Biology 2420 Introduction to Microbiology
Review sheet for Lab Practical II (prepared by Charlie Beaman) (revised 2/05)
Section 5 in your lab manual on differential tests
1. Differential tests are based on biochemical diversity of bacteria. An organismÝs unique biochemical character can be used to identify it.
2. These tests are used on your unknown to specifically identify the organisms given.
EXERCISE 5-2 FERMENTATION TESTS
1. Carbohydrate fermentation tests use a growth medium with a specific carbohydrate as a substrate (ex. lactose or glucose).
2. Lactose is a disaccharide that is broken down into its monosaccharide units glucose and galactose. and then metabolized further. If the organism is a fermenter, it often produces acid waste products and sometimes gas.
3. Glycolysis of sugar produces pyruvic acid, this pyruvic acid can be further broken down to lactic acid and other acids.
4. Phenol red broth is the medium. It has the particular carbohydrate added to it. An indicator, phenol red is added to allow detection of acid end products of fermentation. At neutral ph it is red, below 6.8, it turns yellow. So yellow is positive for fermentation and acid production.
5. A small inverted tube, Durham tube, is placed in the broth, and it collects gas bubbles if the fermentation reaction also produces gas.
6. The indicator can indicate alkaline reactions, if the organisms produce alkaline waste products from deamination of the peptone in the medium. See pg 145. An alkaline reaction is pink ˝ compare to a red control tube. (be careful ˝ color can be subjective)
7. Phenol red broth and fermentation of sugars is a key to identifying gram negative bacilli.
8. Of course, the whole test system is based on the organismÝs ability to produce enzymes to break down the sugars. These enzymes are proteins and they are genetically code for via DNA-RNA as discussed in class. The presence of enzymes, detected by biochemical tests, thus reveal the specific genetic ability of the organisms ˝ thus a key to their id.
Exercise 5-16 ˝ Casease test ˝ testing for the presences of the enzyme, casease, hydrolyzes the protein casein in milk
1. Hydrolytic enzyme, breaks peptide bonds in the milk protein. Remember that hydrolysis is a reaction where water is added to break a bond. (opposite of dehydration synthesis)
2. Medium is skim milk agar, the skim milk added to the medium cause the agar to appear cloudy and white.
3. Bacteria that break down the casein, produce enzymes that diffuse into the medium, hydrolyze the protein, and cause a zone of clearing around the growth.
4. A positive test, thus, is a zone of clearing.
Exercise 5-17 ˝ Gelatinase test ˝ gelatin is a protein and we are testing for the oraganisms ability to hydrolyze the gelatin protein.
1. Bacteria that produce gelatinase can hydrozyze gelatin, a protein found in animal connective tissue (collagen)
2. The bacteria can use the digested end products (amino acids for fuel and for building blocks in biosynthesis)
3. Nutrient gelatin is added to the medium in a concentration high enough to cause the medium to be semi solid at room temperature.
4. If the organism hydrolyzes the gelatin the medium becomes liquified. to be absolutely sure the test is positive, the tubes need to be refrigerated. If upon refrigeration, the test tube is liquid, then the test is positive for presence of gelatinase. It is always good to compare to a sterile uninnoculted control.
Exercise 5-19 ˝Lipase test ˝ this is a differential test to determine if the bacteria has the enzyme to hydrolyze fats (lipids). Tributyrin oil is added to the media. If the organisms have the proper lipase, the lipid is hydrolyzed and the end productrs can be used for fuel or for biosynthesis precursors.
1. Tributyrin oil is added to the medium and emulsified to produce an opaque emulsion.
2. The organisms can grow on the medium and if the organism produces a lipase, the medium exhibits a zone of clearing around the bacterial growth ˝ positive test.
3. No zone of clearing indicates the bacteria lacks the lipase for tributyrin oil.
Exercise 5-13 ˝ starch hydrolysis ˝ this is a differential tests that determines if the bacteria produces an enzyme that hydrolyzes starch (complex polysaccharide) into simpler sugars that can be used for fuel. Glucose is often the product and can be directly used by the cell for fuel.
1. Starch used in this medium is amylose, and the enzyme is amylase. The end product is glucose.
2. The organisms can grow on the medium. However, the medium is clear whether or not the starch is hydrolyzed.
3. To detect the hydrolysis of the starch, iodine is added after the growth of the bacteria. The iodine reacts with the starch producing a dark blue or purple color. The presence of dark color around the growth is a negative result (the starch is intact, not hydrolyzed).
4. A clear zone, then, is positive for starch hydrolysis.
Exercise 5-4 MR-VPtest, Methyl red ˝ Voges Proskauer, two tests in one type of medium.
a. MR test
1. The medium contains only peptone, glucose (fermentable sugr), and potasium phosphate buffer (resists changes in ph)
2. Tests for the organisms ability to produce stable acid end products that overcome the buffer. Incubate for 5 days (other organisms ferment the sugar but over time the end-products are converted to more neutral products.
3. Methyl red indicator is added to the medium after incubation. A positive test is a red color. MR is red at a ph of 4.4 and yellow at a ph of 6.2 (more neutral). Red is positive for MR test. Yellow is negative.
b. VP test ˝ this is a second test using the same medium. VP reagents are added to the medium after incubation.
1. Fermentation of glucose, but quickly convert the end products to acetoin.
2. VP reagents are added to the medium and a positive test is a red color
3. VP positive is red color after adding reagents.
4. Reagents are called BarrittÝs reagents ˝ alpha napthol, and KOH.
These MR-VP tests are part of the set of IMViC test used to differentiate Enterobacteriacceae
Exercise 5-8 Citrate test ˝ this tests for a bacteriaÝs ability to use citrate as its sole carbon source.
1. Bacteria that can use citrate have an enzyme, citrate permease that allows the use of citric acid as a source of energy.
2. Simmons citrate is a defined (not general) medium. It contains only citrate for carbon and only ammonium phosphate for nitrogen.
3. The indicator is BTB (bromthymol blue). It is green in the neutral pH ranges and blue if it becomes alkaline. If organisms can grow then they produce alkaline products changing the medium color to blue. Blue is positive for citrate utilization.
4. Just growth and no blue color is also positive.
5. This test is also part of the IMViC battery of tests for enterics.
Exercise 5-20 SIM medium, Sulfur-Indole-Motility tests, a three in one test
1. This is a combination differential medium. Combines three tests in one medium
2. Reduced agar to show motility.
3. Sulfur waste product, hydrogen sulfide, produced from the breakdown of sulfur containing amino acids, is detected by its combinattion with iron in the medium. The ferric sulfide produces a black precipitate. Black is positive for hydrogen sulfide production
4. The medium contains tryptophan, an amino acid. tryptophan hydrolysis produces indole and when the Kovacs reagent is added a red color is produced. Red color after addition of Kovacs reagent is positive for indole.
5. Also part of IMViC battery for enterics.
Exercise 5-7 Nitrate reduction test ˝ a diificult test to understand ˝ the medium is used to test for a bacteriaÝs ability to reduce nitrate using the nitrate reductase enzyme and even other enzymes to further reduce nitrate to nitrogen gas.
1. Undefined medium with potasium nitrate added.
2. If the organism can reduce nitrate, it is using nitrate as the final electron acceptor in anaerobic respiration.
3. A Durham tube ýmayţ be present to capture any gas produced.
4. The medium does not give direct results. Reagents have to be added. This is where it gets complicated.
5. Look at pg 163 for details on running test
Exercise 5-15 Urease test. This test is designed to detect the presence of the enzyme urease. The enzyme catalyzes the hydrolysis of urea to carbon dioxide and ammonia.
1. We use this medium to detect the organisms that rapidly breakdown urea.
2. Urea in the medium allows for rapid detection of urease activity. phenol red is the indiacator and ammonia raises the ph causing the color to be pink
3. Orange to pink is positive
4. Proteus species are the enterics that rapidly hydrolyze urea
Exercise 5-21 Triple Sugar Iron Agar (TSI) one of my favorites. Contains three sugars - glucose, lactose, and sucrose. The agar is prepared on a slant and growth in the slant and the butt can be differential
1. Phenol red indicator, ferrous sulfate for hydrogen sulfide production
2. Glucose only fermentors produce acid (yellow) reaction very quickly and exaust the glucose, and the slant will change back to red. So acid butt, red slant =glucose only fermentation
3. If the slant and but are yellow throughout, then either lactose or sucrose or both are fermented.
4. Cracks in the agar media show bubbles of gas being formed
5. Nonfermenters turn the medium red (no acid)
6. Fermentation tests are examined after 24 hours of incubation. Return them to incubator and incubate longer to determine hydrogen sulfide production (black precipitate color in medium)
7. Check pg 199 and 201 for proper ways to record data
8. TSI is used to help identify enterics, and also non-enteric gram negative bacilli like Psuedomonas and Alcaligenes
Exercise 5-10 Decarboxylation tests ˝ test for bacterial ability to remove carboxyl group from amino acids, decarboxylase enzymes. Review amino acid structure ˝ carboxyl and amino group
1. See pg 169 for chemical explanation
2. Decarboxylation results in the formation of alkaline end products, mineral oil added to create anaerobic state
3. The indicator in the medium is bromcresol purple (BCP)
4. An alkaline reaction is purple, positive for alakaline end products of amino acid decarboxylation
5. Yellow is negative
6. The base medium can have different amino acids added like lysine, ornithine, arginine (tests again for enterics)
Exercise 5-11 Phenylalanine deaminase test - The amino acid phenylalanine is deaminated (amino group removed) by the enzyme, phenylalanine deaminase. The amino group is removed and converted to ammonia. The end product phenylpyruvic acid can then be detected.
1. Medium is rich in phenylalanine.
2. After growth a reagent is added. Ferric chloride reacts with the phenylpyruvic acid to form a dark green color. Dark green is positive test for the enzyme.
3. Yellow color is negative
4. Test is helpful with enteric differentiation.
Exercise 5-18 DNase test ˝ DNA is broken down by an enzyme, DNase.
1. DNA medium is rich in DNA.
2. The indicator is methyl green. DNA and the dye cause the medium to appear blue-green color
3. Growth of bacteria that have the DNase produce a clearing around the colonies. Clearing is a positive test.
4. Saph and Strep and Serratia produce DNase.