen to 6.2 or above.
- Orange color: Record as an "equivocal" reaction.
- The Voges-Proskauer (VP) Test: (This test is not run routinely in this course, but the procedure is given here for those interested or in case it is done optionally.) A second tube of MR-VP Broth is inoculated and incubated. Then, 12 drops of alpha-naphthol reagent and 4 drops of 40% KOH are added. Results are noted after 10-30 minutes:
- Red color: Positive reaction for the presence of neutral products (acetoin and/or 2,3-butanediol), indicative of butanediol fermentation. Such organisms are usually methyl red-negative.
- Yellowish color: Negative reaction, indicative of mixed-acid fermentation. Such organisms are usually methyl red-positive.
- Motility Indole Ornithine (MIO) Medium.
- MIO - Motility. Observe for cloudiness in the medium (growth away from the stab line). For a non-motile organism, growth may be seen along cracks in the medium caused by gas production, but there will be clear pockets of no growth. Figure 14-8 shows various reactions for MIO medium.
- MIO - Ornithine Decarboxylation. Observe the lower three-quarters (anaerobic region) of the medium for change in color of the pH indicator; growth must be present in this part of the tube.
- Gray, blue or purple color: Positive reaction for ornithine decarboxylation - formation of a highly alkaline product, overneutralizing the acid produced from glucose fermentation.
- Yellow color: Negative reaction. Yellow color is due to acid production from glucose fermentation.
- MIO - Indole Production. As for the Tryptone Broth (Exp. 7), add about one-half dropperful of Kovacs reagent to the medium. A red ring indicates production of indole from the breakdown of tryptophan.
- Simmons Citrate Agar, Figure 14-9. If utilization of citrate (the sole carbon and energy source in the medium) has taken place, an alkaline reaction results. The pH indicator turns bright blue in the slant region.
- Decarboxylase Control Broth, Figure 14-10. This medium is identical to the following medium except that that lysine is not included. All enterics should be able to grow in this anaerobic medium, fermenting the glucose and producing acid, resulting in a yellow color. A strict aerobe such as Pseudomonas will not grow, in which case the medium will remain somewhat purple in color.
- Lysine Decarboxylase Broth Figure 14-10. (If growth and acid production are not seen in the control broth, results in this medium are meaningless.)
- Gray, blue or purple color (a color "darker" than that of the corresponding control): Positive reaction for lysine decarboxylation - formation of a highly alkaline product, overneutralizing the acid produced from glucose fermentation.
- Yellow color (identical to the corresponding control): Negative reaction.