ExerciseⅦ ldentification of Enterobacterium
Bacteria accomplish their various biochemical activities（growth and multiplication）using raw materials（nutrients）obtained from the environment.The biochemical transformations that occur both inside and outside of bacteria are governed by biological catalysts called enzymes.
This part of the laboratory manual presents exercises that have been designed to experimentally demonstrate or test for some of the biochemical activities of bacteria.This will be accomplished by observing the ability of bacteria to use enzymes and degrade carbohydrates，lipids，proteins，and amino acids.The metabolism，or use，of these organic molecules often produces by-products that can be used in the identification and characterization of bacteria.
Materials
24 to 48 hour tryptic soy broth cultures of Escherichia coli；Shigella，Salmonella；5 triple sugar iron agar slants；Bunsen burner；DifcoTM SS Agar；inoculating needle；test-tube rack；3 SIM（sulfide-indole-motility）agar deeps；3 motility test medium deeps；Kovacs'reagent；incubator set at 35℃；urea disks or urease test tablets；4 sterile test tubes；wax pencil；sterile forceps.
Learning Objectives
Each student should be able to：
（1）Prepare the SSagar and Identification of pathogenic Bacteria.
（2）Understand the biochemical reactions involved in the triple sugar iron agar test.
（3）Differentiate among members of the family Enterobacteriaceae.
（4）Distinguish between the Enterobacteriaceae and other intestinal bacteria SS Agar and Salmonella Shigella Agar are moderately selective and differential media for test.
（5）Perform a TSI test.
Salmonella Shigella Agar for testing bacillus
Principles
He isolation of pathogenic enteric bacilli，especially those belonging to the genus Salmonella.This formulation is not recommended for the primary isolation of Shigella.
The culture media that have been developed for the selection and differentiation of enteric microorganisms from clinical and nonclinical materials inhibit the growth of gram-positive species to a varying degree due to the presence of either pure bile salts，mixtures of bile salts or dyes.SS Agar and Salmonella Shigella Agar are examples of media used in the plating of samples for the detection of enteric pathogens that contain bile salt mixtures.This formulation is essentially a modification of the Desoxycholate-Citrate Agar described by Leifson.
SS Agar and Salmonella Shigella Agar are designated as moderately selective media based upon the degree of inhibition of gram-positive microorganisms that they inhibit due to their content of bile salts，brilliant green and citrates.Differentiation of enteric organisms is achieved by the incorporation of lactose in the medium.Organisms that ferment lactose produce acid which，in the presence of the neutral red indicator，results in the formation of red colonies.Lactose nonfermenters form colorless colonies.The latter group contains the majority of the intestinal pathogens，including Salmonella and Shigella.The sodium thiosulfate and ferric citrate enable the detection of hydrogen sulfide production as evidenced by colonies with black centers.
Procedure
1.Suspend the powder in 1 L of purified water.Mix thoroughly.
2.Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.do not autoclave.
3.Cool the medium to approximately 45℃～50℃and pour into Petri dishes.
4.Allow the plates to dry for approximately 2 hours with the covers partially removed.
5.Test samples of the finished product for performance using stable，typical control cultures.
6.Use standard procedures to obtain isolated colonies from specimens.A nonselective medium should also be streaked to increase the chance of recovery when the population of gram negative organisms is low and to provide an indication of other organisms present in the specimen.
7.Incubate plates，protected from light，at（35±2）℃for 18 to 24 hours.If negative after 24 hours，reincubate an additional 24 hours（Figure 1，2）.
Figure 1 Expected Results Typical colonial morphology on Salmonella Shigella Agar is as follows：NO.62 or 63
Figure 2 Typical colonial morphology on Salmonella Shigella Agar
Carbohydrates I:Fermentation of Sugar
Principles
Fermentations are energy-producing biochemical reactions in which organic molecules serve both as electron acceptors and donors.The ability of microorganisms to ferment carbohydrates and the types of products formed are very useful in identification.A given carbohydrate may be fermented to a number of different end products depending upon the microorganism involved.These end products（alcohols，acids，gases，or other organic molecules）are characteristic of the particular microorganisms.For example，if fermenting bacteria are grown in a liquid culture medium containing the carbohydrate glucose，they may produce organic acids as by-products of the fermentation.These acids are released into the medium and lower its p H.If a p H indicator such as phenol red or bromcresol purple is included in the medium，the acid production will change the medium from its original color to yellow.
Some microorganisms，such as E.coli，can use lactose as their sole source of carbon.An essential enzyme in the metabolism of this sugar isβ-galactosidase.βgalactosidase hydrolyzes lactose to galactose and glucose.
As originally described in 1911 by F.F.Russell，the triple sugar iron（TSI）agar test is generally used for the identification of enteric bacteria（Enterobacteriaceae）.It is also used to distinguish the Enterobacteriaceae from other gram negative intestinal bacilli by their ability to catabolize glucose，lactose，or sucrose，and to liberate sulfides from ferrous ammonium sulfate or sodium thiosulfate.TSI agar slants contain a 1% concentration of lactose and sucrose，and a 0.1% glucose concentration.The p H indicator，phenol red，is also incorporated into the medium to detect acid production from carbohydrate fermentation（Figure 3）.
Figure 3 Carbohydrate Fermentation NO.64
（a）Possible carbohydrate fermentation patterns of microorganisms，with phenol red as the p H indicator.（b）The tube on the left is the control.The next tube shows alcohol fermentation.Notice the gas bubble at the top.The third tube from the left shows no carbohydrate fermentation（negative）.The tube on the right shows acid and gas production.
Often Kligler Iron Agar（named after I.J.Kligler in 1917），a differential medium similar to TSI，is used to obtain approximately the same information.
TSI slants are inoculated by streaking the slant surface using a zig-zag streak pattern and then stabbing the agar deep with a straight inoculating needle（Figure 4）.
Figure 4 Transferring Techniques
（a）-（c）Stab technique for transferring bacteria.Notice that the inoculating needle is moved into the tube without touching the walls of the tube，and the needle penetrates medium to i its depth.（d）Technique for streaking the surface of a slant with a loop.
Incubation is for 18 to 24 hours in order to detect the presence of sugar fermentation，gas production，and H2 S production.The following reactions may occur in the TSI tube（Figures 5 -7）
1.Yellow butt（A）and red slant（A）due to the fermentation of glucose（phenol red indicator turns yellow due to the persisting acid formation in the butt）.The slant remains red（alkaline）（K）because of the limited glucose in the medium and，therefore，limited acid formation，which does not persist.
2.A yellow butt（A）and slant（A）due to the fermentation of lactose and/or sucrose（yellow slant and butt due to the high concentration of these sugars）leading to excessive acid formation in the entire medium.
3.Gas formation noted by splitting of the agar.
4.Gas formation（H2 S）seen by blackening of the agar.
Figure 5 Triple Sugar Iron Reactions（TSI-1）and Their Interpretation NO.65
（a）The tube on the left has a yellow butt（acid），red slant（alkaline），H2 S production as indicated by blackening of the agar，and no gas production.（b）The tube on the right shows no H2 S formation，a yellow slant（acid），gas production，and an acid butt.Note that the gas on the bottom has lifted the agar.
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Figure 6 Triple Sugar Iron Reactions（TSI-2）and Their Interpretation NO.66
（a）The tube on the left has a red butt（alkaline），red slant（alkaline），and no acid or H2 S production.（b）The tube on the right has a yellow slant（acid），yellow butt（acid），and no gas or H2S production.
Figure 7 Triple Sugar Iron Reactions（TSI-3）and Their Interpretation NO.67
（a）The tube on the left is an uninoculated control.Notice the red color.（b）The second tube from the left has a yellow slant（acid），yellow butt（acid），gas production at the bottom of the tube，and no H2 S production.This would indicate a weak lactose fermenter.（c）The third tube from the left has a red slant（alkaline），red butt（alkaline），and the black indicates H2 S production，but no gas.（d）The tube on the right has a red slant（alkaline），yellow butt（acid），H2 S production，but no gas production.This would indicate a nonlactose fermenter.
5.Red butt（K）and slant（K）indicates that none of the sugars were fermented and neither gas nor H2S were produced.Table 1 gives reactions usually expected from some of the more frequently encountered genera of the Enterobacteriaceae.Figure 6 summarizes the possible reactions and results in TSI for the various bacteria used in this experiment.
Table 1 Results of TSI Reaction
Procedure
1.Label each of the TSI agar slants with the name of the bacterium to be inoculated.Use one of the tubes as a control.Place your name and date on each tube.
2.Using aseptic technique streak the slant with the appropriate bacterium and then stab the butt.Screw the caps on the tubes but do not tighten！
3.Incubate for only 18 to 24 hours at 35℃for changes in the butt and on the slant.Tubes should be incubated and checked daily for up to seven days in order to observe blackening.
4.Examine all slant cultures for the color of the slant and butt，and for the presence or absence of blackening within the medium.
5.Record your results in the report.
Hydrogen Sulfide Production and Motility
Principles
Many proteins are rich in sulfur-containing amino acids such as cysteine.When these proteins are hydrolyzed by some bacteria，the amino acids are released and taken up as nutrients.Cysteine，in the presence of cysteine desulfurase，loses its sulfur atom through the addition of hydrogen from water to form hydrogen sulfide gas.
In this exercise，the SIM medium（named after J.S.Simmons in 1926）contains peptones and sodium thiosulfate as substrates，and ferrous ammonium sulfate，Fe（NH4）SO4，as the H2Sindicator.Cysteine is a component of the peptones used in SIM medium.Sufficient agar is present to make the medium semisolid.Once H2Sis produced，it combines with the ferrous ammonium sulfate，forming an insoluble，black ferrous sulfide precipitate that can be seen along the line of the stab inoculation.If the organism is also motile，the entire tube may turn black.This black line or tube indicates a positive H2 S reaction；absence of a black precipitate indicates a negative reaction（Figure 8）.
Figure 8 Hydrogen Sulfide Production
It should be noted that not all bacteria are either H2 S positive with motility or H2 S negative and no motility.Many other possible combinations exist.
Motility is present when the growth of the culture is not restricted to the stab line of the inoculation.Growth of nonmotile bacteria is confined to the line of inoculation.One can also use semisolid media（motility test medium deeps）to determine whether a bacterial strain is motile.During growth，motile bacteria will migrate from the line of inoculation to form a dense turbidity in the surrounding medium；nonmotile bacteria will grow only along the line of the inoculation.
Procedure
1.Label each of the SIM agar deep tubes with the name of the bacterium to be inoculated，your name，and date.
2.Using aseptic technique inoculate each tube with the appropriate bacterium by stabbing the medium 3/4 of the way to the bottom of the tube.Do the same for the three motility test medium deeps.
3.Incubate the cultures for 24 to 48 hours at 35℃.
4.Examine the SIM cultures for the presence or absence of a black precipitate along the line of the stab inoculation.A black precipitate of FeS indicates the presence of H2S.
5.Based on your observations，determine and record in the report whether or not each bacterium was capable of H2 S production，and the presence（+）or absence（-）of motility.
6.If desired，one can also test for indole production by adding 5 drops of Kovacs'（named after the German bacteriologist，Nikolaus Kovacs，in the early 1900s）reagent to the SIM cultures and looking for the development of a red color at the top of the deeps.
Proteins,Amino Acids,and Enzymes:Urease Activity（for Optional）
Principles
In the clinical laboratory，members of the genus Proteus can be distinguished from other enteric nonlactose-fermenting bacteria（Salmonella，Shigella）by their fast urease activity.P.mirabilis is a major cause of human urinary tract infections.
Some bacteria are able to produce an enzyme called urease that attacks the nitrogen and carbon bond in amide compounds such as urea，forming the end products ammonia，CO2，and water（Figure 9）.Urease activity（the urease test）is detected by growing bacteria in a medium containing urea and using a p H indicator such as phenol red.When urea is hydrolyzed，ammonia accumulates in the medium and makes it alkaline.This increase in p H causes the indicator to change from orange-red to deep pink or purplish red（cerise）and is a positive test for urea hydrolysis.Failure of a deep pink color to develop is a negative test.
Figure 9 Urea Hydrolysis NO.68
（a）Uninoculated control.（b）Weakly positive reaction（delayed positive）.（c）Very rapid positive reaction.（d）Negative reaction.
Procedure
First Period
1.Label each of the urea broth tubes with the name of the bacterium to be inoculated，your name，and date.
2.Using aseptic technique inoculate each tube with the appropriate bacterium by means of a loop inoculation.
3.Incubate the tubes for 24 to 48 hours at 35℃.
Urea Disks or Tablets：
1.Add 0.5 ml（about 20 drops）of sterile distilled water to four sterile test tubes for the Difco disk or 1 ml distilled water for the KEY tablet.
2.Transfer one or two loopfuls of bacterial paste to each tube.Label with your name and date.
3.Using sterile forceps，add one urea or urease disk tablet to each tube.
4.Incubate up to 4 hours at 35℃.Check for a color change each hour.（The KEY test may be incubated up to 24 hours if necessary.）
5.Examine all of the urea broth cultures and urea disk or urease tablet tubes to determine their color（Figures 10）.
Figure 10 KEY Test for Urea NO.69
After incubation，a pink to red color constitutes a positive test（tube on the left）.If the original straw color persists，the test is negative（tube on the right）.
6.Based on your observations，determine and record in the report for exercise 31 whether each bacterium was capable of hydrolyzing urea.