Introductory Microbiology Lab Week 5 (April 7)

Biochemical tests for identification of bacteria

In this and next weeks lab, you will be performing a set of tests to detect various metabolic and physiologic processes that can be used to identify bacteri, including your unknowns in many cases. In this week's lab, you will be attempting to detect: different carbohydrate fermentations (using Durham tubes) - can detect acid & gas production from fermentations of various "sugars" fermentation of the monomer, glucose, to produce energy lactose (a dimer) hydrolyzed by the enzyme, beta-galactosidase, to produce the monomers glucose and galactose (the glucose can then be fermented) - thus, will detect ability to hydrolyze lactose andferment the resulting glucose monomers sucrose (another dimer) hydrolyzed by the enzyme, beta-fructosidase, to produce the monomers glucose and fructose (both of these monomers can often be fermented by many types of bacteria). Thus, will detect the ability to hydrolyze sucrose and ferment glucose or fructose or both

gelatin hydrolysis - bacteria produce gelatinase to hydrolyze the protein polymer, gelatin, to amino acids urease enzyme activity - hydrolyzes urea and produces ammonia (NH4) citrate utilization - citrate, a 6-carbon compound, can be utilized as the sole source of carbon starch hydrolysis - hydrolysis of starch (a polymer of glucose) to produce glucose monomers (a bacterium could then ferment the monomers to obtain energy) lipid (fat) hydrolysis - the results of lipid hydrolysis can then be converted to molecular forms that can be used to obtain energy (e.g., 2-carbon compounds that can be converted into acetylCoA to enter the Kreb's cycle) catalase enzyme activity - degradation of hydrogen peroxide

oxidase enzyme activity - detects activity of cytochrome oxidase (one of the enzymes in the bacterium's electron transport system - assumes the bacterium is an aerobe or facultative anaerobe) nitrate reduction - a very common reaction of many bacteria that reside in soils

You will also inoculate four media that can detect multiple reactions: litmus milk lactose fermentation (acid & gas) acid curd formation from lactic acid produced as an end product of lactose fermentation - acid produces a rather "stiff" insoluble form ("curd") of casein (milk protein) that does not move when the tube is tilted (compare to softer rennin curd below) rennin curd formation from the activity of the enzyme, rennin - produces a "softer" curd that flows slowly when the tube is tilted casein hydrolysis with release of ammonia (produces a basic pH in the medium that turns the litmus a blue/purple color in the upper portion of the tube (uninoculated medium is pink) triple sugar iron agar slants glucose, lactose, sucrose fermentations (acid & gas) production of H2S (from sodium thiosulfate) SIM agar stabs hydrogen Sulfide production (from sodium thiosulfate) Indole production from the amino acid, tryptophan Motility (does the bacterium have one or more flagella that propel the cell)

MR-VP broth Methyl Red test - red color (methyl red is a pH indicator) red color indicates production of large quantities of acid in the medium resulting from fermentation of glucose; yellow color indicates little or no organic acid remains in the medium (the acids have been converted into non-acidic or neutral end products that are then detected in the VP test that follows) Voges-Proskauer test - detects the production of non-acidic or neutral end products at the end of glucose fermentation; butanediol and acetoin (acetylmethylcarbinol) are examples of such end products

Each test will give a result that is quite easily viewed, either directly or after adding one or more reagents. Quite often, individual bacterial species have a unique set of reactions that distinguishes that bacterium from other species. Sometimes even a single result from one test identifies a bacterial genus and species.

Since you will know the genus and species of the bacteria you are using, the class as a whole will be gathering useful information for the entire set of known bacteria used in these two labs. Many of these data can often be used to identify one or both of your unknowns. In todays lab, you will simply be inoculating the assigned known bacteria, into or on each of the test media provided. It takes 24-48 hours (or sometimes even longer) for the bacteria to grow sufficiently to give reliable results. Thus, you will have to return to the lab at time(s) convenient to you to gather the data. However, you should not attempt to gather data before the minimum incubation time or much after the maximum incubation time. Students are welcome to organize themselves to visit the lab and gather each others data. Each student will be required to record the entire set of results and interpretations of those results for her or his assigned known bacteria in the Microorganism Data Notebook. The entire 10 points for this lab will be based on your notebook results/interpretations. Your lab instructor may ask that you hand in your notebook for scoring after you have recorded the results and interpretations.

Procedures: LABELING of each tube/plate of medium is very important, as you will be inoculating several different assigned bacteria into or on several different media. Type of medium (if not obvious); organism inoculated (remember to label separate sections of agar plates with the name of the bacterium inoculated in each section); your name (or initials, IF you are the only person in the entire class with those intials). Make sure that incubation times and temperatures are followed. Most, but NOT all, will be for 24-48 hours at 37oC. Make sure that you add any required reagent(s) in the order specified (if more than one reagent will be used). All reagents that you may need to add should be considered hazardous and used ONLY in the fume hood. Wear gloves and protective eyewear. Compare results with uninoculated controls (provided by your lab instructor) when needed. Record all of your results/observations for each of your assigned bacteria in your Microorganism Data Notebook. Inoculate the bacterium you have been assigned into or on each of the media listed below. Your lab instructor will provide one set of uninoculated control against which you may be able to compare some (but not all) of your inoculated media results. Incubate the inoculated media at the indicated temperature for the indicated time. Then perform any additional steps, if given, for each medium. All reagents that you may need to add should be considered hazardous and used ONLY in the fume hood. Wear gloves and protective eyewear. You can use the refrigerator in the lab to cool your geletin agars (to test for gelatin hydrolysis).. Make your observations and record your results/interpretations in your Microorganism Data Notebook. There will be several

resources (atlases, etc.) in the lab, and a few on-line resources linked to this lab writeup, that you can use to help you interpret your results. When you are confident that you have accurately recorded all results, you can either keep your cultures in the cold room (no need to parafilm) in case you want to look at them again for any reason, OR remove any tape from tubes and tub caps and any parafilm from agar plates and place all inoculated media in the washroom (tubes in racks and plates in the plastic bins). Please do NOT discard the uninoculated controls, as other students need to use them. The following tests used tubed media: Fermentation of glucose, lactose and sucrose In this set of tests, you will be able to determine if the bacterium can ferment glucose, can hydrolyze lactose (into glucose and galactose and then ferment either of the monomers released, usually only the glucose), and can hydrolyze sucrose (into glucose and fructose and then ferment either of the monomers released). Fermentation simply uses an organic molecule as an electron acceptor, with the result being the production of organic acids (and a pH change in the medium). You will also be able to determine if the bacterium can produce a gas (usually CO2) during the fermentation process. 1. Using aseptic technique, transfer a small inoculum of each of your assigned bacteria into each of the three broths (glucose, sucrose, lactose). 2. Incubate the inoculated broths at 37oC for 24-48 hours. 3. No additional steps need to be performed before making the observations. 4. Observe each broth for each of the following results (scroll down at this website and also compare to the uninoculated controls) and record your observations for your assigned bacteria. Remember that you are checking for several characteristics in each tube. a. growth or no growth b. growth with red color (i.e., no change in color compared to the uninoculated control) indicates the bacterium can NOT ferment the sugar in the tube (either can not ferment either of the monomers or can not hydrolyze the dimer to release monomers) or does not produce any organic acids if fermentation does take place. c. growth with yellow color acid produced (lower pH changes the phenol red pH indicator in the broth to yellow); this indicates the bacterium CAN ferment the sugar and, if the sugar is sucrose or lactose, can also hydrolyze that sugar to release fermentable monomers. d. no bubble trapped in the small tube inverted in the broth NO gas produced. e. bubble trapped in the small tube Gas (usually CO2) IS produced.

Gelatin hydrolysis Unlike agar, gelatin CAN be used as a nutrient source (complex protein). However, in order to use gelatin, this complex polymer must first be broken down (hydrolyzed). This test will allow you to determine if a bacterium has the enzyme necessary to perform this hydrolysis. 1. Using sterile technique and an inoculating needle, stab a small inoculum of the bacterium about of the way to the bottom of a tube of deep agar. Repeat with separate stab tubes for each of your assigned bacteria. 2. Incubate the inoculated media at 37oC for 24-48 hours. 3. Place the incubated inoculated stab and the uninoculated control into the refrigerator for approximately 30 minutes. This step is necessary, as even non-hydrolyzed gelatin is rather fluid. 4. Gently tip the cooled tubes. Compare your inoculated stab with the control. If the gelatin has been hydrolyzed, the medium will still be rather fluid after cooling (upper tube). If the gelatin has NOT been hydrolyzed, the cooled medium will remain in its gelled condition (lower tube). Record the results for your assigned bacteria. Please be sure to return the uninoculated control so other students can use it. Urea hydrolysis another test for the ability to break down a molecule (urea). In this case it is not hydrolysis of a complex polymer, but simply the splitting of urea into ammonia and CO2. 1. Using aseptic technique, inoculate tubes of urea broth with your assigned bacteria. 2. Incubate the inoculated broth and the control at 37oC for 48 hours. 3. Compare the inoculated broth to the control. Observe the inoculated broth for a change in color to PINK. This indicates that the bacterium was able to degrade urea (with the enzyme urease). Record the results for your assigned bacteria. Citrate utilization this test determines if a bacterium can use citrate as the sole source of carbon. 1. Using aseptic technique, inoculate the surface of slants of Simmons Citrate agar with your assigned bacteria. Use only a small amount of inoculum. 2. Incubate slants at 37oC for 48 hours. 3. Compare your inoculated slant with the control. If the color of the medium has changed from green to BLUE, the bacterium IS able to use citrate as the sole energy and carbon source. If the color remains green, then the bacterium can not use citrate as the sole source of carbon. Usually a bacterium that can not utilize citrate will also not grow. However, there are rare circumstances

in which a bacterium might grow a little but not produce the blue color. This sometimes does occur if a large amount of inoculum is placed on the slant. Record the results for your assigned bacteria.

Catalase enzyme activity (degradation of hydrogen peroxide into water and oxygen). 1. Using aspetic technique, place a drop of culture onto a clean glass microscope slide 2. Add one drop of 3% hydrogen peroxide to the drop of culture 3. Observe for presence or absence of bubbling or foaming. Presence indicates that the hydrogen peroxide is being broken down (by catalase), with the release of oxygen gas (causes the bubbling). The absence of foaming/bubbling indicates that catalase is not present (or is not active for some reason).

Nitrate reduction (a very common reaction of many bacteria that reside in soils). 1. Inoculate separate tubes of trypticase nitrate broth with each of your assigned bacteria. 2. Incubate the tubes at 37oC for 24-48 hours. 3. After incubation, add five drops of sulfanilic acid and then five drops of alpha-naphthylamine to each tube. 4. Observe whether or not a red coloration develops in the cultures (2nd tube from the left). The development of a red color indicates the reduction of nitrates to nitrites. If no color develops, either the bacterium can not reduce nitrates to nitrites OR any nitrites produced were rapidly further reduced to ammonia or other end products (that would not impart the red color). 5. To determine if nitrites were produced, but then some or all were reduced past the nitrite stage, add a minute quantity of powdered zinc to any tubes that are colorless after the sulfanilic acid and alpha-naphthylamine were added. 6. If a red color then appears after the addition of the zinc (far right tube), this is interpreted as NO reduction of nitrates (can't tell if the other result, further reduction of all nitrites, has occured). The zinc actually reduces the nitrates to nitrites, which then produce the red color in the presence of the sulfanilic acid and alpha-naphthylamine.

Litmus milk 1. Inoculate separate tubes of litmus milk medium with each of your assigned bacteria. 2. Incubate all tubes at 37oC for 24-48 hours. 3. After incubation, observe your tubes for one or more of the following results. You will likely need to compare your inoculated litmus milk tubes to an uninoculated control. *Distinctive pink color throughout the medium (middle tube labeled "acid" ) - lactose fermentation (with the production of copious amounts of organic acids) *Distinctive pink color as a band at the top of the tube, but a white color throughout the remaining portion of the medium (below the band all the way to the bottom) and solids in part or most of the tube (usually below any banding of color at the top)- (2nd tube from right, labeled "acid, curd reduction") - acid produced and the litmus (which has acted as a pH indicator) was reduced - reduced litmus is white; if the curd is rather soft and flows when the tube is tipped, this in interpreted as acid curd production (2nd tube from right side); if the curd is rather hard and does not flow when the tube is tipped, this is likely the result of rennin curd formation *If "cracks" appear in any curd that is formed - interpreted as gas production (one possible result during fermentation - could be CO2 and/or H2) *Purple band at the top with the remaining color being white - litmus has been reduced (the white color); the purple color may not mean anything, OR casein has been hydrolyzed with the accompanying production of ammonia, which will turn litmus purple *Deep purple band at the top AND the medium begins to lose body and produces a translucent, brown wheylike (partially liquid (tube at far right side), this is usually interpreted as significant casein breakdown (by several proteolytic enzymes) with the accompanying production of ammonia which turns the litmus purple (indicates alkaline conditions) *Color of the inoculated medium throughout is blue indicates some type of alkaline reaction (tube labeled "alkaline") - may or may not be related to protein (casein) degradation and ammonia production 4. Record the complete set of results for each of your assigned bacteria.

Triple sugar iron - various sugar fermentation "patterns" (acid production) and presence or absence of gas production is used to distinguish between bacteria in the family Enterobacteriaceae and other groups of intestinal bacilli. While not quite as easy to interpret as are separate tubes of glucose, lactose and sucrose (Durham tubes), the time and cost reduction of this "combination" media (which also allows for detection of H2S) makes the use of this medium quite efficient when a lot of tests for enteric bacteria are necessary. 1. Using an inoculating NEEDLE, do a stab inoculation of each TSI slant, followed by a streaking of the inoculum across the surface. Do BOTH types of inoculation in each tube for each of your assigned bacteria. If you are not sure any inoculum remained on the needle to give good inoculation of the slant surface (after the stab), then simply add gather more inoculum (remember to use aseptic technique) and inoculate the surface.

2. Incubate all tubes at 37oC for 18-24 hours (note the shorter incubation time - this allows for easier observations of the various colors and locations of colors in the medium). 3. After incubation, observe the entire slant (within the agar and at the surface) for (hopefully) one of the following results - scroll down to Triple Sugar Iron (TSI) Agar (NOT Kligler's Iron Agar) *red (alkaline) slant and yellow (acid) "butt" (within the agar, often at or near the bottom) - only glucose has been fermented *yellow (acid) slant and yellow (acid) butt - lactose and/or sucrose has been hydrolyzed and then the monomers (e.g., glucose) have been fermented (however, you can not determine which of the dimers has been used) * red (alkaline) slant and red (alkaline) butt or no change in butt color (remains an orange-red color - compare to uninoculated control) - NO fermentation of any of the carbohydrates (glucose, lactose or sucrose) has taken place *cracks/breaks in the agar butt - gas production (CO2 or H2, can't determine which); gas production should be accompanied by either glucose fermentation or lactose/sucrose fermentation - if cracks appear and there is no indication of acid production (i.e., yellow color does not appear), then the cracks should not be interpreted as indicative of gas production SIM a combination of three tests in one tube. These tests are: Sulfur reduction ability of the bacterium to reduce a sulfur metabolite in the medium, producing H2S (a colorless gas that generates a black color in the agar medium); sulfates serve as an inorganic source of energy (chemolithotroph); medium contains ferrous (iron) sulfate to detect the production of H2S (which is colorless gas) Indole production ability of the bacterium to convert tryptophan (an amino acid in the medium) into indole Motility determines if the bacterium is motile or not 1. Using aseptic technique and an inoculating needle, stab a small inoculum of each of your assigned bacteria approximately into separate tubes of SIM agar stab.tubes (sometimes called "deeps") 2. Incubate the deep stabs at 37oC for 24-48 hours. 3. Add a few drops of Kovacs reagent to the top of each of the stabs. The reagent will simply stay there (dont attempt to mix it into the agar). 4. Compare the inoculated deep stab to the control and observe for: Sulfur reduction produces a rather distinct black color in the medium (far right tube) - if H2S is produced (sulfur has been reduced), it reacts with a medium component to produce this black color

Indole production if a red color develops in the Kovacs reagent at the top of the agar (tube on the left) , the bacterium IS able to convert tryptophane to indole (Kovacs mixed with indole turns red); if the Kovac's reagent color does not change (remains a golden/yellow - tube on the right) then indole is not produced Motility if there is a distinct feathering out pattern that extends from the original stab (this is not SIM medium), then the bacterium is motile. Sometimes the black color (if the bacterium can reduce the sulfur-containing substrate) makes it difficult to see the feathering pattern (if it is there). 5. Record the results for your assigned bacteria.

MR-VP 1. Inoculate separate tubes of MR-VP broth with each of your assigned bacteria. 2. Incubated the tubes at 37oC for 24-48 hours. 3. After incubation, transfer approximately 1/3rd of each culture into empty glass test tubes and set aside for VP test. 4. Add five drops of the methyl red indicator to the remaining broths in the original tubes. 5. A red color, (MR positive) indicates the production of large quantities of organic acids are in the medium as the result of glucose fermentation (most often used to detect high lactic-acid producing E. coli). A yellow color (recorded as MR negative) indicates that little or no acid remains in the medium (has likely been converted into non-acidic or neutral end products that can often be detected by the VP test - see the following steps). Record MR the results for each of your assigned bacteria. 6. Add 10 drops of Barritt's reagent A to each of the broths that were set aside for the VP test. 7. Shake each culture to mix the reagent with the rest of the broth. 8. Immediately add 10 drops of Barritt's reagent B and shake the cultures again. 9. Shake the cultures again every 3-4 minutes until approximately 15 minutes has passed. 10. Observe the color appearing in each tube. The presence of a deep "rose" color (right side tube) indicates the presence of acetoin (one of the non-acidic end products). This would be recorded as VP positive. If the deep rose color does not appear after approximately 15 minutes, the bacterium would be considered VP negative. 11. Record the VP results (positive or negative) for each of your assigned bacteria.

The following tests use plates of media (inoculate the surface - however, streaking for isolation of colonies is NOT required, as you are using pure cultures). You should inoculate each bacterium in a small area on the surface of each medium. You can inoculate your assigned bacteria into separate sections of each plate until you have inoculated all of your assigned bacteria. You can use one section of one of the plates as the uninoculated control. Starch hydrolysis determines of a bacterium can hydrolyze starch (a polysaccharide) into maltose and glucose. This is NOT a test to see if the bacterium can ferment either sugar, only if it can hydrolyze starch. 1. Using aseptic technique, inoculate separate sections of plates of starch agar with each of your assigned bacteria. 2. Incubate the plates at 37oC for 24-48 hours. 3. Drip a small amount of Grams iodine on the plate around the inoculated area, and a small amount in an uninoculated area away from the inoculum (your control quadrant). 4. If starch has been hydrolyzed, a CLEAR zone will form around the inoculum. If starch has not been hydrolyzed, no clear zone will form and a blue-black color will result (left side). The iodine reacts with unhydrolyzed starch to produce the color. 5. Compare the inoculate area with the uninoculated area, and record the results for your assigned bacteria. Lipid (fat) hydrolysis the Spirit Blue agar medium detects the ability of a bacterium to break down (hydrolyze) triglycerides in animal or plant fats (lipids). 1. Using aseptic technique, inoculate separate sections of plates of Spirit Blue agar with each of your assigned bacteria 2. Incubate the plates at 37oC for 24-48 hours. 3. Observe for the development of a distinctive, rather intense BLUE zone associated with or around any colonies (left side of the plate) that is darker blue than the medium itself that grow on this medium . The blue color indicates that lipid hydrolysis has occurred. If the color remains a pale blue (right side of plate - compare each different bacterium to an uninoculated medium), or the medium becomes colorless (plate of left side - compare to uninoculated plate on the right) the bacterium is NOT able to hydrolyze the triglycerides in the medium. 4. Record the results for your assigned bacteria.

Oxidase enzyme activity - detects activity of cytochrome oxidase (one of the enzymes in the bacterium's electron transport system - assumes the bacterium is an aerobe or facultative anaerobe) 1. Using aspetic technique, Inoculate separate sections of Tryptic Soy (TS) agar plates with each of your assigned bacteria. 2. Incubate the plates at 37oC for 24-48 hours. 3. After incubation, add 2-3 drops of p-aminodimethylaniline oxidate to each of the inoculated areas. 4. Observe for the presence or absence of a color CHANGE from pink to maroon and finally to purple (lower portion of the plate) that appears within 10-30 seconds after adding the reagent the bacterium is considere positive for oxidase enzyme activity. If no color change takes place, or the change is just slightly darker pink, the bacterium is considered negative for oxidase activity. 5. Record the results for each of your assigned bacterium.