ANDZINC✦Finally, a pinch of zinc is added to tubes 2, 3, and 4 because they have been colorless up to this point. Tube 2 (the control tube) and tube 3 turned red. This is a negative result because it indicates that nitrate is still present in the tube.
Tube 4 did not change color, which indicates that the nitrate was reduced by the organism beyond nitrite to some other nitrogenous compound. This is a positive result.
✦ Application
Virtually all members of Entero bacteriaceaeperform a one-step reduction of nitrate to nitrite. The nitrate test differentiates them from Gram-negative rods that either do not reduce nitrate or reduce it beyond nitrite to N2or other compounds.
✦ Materials
Per Student Group
✦four Nitrate Broths
✦nitrate test reagents A and B
✦zinc powder
✦fresh cultures of:
⽧Erwinia amylovora
(Alternative: Enterococcus faecalis)
⽧Escherichia coli
⽧Pseudomonas aeruginosa(BSL-2)
✦ Medium and Reagent Recipes
Nitrate Broth
⽧Beef extract 3.0 g
⽧Peptone 5.0 g
⽧Potassium nitrate 1.0 g
⽧Distilled or deionized water 1.0 L pH 6.8–7.2 at 25°C
Reagent A
⽧N,N-Dimethyl-1-naphthylamine 0.6 g
⽧5N Acetic acid (30%) 100.0 mL
Reagent B
⽧Sulfanilic acid 0.8 g
⽧5N Acetic acid (30%) 100.0
Procedure
Lab One
1 Obtain four Nitrate Broths. Label three of them with the names of the organisms, your name, and the date. Label the fourth tube “control.”
2 Inoculate three broths with the test organisms. Do not inoculate the control.
3 Incubate all tubes at 35Ⳳ2°C for 24 to 48 hours.
Lab Two
1 Examine each tube for evidence of gas production.
Record your results in the chart provided on the Data Sheet. Refer to Table 5-7 when making your interpretations. (Be methodical when recording your results. Red color can have opposite interpretations depending on where you are in the procedure.)
2 Using Figure 5-28 as a guide, proceed to the addition of reagents to alltubes as follows:
a. Add eight drops each (approximately 0.5 mL) of reagent A and reagent B to each tube. Mix well, and let the tubes stand undisturbed for 10 minutes.
Using the control as a comparison, record your test results in the chart provided in the Data Sheet.
Set aside any test broths that are positive.
b. Where appropriate, add a pinch of zinc dust. (Only a small amount is needed. Dip a wooden applica - tor into the zinc dust and transfer only the amount that clings to the wood.) Let the tubes stand for 10 minutes. Record your results in the chart pro- vided on the Data Sheet.
Result Interpretation Symbol
Gas (Nonfermenter) Denitrification—production of nitrogen gas Ⳮ
(NO3씮NO
2씮N
2)
Gas (Fermenter, or status is unknown) Source of gas is unknown; requires addition of reagents Red color (after addition of reagents Nitrate reduction to nitrite (NO
3씮NO
2) Ⳮ
A and B)
No color (after the addition of reagents) Incomplete test; requires the addition of zinc dust
No color change (after addition Nitrate reduction to nongaseous nitrogenous compounds Ⳮ
of zinc) (NO
3씮NO
2씮nongaseous nitrogenous products)
Red color (after addition of zinc dust) No nitrate reduction ⳮ
TABLE 5-7Nitrate Test Results and Interpretations
T A B L E O F R E S U L T S
References
Atlas, Ronald M., and Richard Bartha. 1998. Pages 423–425 in Microbial Ecology—Fundamentals and Applications, 4th ed. Benjamin/Cummings Science Publishing, Menlo Park, CA.
Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. 2002. Page 277 in Bailey & Scott’s Diagnostic Microbiology, 11th ed. Mosby, St. Louis.
Lányi, B. 1987. Page 21 in Methods in Microbiology, Vol. 19, edited by R. R. Colwell and R. Grigorova. Academic Press, New York.
MacFaddin, Jean F. 2000. Page 348 in Biochemical Tests for Identification of Medical Bacteria,3rd ed. Williams & Wilkins, Philadelphia.
Moat, Albert G., John W. Foster, and Michael P. Spector. Chapter 14 in Microbial Physiology, 4th ed. Wiley-Liss, New York.
Zimbro, Mary Jo, and David A. Power, Eds. 2003. Page 400 in Difco™
and BBL™ Manual—Manual of Microbiological Culture Media.Becton Dickinson and Co., Sparks, MD.
1. Inoculate with pure culture.
2. Incubate at 35±2°C for 24 hours
4. If no bubble, add 8 drops Reagent A and 8 drops Reagent B—Mix well.
If red, organism reduces nitrate to nitrite.
Test is completed.
5. If not red, add zinc.
If red, organism does not reduce nitrate.
Test is completed.
If not red, organism reduces nitrate to
something other than nitrite.
Test is completed.
3. Examine the Durham tube
If there is a bubble and organism is NOT a fermenter, organism reduces nitrate to N2.
Test is completed.
5-28 PROCEDURALDIAGRAM FOR THENITRATEREDUCTIONTEST✦
✦ Theory
In many bacteria, citrate (citric acid) produced as acetyl coenzyme A (from the oxidation of pyruvate or the
-oxidation of fatty acids) reacts with oxaloacetate at the entry to the Krebs cycle. Citrate then is converted back to oxaloacetate through a complex series of re - actions, which begins the cycle anew. For more infor - mation on the Krebs cycle and fatty acid metabolism, refer to Appendix A and Figure 5-54.
In a medium containing citrate as the only available carbon source, bacteria that possess citrate-permeasecan transport the molecules into the cell and enzymatically convert it to pyruvate. Pyruvate then can be converted to a variety of products, depending on the pH of the environment (Figure 5-29).
Simmons Citrate Agar is a defined medium that contains sodium citrate as the sole carbon source and ammonium phosphate as the sole nitrogen source.
Brom thymol blue dye, which is green at pH 6.9 and blue at pH 7.6, is added as an indicator. Bacteria that survive in the medium and utilize the citrate also convert the ammonium phosphate to ammonia (NH3) and am- monium hydroxide (NH4OH), both of which tend to alkalinize the agar. As the pH goes up, the medium changes from green to blue (Figure 5-30). Thus, con ver- sion of the medium to blue is a positive citrate test result (Table 5-8).
Occasionally a citrate-positive organism will grow on a Simmons Citrate slant without producing a change in color. In most cases, this is because of incomplete in- cubation. In the absence of color change, growth on the
Nutrient Utilization Tests
In this unit you will per form tests using two examples of utilization media—Simmons Citrate Medium and Malonate Broth. Utilization media are highly defined formulations designed to differentiate organisms based on their ability to grow when an essential nutrient (e.g.,carbon or nitrogen) is strictly limited. For example, citrate medium contains sodium citrate as the only carbon-containing compound and ammonium ion as the only nitrogen source. Malonate broth contains three sources of carbon but prevents utilization of all but one by competitive inhibition of a specific enzyme. ✦
E X E R C I S E
5-8 Citrate Test
C CH2 OH
COO- Citrate CH2
COO- COO-
C O
COO- CH2
COO-
Oxaloacetate
COO- CH3
Acetate
C CH3
O COOH
Pyruvate Citrase +
Alkaline pH
COO- CH3
Acetate
CHOO- +
Formate
Pyruvate
Acidic pH CH3 COO-
Acetate
CHOH CH3
COO- Lactate C
CH3 O HCOH
Acetoin CH3
+ CO2
CO2 2CO2
5-29 CITRATECHEMISTRY✦In the presence of citrate-permease enzyme, citrate enters the cell and is conver ted to pyruvate. The pyruvate then is conver ted to a variety of products depending on the pH of the environment.
slant indicates that citrate is being utilized and is evi dence of a positive reaction. To avoid confusion between actual growth and heavy inoculum, which may appear as growth, citrate slants typically are inoculated lightly with an inoculating needle rather than a loop.
✦ Application
The citrate utilization test is used to determine the ability of an organism to use citrate as its sole source of carbon.
Citrate utilization is one part of a test series referred to as the IMViC (Indole, Methyl Red, Voges-Proskauer and Citrate tests) that distinguishes between members of the family Enterobacteriaceaeand differentiate them from other Gram-negative rods.
✦ In This Exercise
You will be inoculating Simmons Citrate slants with a citrate-positive and a citrate-negative organism. To avoid confusing growth on the slant with a heavy inoculum, be sure to inoculate using a needle instead of a loop.
✦ Materials
Per Student Group
✦three Simmons Citrate slants
✦fresh slant cultures of:
⽧Enterobacter aerogenes
⽧Escherichia coli
✦ Medium Recipe
Simmons Citrate Agar
⽧Ammonium dihydrogen phosphate 1.0 g
⽧Dipotassium phosphate 1.0 g
⽧Sodium chloride 5.0 g
⽧Sodium citrate 2.0 g
⽧Magnesium sulfate 0.2 g
⽧Agar 15.0 g
⽧Bromthymol blue 0.08 g
⽧Distilled or deionized water 1.0 L pH 6.7–7.1 at 25°C
Procedure
Lab One
1 Obtain three Simmons Citrate tubes. Label two with the names of the organisms, your name, and the date. Label the third tube “control.”
2 Using an inoculating needle and light inoculum, streak the slants with the test organisms. Do not inoculate the control.
3 Incubate all tubes at 35Ⳳ2°C for 48 hours.
Lab Two
1 Observe the tubes for color changes and/or growth.
2 Record your results in the chart provided on the Data Sheet.
References
Collins, C. H., Patricia M. Lyne, and J. M. Grange. 1995. Page 111 in Collins and Lyne’s Microbiological Methods, 7th ed. Butterworth- Heinemann, Oxford, United Kingdom.
Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. 2002. Page 266 in Bailey & Scott’s Diagnostic Microbiology, 11th ed. Mosby, St. Louis.
MacFaddin, Jean F. 2000. Page 98 in Biochemical Tests for Identification of Medical Bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia.
Smibert, Robert M., and Noel R. Krieg. 1994. Page 614 in Methods for General and Molecular Bacteriology, edited by Philipp Gerhardt, R. G. E.
Murray, Willis A. Wood, and Noel R. Krieg, American Society for Micro- biology, Washington, DC.
Zimbro, Mary Jo, and David A. Power, Eds. 2003. Page 514 in Difco™
and BBL™ Manual—Manual of Microbiological Culture Media.Becton Dickinson and Co., Sparks, MD.