32 STUDY OF METABOLIC ACTIVITIES OF BACTERIA METABOLIC DIVERSITY Different species of bacteria show a tremendous range in the types of metabolic activities they are able to carry out. They vary in their ability to hydrolyze or digest large molecules like carbohydrates (polysaccharides), proteins or fats. These variations are due to the differences in the types of enzymes that bacteria possess. This biochemical diversity found among bacteria has two consequences in nature. 1, It makes possible the decomposition of all naturally occurring organic molecules, and thereby exploitation of most natural habitats. 2. Due to this variation there is a close network of interactions and interdependence among different species. One species can utilize the breakdown substances that another species produces. Biochemical diversities among bacteria has been exploited by microbiologists as a means of identifying and classifying bacterial species. BIOCHEMICAL TESTS The presence of a particular enzyme in a microorganism can be tested by incorporating a specific substrate in a medium, (if necessary), and then detecting the products formed or even checking the disappearance of the substrate from the medium. These biochemical tests employ various media (having different substrates) which when in- oculated with a particular species of bacteria will follow a specific metabolic pathway to hydrolyze the substrates available to them. Some of the routine biochemical tests used for determining metabolic activities of bacteria can be broadly classified as follows. Utilization of carbohydrates and organic acids 1. Carbohydrate fermentation test. 2. Oxidation—fermentation test. 3. Methyl red test. 4. Voges-Proskauer test. 5. Citrate utilization test. Utilization of nitrogenous compounds 1. Indole production test. 2, HS production test. 3. Decarboxylation test . 4, Deamination test, 5. Urea hydrolysis test. 6. Nitrate reduction test. 7. Ammonia production test. Decomposition of large molecules 1. Starch hydrolysis test. 2. Gelatin hydrolysis test. 3. Casein hydrolysis test. 4. Lipid hydrolysis test. Miscellaneous tests 1, Catalase test. 2. Coagulase test. 3. Oxidase test. Combined tests using composite test media 1. Triple sugar iron agar test. 2. Litmus milk test. 110BIOCHEMICAL TESTS CARBOHYDRATES FERMENTATION (SUGAR UTILIZATION) TEST Microorganisms can catabolize many different types of s fructose, mannose, galactose) can easily enter glycolytic pathway either dir Phosphorylation. However, di accharides are first broken down to monosa gars. Monosaccharides (glucose, tly or after initial iecharides by direct hydrolysis or by phosphorolysis Maltose + H,O ———allase __> 2 glucose Maltose + Pj —malinse phosphorylase > p.D.glucose-I-P + glucose Sucrose + H,0 —sucrase > glucose + fructose Sucrose + Pi —Sustase_phosphorvlas: __> q-D-glucose-I-P + fructose Lactose + H,0 —L-stlactosidase __ giucose + galactose The monosaccharides so formed then enter glycolytic pathway. Pentose sugars like ribose and xylose are phosphorylated and then are broken down via, pentose phosphate pathway. Principle Sugars are metabolized through different metabolic pathways (depending on type of species and aerobic or anaerobic environment) to form various acids like pyruvate, lactate, succinate, formate, acetate etc. These acids so formed may further break down to gases (formic hydrogenlyase will split formic acid to H, and CO,). Due to acid formation the pH of the medium is lowered and Andrade’s indicator turns pink. Gas formation can be demonstrated by the use of Durham's tube (a small tube placed verted in the sugar tube), which collects the gas. Requirements 1. Test culture. 2. Nutrient sugar broth. Procedure J. Inoculate a loopful of culture into the sugar broth and incubate 37 °C for overnight, 2. Observe the tube for acid and gas production. Interpretation Acid production changes the color of the medium to pink and gas produced is collected in Durham's tube as a small bubble. OXIDATION—FERMENTATION {HUGH AND LEIFSON) TEST This is also known as oxferm’ test or O-F test. Saccharolytic organisms degrade glucose oxidatively or fermentatively. The end products of fermentation are relatively strong acids that can be detected in a conventional fermentation test medium. However, the acids formed during the oxidative degradation of glucose are extremely weak, and the more sensitive Hugh and Leifson medium is required for the test. Principle Hugh and Leifson medium has peptone and carbohydrate in the ratio of 0.2 :1, in contrast to the 2:1 ratio found in media used for carbohydrate fermentation. The decrease in peptone minimizes the formation of alkaline amines that may be formed from oxidative metabolism. The relatively larger amount of carbohydrate serve to increase the amount of acid that can be formed. The semisolid consistency of agar permits the acids that form on the surface of gar to penetrate throughout the medium, making the interpretation of the pH shift of the oa 4141 bromothymol blue easier to visualize. e ion re trappe Production of gas during the metabolism can also be detected as the gas bubbles are trapp* in the medium due to the semisolid consistency of the medium Motility can also be observed in this medium. : i ed with In order to create the environment for fermentative metabolism, the medium is cover 2 em of sterile paraffin oil Requirements 1. Hugh and Leifson medium (two tubes), 2. Test culture. 3. Sterile paraffin oil. Procedure {Heat wo tubes of the Hugh and Leifson medium in the boiling water bath for 10 minutes to drive off the oxygen 2. Cool and inoculate (stab inoculation) media with the test culture. 3. Overlay one tube with sterile paraffin oil up to 2 cm above medium. 4, Incubate both the tubes at 37 °C for overnight. 5. Observe for the acid &/or gas production in both tubes. Interpretation |. Acid &/or gas in aerobic tube only: oxidative metabolism positive. 2. Acid &/or gas in both the tubes: fermentative metabolism positive. 3. Acid &/or gas absent in both the tubes: non-saccharolytie organisms. METHYL RED (M-R) TEST There are several different types of fermentative pathways, Members of the family Entero- bacteriaceae, can metabolize glucose to pyruvic acid to formic acid in, a process sometimes referred to as “formic acid fermentation’. There are two types of formic acid fermentations, MIXED ACID FERMENTATION Fr results in formation of ethanol and a complex mixture of various acids like acetic, lactic, Guseinic and formic acids. If formic hydrogenlyase is present, formic acid will split to H, and Oz, This pattern is seen in Escherichia, Salmonella, Proteus and other genera, BUTANEDIOL FERMENTATION sre pyruvate is converted to acetoin (acetyl methyl earbinol) which is then reduced to 2,3+ butanediol with NADH. A large amount of ethanol is also produced, together with smaller amounts of acids found in mixed acid fermentation. This is the characteristic feature of Enterobacter, Serratia and few species of Bacillus. Thus mixed acid fermenters produce four times more acidic products, whereas butanediol fer- menters form mainly neutral products. Hence, mixed acid fermenters acidify incubation media to a much greater extent. This is the basis of methyl red test. Principle tor. This is because of the quantities of acids if produ over, methyl red is a pH indicator having a range between 6.2 (yellow) to 4.4 (red), so the PH at which methyl red detects acid is considerable lower than the pH for other indicators used in bacteriological media. Requirements }; Glucose phosphate broth (GPB), methyl red indicator. 2. Test’ culture, fact that the medium (GPB) is strongly buffered, hence, minute iced, will not permit the pH of the medium to drop down. More-es Procedure °C for 48-72 hours. 1 2 3. Inoculate GPB with the test culture and incubate the broth at 37 After incubation, add about 5 drops of methyl red indicator to the medium. Observe for the development of the red color. Interpretation . broth indicates the methyl red test positive The development of stable red color in the VOGES-PROSKAUER (V-P) TEST fa (acetyl methyl earbinol), the precursor of butanediol which is The V-P test detects acet produced during the buta tails refer M-R test). id fermentation (for de- ol fermentation but not during mixed 2 Principle Requirements In presence of alkali and air (vigorons shaking) acetoin is oxidized to diacetyl, which reacts with the guanidine nucleus of arginine present in proteins of peptone to produce pink color. At times a pinch of creatine is added to provide an additional source of guanidine nucleus and thus accelerate pink color formation. Test is made sensitive by adding a-naphthol, which serves as catalyst.» C20, CH,.CO.CO.CH, + NH;.CNH.CH,N.CH; COOH —___—> pink-red product > CH;.CHOH.CO.CH, —————> CH,.CO.CO.CH,; 1. Glucose phosphate broth (GPB). .2. 5% alcoholic x-naphthol and 40% KOH solution. 3. Test cuiture. Procedure 1. Inoculate the medium (GPB) with culture and incubate the medium at 37 °C for 24-48 hours. 2. After incubation, add 0.6 ml of x-naphthol and 0.2 ml of KOH solution per ml of culture broth (reagents should be added in this order only because a-naphihol exerts catalytic effect only if added before the KOH). 4, Shake well after addition of each reagent and slope the tube to increase the aeration. Read results after 15-60 minutes. ( Interpretation CITRATE UTILIZATION TEST Development of red color within 15 minutes or more, after addition of reagents indicate the presence of diacetyl. The test should not be read after standing for over 1 hour. Sodium citrate is a salt of citric acid, a simple organic compound found as one of the metabolites in the tricarboxylic acid (TCA) or Kreb’s cycle. Some bacteria can obtain energy and the carbon by utilizing citrate. This characteristic is an important identification criteria among many members of Enterobacteriaceae. Any medium used to detect citrate utilization must be devoid of proteins and carbohydrates as sources of carbon. Principle ‘The test determines the ability of bacteria to use citrate as a sole of carbon and energy. This ability depends on the presence of a citrate permease that facilitates transport of citrate into the bacterium. Once inside the cell, citrate is converted to pyruvate and CO,. Citrate agar slant contain sodium citrate as the sole source of carbon, ammonium phosphate as a sole source of nitrogen, and bromothymol blue as a pH icator [pH 6 (yellow)—pH 7.6 (blue)]. This test is done on slants since O, is necessary for citrate utilization. When bacteria oxidize citrate, ‘they remove it from the medium and liberate CO;. This CO, combines with sodium (supplied 413Sie once : ct. Similarly by Sodium citrate) and water to form sodium carbonate—an alkaline Dee eet ppdce teria that utilize citrate can also extract nitrogen from the ammonium salt, roducts ; ‘hese alkaline pr‘ ton of ammonia, which is converted to ammonium hydroxide (NH,OH). Tl ‘ve citrate test. Taise pH, and turn pH indicator to a blue color and represents a posit Requirements 1. Simmons’s citrate agar slant. 2. Test culture Procedure 4 24-48 hours. 1, Streak heavily on the surface of the agar slant and incubate the slant at 37 °C for 24 2. Record the color change of the slant after incubation Interpretation Rca test is represented by the development of a deep blue color within mea indicating that the test organism has been able to utilize the citrate contained in the medi with the production of alkaline products.«4 positive test may also be read ene rata if there is visible colonial growth along the inoculation sireak line. This is possible becat me for growth to be visible, the organism must enter the log phase of the growth and should pal carbon and nitrogen. A positive interpretation from reading the streak line can be confirme: by incubating the tube for additional 24 hours, when a blue color usually develops. INDOLE PRODUCTION TEST This is one of the test based on protein utilization. It forms one of the four test (IMViC) widely used in identification of coliforms. Test can be performed in any protein containing medium (e.g. peptone broth) because all proteins contain tryptophan (a parent molecule from which indole is formed). However, the use of tryptone is recommended instead of peptone because it contains higher amount of tryptophan. It is also advisable to use non carbohy- drate protein medium so as to avoid protein sparing effect. Principle Indole, a benzy! pyrrole, is one of the metabolic degradation products of the amino acid tryptophan. Organisms that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvate and ammonia. Indole so produced reacts with the aldehyde group of a weakl dimethylaminobenzaldehyde (Ehrlich’s or Kovac’s reagent) in colored rose-indole complex. The reaction can also occur with pared with HCl. Indole is a substance which reduces surface tension and hence it is concentrated in the surface layer of the medium. Moreover because indole is soluble in orgaaic compounds, it is rec. ommended that chloroform or xylene be added prior to adding Ehrlich’s reagent. This serves {wo Purposes, firstly it extracts indole from whole of the medium and secondly it forms a Separate layer above the medium, As a result reagent reacts with the indole extracted in xylene and forms pink color. Organic solvents like chloroform, ether, and light petroleum can be used instead of xylene. ir suis step-is-not necessary with Kovac’s reagent because the amvl alcohol is _used for the diluent is capable of extracting sufficient indole from the aqueous medium to Produce a positive reaction. fo Requirements 1. 1% Tryptone broth, and Ehrlich’s or Kovac’s reagent. Procedure \. Inoculate the tryptone broth with a loopful of test culture 2. After incubati ion add 3-4 drops of xylene in the medium 3. Allow the two layers to separate. ly acid alcoholic solution of p~ presence of heat to form pink out heat, if the reagent is pre- 2. Test culture, & incubate at 37 EC endihaie for 24 hours. it vigorously.4. Add slowly, 1 ml of Ehrlich’s reagent so as to form the layer on the surface of xylene. Observe the formation of pink colored ring at the lower surface of the xylene layer. 6. If Kovac’s reagent is to be used (do not add xylene), add | ml of reagent on top of the broth and observe for the pink ring, Interpretation Development of bright fuc! seconds after adding the reag red color at the interface of the reagent and the broth within ent is indicative of the presence of indole and is a positive test. HYDROGEN SULPHIDE PRODUCTION TEST Microorganisms produce HS by two different ways; 1. By reduction of thiosulphate, and 2. Formation of H,S from sulphur containing amino acids by the enzyme desulphurase Therefore two kinds of test exist for detection of HS production, THIOSULPHATE IRON TEST This test detects the reduction of thiosulphate ($,0,*), sulphate (SO,*), or sulphite (SO,*) Principle Thiosulphate present in the medium is reduced, resulting in production of H,S, this H,S forms a black ferrous sulphide Precipitate with the iron salt indicator. 38,0% + 4H- > 280; + 2H,S WS + FeSO, ————_> es 4. 80, Anaerobic as well as acidic environments are optimum for reduction of thiosulphate. There fore H,S production occurs inside but not on the surface of the standard H.S stab agar. Similarly in the butt of TSI-type media fermentation of glucose creates oxidation-reduction Potential favorable for H;S_ production. = On media like Salmonella—Shigella agar, uth sulphite agar, deoxycholate citrate agar etc., black FeS precipitates appears at the sites (je. at the center of the colony, because center of the colony is more anaerobic as compared to its periphery) which provide suitable envi- ronment for its accumulation. Requirements 1. Standard thiosulphate iron agar stab medium. 2. Test culture. Procedure 1. Stab the medium with the test culture and incubate the medium at 37 °C for 24 hours. 2. After incubation look for the black color in the lower portion of the stab agar medium. Interpretation Formation of black FeS precipitates in the medium is indication of thiosulphate reduction LEAD ACETATE PAPER-STRIP TEST This test is for detection of H,S production from sulphur containing amino acids, Principle There are about twenty amino acids commonly found in proteins. Out of these methionine and cysteine contain sulphur in their structure. Cysteine often occurs in proteins in its oxi- dized form called cystine. Organisms possessing the enzyme amino acid desulphurase can remove the sulphur found in the amino acid as H,S according to the following reactions. Cysteine + HO ——#istine desuiphurase > Pyruvate + H,S + NH,‘Oth \ ine as follows. “t {WO sulphur containing amino acids are first converted to cysteine #5 foll i > cysteine > a-ketoglutarate urase 8) Cystine —cvstine reductase 5) Methionine —> cysteine —> homocysteine ———> eystathione —— . iphi Cysteine so formed can liberate H,S when acted upon by cysteine desulp! bismuth; : ‘ iron or HGS can be detected by reacting it with salts of certain metals like lead, f Tesulting in formation of dark sulphide of these metals. fe cuitaeetietianec Bacteria that give a positive thiosulphate-iron test are also positive for tl test Requirements iced 1, 2% Peptone broth. 2. Saturated solution of lead acetate. 3. Test culture. Procedure 1. Inoculate a loopful of test culture in 2% peptone broth ‘ ti acetate. Soak a white filter paper strip (5 mm x 50 mm) in saturated solution of een aii Place lead acetate filter paper strip in neck of the tube in such a position ti is of the strip projects below the cotton plug. Incubate the medium at 37 °C for 4. After incubation observe for the blackening of the filter paper. Interpretation Blackening of filter paper strip is due to the formation of lead sulphide, which indicates HS production by the organism. DECARBOXYLATION (MOELLER’S) TEST Decarboxylases are substrate specific enzymes that are capable of removing carboxy! (COOH) group of amino acids as carbon dioxide together with the formation of alkaline amines. Each decarboxylase enzyme is specific for an amino acid and requires pyridoxal phosphate as coenzyme. Amino acid decarboxylases are produced at low pH, and show their optimal ac- tivity at pH 3-5, The general reaction for all of these enzymes is as follows: R-CH-NH,-COOH —4decatbawlases > R-CH,-NH, + CO, Lysine, ornithine, arginine are the three amino acids routinely tested for identification of bacteria, The specific products are as follows: lysine —> cadaverine, ornithine ——> putrescine, Principle The pH of the Moeller’s medium is adjusted to 6.0. This low pH is essential because de- carboxylases ate not optimally active until the pH of the medium drops below 5.5. This is achieved by the fermentation of small amounts (0.05%) of glucose to A yellow color change in bromoc: arginine ——> citrulline Since the decarboxylation reaction proceeds opti s . ptimally under anaerobi iti i is overlayered with mineral oil after inoculation, conditions the medium Requirements |. Moeller’s decarboxylation broth (with and without amino aci 2. Sterile mineral oil, 3. nner). Test culture.Procedure 1. Inoculate @ loopful of test culture into two tubes of Moeller’s decarboxylase broth, one con- taining the amino acid to be tested and, the other to be used as a control tube which is devoid of amino acid. 2, Overlay both the media tubes with sterile mineral oil to cover about 2 cm above the surface of the medium and incubate both tubes at 37 °C for up to 72 hours, 3. Observe the change of color of the medium in both the tubes up to 72 hours. interpretation ; Conversion of control tube to yellow color indicates that the organism is viable and that the pH of the medium has been lowered sufficiently to activate decarboxylase enzyme® Rever Fon of the tube containing the amino acid, to a purple blue color indicates a positive test due to the release of amines from the decarboxylation reaction PHENYLALANINE DEAMINATION TEST Phenylalanine is an amino acid that upon deamination forms phenylpyruvic acid (a keto acid), ‘Among Enterobacteriaceae, only certain members of genera like Proteus possess the enzyme necessary for this conversion. Principle f Organisms capable of producing the enzyme deaminase. can remove the amino group from the amino acid phenylalanine, and forms phenylpyruvic acid. Pheaylpyruvic acid so formed react with ferric chloride to form green colored complex. Requirements 1. Phenylalanine agar slant. 2. 10% aqueous ferric chloride solution and test culture. Procedure |. Streak the slant with a loopful of test culture, and incubate at 37 °C for 18-24 hours. 2. After incubation add 4-5 drops of ferric chloride reagent on to the surface of the agar. 3. Observe for the appearance of an intense green color. Interpretation Immediate appearance of an intense green color indicates the presence of phenylpyruvic acid and it is positive test. UREA HYDROLYSIS TEST |. _ Urea is a diamide of carbonic acid. Amides are hydrolyzed with the release of ammonia. Principle A strongly buffered medium (Stuart’s urea broth, pH 6.8), in which urea is the only nitrogen source is used for the test. Urease is an enzyme possessed by many species of mi- eroorganisms that can hydrolyze urea as follows. NH-CNH, + 2H,o —H## > co, + 2NH, ——————> (NH)),CO, The ammonia so produced reacts in solution to form ammonium carbonate, resulting in alkalinization and an increase in the pH of the medium. This is indicated by change in color of the indicator phenol red (pH 6.8-8.4, yellow-purple red) Due to high buffering capacity of the medium, only those organisms possessing vigorous urease activity (Proteus vulgaris) can give the test positive. Requirements 1, Stuart's urea broth. 2. Test culture. 3. Chnstensens Urea. knediimnProcedure °C for 24 hours. i. inoculate a loopful of test culture in ur 3. Observe for the change in color of the broth after incubation ea broth, and incubate at 37 Interpretation ’ skate Peale red color throughout the medium indicates alkalinization and urea hy NITRATE REDUCTION TEST SI ce nitrate under two different gr Microorganisms reduce nitrate under tw ‘ i aun 1. Certain bacteria catty out nitrate respiration under anaerobic / microaerophilic co use nitrate as the terminal electron acceptor, thereby reducing it. uma i aerobic s 2. In contrast to this, assimilatory nitrates reduction generally occurs under aerol in absence of sources of nitrogen. ‘ F 4 aaeacen latory nitrate reduction, nitrate is incorporated into organic material and jot gi, and algae owth conditions with different purpose. and Thus in assi eave participate in energy production. This process is wide spread among bacteria, which can use nitrate as the sole source of nitrogen. In the first step nitrate is reduced to nitrite by nitrate reductas NOx + NADPH + H" —Mivete reductase > NO, + NADP + HO Nitrite is next reduced to ammonia with a series of two electrons addition catalyzed by nitrite reductase, Hydroxylamine may be an intermediate. NO; —————_> (NH,OH) ——22.2#._#20 _> NH, Ammonia is then assimilated by the reaction, o-ketoglutarate + NH, —slilamale detydrogenase > glutamate Test for nitrate reduction is performed using nitrate broth. Principle Organisms possessing nitrate reductase when grown in a medium containing nitrate as the sole source of nitrogen will reduce nitrate to nitrite. The formation of nitrite can be detected by adding sulphanilic acid, which forms a diazonium salt, which in turn reacts with o-naph- thylamine thereby leading to formation of soluble red azo dye (p-sulphobenzene-azo-c-napth- lamine). Requirements 1. Peptone nitrate broth (PNB). 2. Test culture 3. Zine dust 4. a-napthylamine reagent (reagent A). 5. Sulphanilic acid reagent (reagent B). Procedure. |. Inoculate PNB with a loopful of test culture and incubate the medium at 37 °C. 2. Add 0.5 ml of the reagent A and B each to the test medium in this order. 3. Observe the development of red color within 30 seconds after adding test reagent. 4. If no color develops, add a pinch of zine dust, mix them well and observe the development of red color. Interpretation The development of red color within 30 seconds after adding the test Teagent indicates the Presence of nitrites and is positive nitrite reduction test. If no color devel i test reagents, this may indicate that; gence Nitrates have been not reduced, (a true negative reaction) OR + They have been reduced to products other than nitrites, such as ammonia, molecular nitrogen(denitrification), nitric oxide (NO), or nitrous oxide (N,O) and hydroxylamine. Since ae reagents detect only nitrites the latter process would lead to @ false negative reading. i) us it is necessary to add a small quantity of zinc dust to all negative reactions. Zine ions reé be nitrates to nitrites and the development of a red color after adding zine dust indicates the presence of residual nitrates and confirms a true negative reaction AMMONIA PRODUCTION 4 As explained earlier certain organisms possessing the enzymes nitrate reductase and nitrite reductase can reduce nitrate and nitrite to ammonia This ammonia can be assimilated to or- Principle When nitrates or nitrites are reduced by microorganisms, ammonia is liberated. This ammonia while trying to escape ftom the tube converts the litmus paper hanging from the neck of the tube from red to blue. Requirements 1, Peptone nitrate broth 2. red litmus paper. 3. Test culture Procedure 1. Inoculate a loopful of test culture to peptone nitrate broth, n 2 Place a red litmus paper strip in the mouth of the culture tube in such a position that 1/4 to 1/2 of the strip projects below the cotton plug. Incubate the medium at 37 °C for 24 hours. After incubation observe for the change of red litmus to blue color. Interpretation The change of red litmus to purple or blue indicates the ammonia production and can be read as positive test. STARCH HYDROLYSIS TEST Starch is a composite polysaccharide consisting of amylose and amylopectin usually in a ratio of 1:4 or 1:5. Amylose is a linear a-(I—>4)- linked polymer of D-glucose; amylopectin is 2 branched a-(1—>4)- linked polymer of D-glucose linked with a-(I—>6) branched points. Starch when added in hot water, amylose fraction diffuses into the solution whereas amylopecti remains insoluble. Enzyme amylase act upon the starch and catalyze the hydrolysis of a-(1—>4)- glycosidic linkage resulting into formation of dextrins, and then into maltose and finally to glucose. Principle 1. Starch hydrolysis test is based on a color reaction of non-hydrolyzed starch with Lugol’s iodine. Starch gives a deep-blue color, whereas its breakdown products as hydrolysis progresses, gradually becomes violet, brownish red and finally colorless. Organisms producing amylase utilize starch in the vicinity of the colony, hence when the medium is flooded with iodine solution, colorless zone is seen surrounding the colony, the remaining portion of the medium turns blue, When the starch is added in hot water, amylose fraction being soluble diffuses in it; while amylopectin remains insoluble. As a result, milky white colloidal solution is formed. Hence the starch agar plate has a opaque, milky white appearance. If the organisms growing on such a medium produce amylase, the starch from the vicinity of the colony is utilized gi ing a clear transparent zone surrounded by opaque medium. Requirements ~ 1. Starch agar plate, 2. Lugol’s iodine, 3. Test culture. ae 119Procedure F i Dee epitowt 1. Inoculate the test culture on the plate as spot or line, and incubate at 37 °C 3. Observe for transparent zone surrounding the colony. a ctgel ae aealy 4. Flood the plate with Lugol’s iodine and read immediately, because the blue Interpretation reerenac: Clear colorless zone around the growth is due to starch hydrolysis (due to amy'as Pi tion) and test is positive. While the blue color throughout the medium and arou is a negative test. CASEIN HYDROLYSIS TEST 5 Many organotrophs (heterotrophs) can utilize exogenous proteins as a source of carbon and nitrogen. Since the protein molecules are too large to enter the cell, microorganisms sect extracellular enzymes called proteases, which hydrolyze peptide bonds to form peptides Peptides are short chains made up of few amino acids, which can be further broken down to the individual amino acids by peptidases. The sequence of protein breakdown can be summarized as follows. he growth Proteins ——> proteoses > peptones > peptides ——> amino acids. Peptides and amino acids being smaller in size can enter the cell where the amino acids undergo oxidation to form the compounds that may enter the TCA cycle. Principle ; Casein is sparingly soluble in water and hence medium formed due to its incorporation is opaque (milky white). Caseolytic organisms produce casease which hydrolyze casein to soluble form paracasein. Hence the clear zone is observed in the medium surrounding the growth. Requirements 1 Test culture. 2. Nutrient casein agar plate / skim milk agar plate. Procedure 1. Inoculate the test culture on the plate as spot or line, and incubate at 37 °C for 24-48 h 2, Observe for a clear zone of casein solubilization surrounding the growth of organisms. Interpretation Development of clear zone surrounding the growth indicates the formation of paracasein due to hydrolysis of casein by enzyme casease and the test is positive, GELATIN HYDROLYSIS TEST Gelatin is a bone protein obtained from bones from which fats, and minerals like phosphates have been removed. Gelatin is an unusual kind of protein which in aqueous solution forms a solid gel at room temperature but changes to liquid above 25-28 °C. It can tolerate heating at 100-121 °C without being coagulated, and therefore, unlike other proteins, it can be ster. ilized by autoclaving, Gelatin is also unique in a sense that very few organisms like species of Pseudomonas, and Proteus wbich possess enzyme gelatinase can hydrolyze it. Production of enzyme gelatinase can be demonstrated by tube test, and plate test. Tube Test Principle Gelatinase is an extracellular proteolytic enzyme capable of hydrolyzing gelatin, lyzed product do not gel at a temperature below 25-28 °C. This is known as of gelatin. As the cultures are usually incubated at a temperature above the mel gelatin (37 °C), it is necessary to cool the medium for 30 minutes prior to readi Such hydro- liquefaction Iting point of ing the results, 120: Requirements ater bath with ice: T vies nutrientielatin’agar tubs aeiTeatvgniture.. 3+ Refrigerator oF #20 eee Procedure e is left uninocu- 1. Tnoculate a loopful of test culture into one of the tube, and the second tube is left ur lated (control), Incubate both the tubes at 37 °C for 24-72 hours. + in refrigerator or in ice water bath, 2. After incubation place both the tubes at 5-10 °C either for 30-60 minutes. ' 3. After refrigeration slightly tilt tubes, so as to check the liquefaction of gelatin: Interpretation ia in test is positive (indicati e production) if the inoculated medium remains in liquid state even after reftigeration; while the control medium solidifies Frazier’s Plate Test . Principle ‘Ont of the properties of proteins is to coagulate under acidic conditions As eller presence of heavy metals. Gelatin being protein, when the plate is flooded with acide mer furie chloride solution, the unhydrolysed gelatin is precipitated as cloudy white precipitate. Requirements 1. Nutrient gelatin agar plate, 2. Frazier’s reagent. 3. Test culture. Procedure 1. inoculate the test culture on the plate as spot or line, and incubate at 37 °C for 24-72 hours, 2 After incubation flood the plate with Frazier's reagent (acidic mercuric chloride). Observe for clear zone around the growth, surrounded by the cloudy white prec! Interpretation ; f Formation of clear zone around the colony is due to gelatinase production by microorganisms, which hydrolyze the gelatin indicating the positive test. While cloudy white precipitates throughout the medium and around the growth is an indication of negative test tes, LIPID HYDROLYSIS TEST Fatty acid esters of the alcohol glycerol are called acylglycerol of glycerides or neutral lipids/fats, Triacylglycerols are the most common of all lipids and are mainly found in plant ‘and animal cells. Triacylglycerols that are stored at room temperature are often referred to as ‘fats’ and those which are liquid as ‘oils’. Few organisms can use lipids as alternative source of energy (main being carbohydrates like glucose & sucrose). The break down of lipids or fats begin with the cleavage of triglycerides by addition of water to form glycerol and fatty acids by means of enzymes called lipases. The glycerol liberated is oxidized to glycerol-3-phosphate and dihydroxy acetone phosphate which then enters EMP pathway. Fatty acids are oxidized by the successive removal of 2- carbon fragments in the form of acetyl-CoA, a process known as B-oxidation. The acetyl-CoA formed can then enter the TCA cycle. When lipase-producing bacteria contaminate food products, the lipolytic bacteria hydrolyze the lipids, causing spoilage termed rancidity. Principle Tributyric acid (tributyrene) is commonly used to check the lipase activi i i Tributyrene is hydrolyzed as follows: , See ee ee aE Tributyrene + 3H,0 —limuss_—> Glycerol + butyric acid 124alcium butyrate: carbonate to form solubilized calcium buty ‘The butyric acid so formed reacts with eatei CH,COOH + Caco, — —> CH,COOCa + CO, By adding a pH indicator to the culture medium, it is also of lipids by a color change. For example, Nile blue sulphite has a | royal blue around lipolytic bact colonies due to the acid pH. Requirements possible to detect the hydrolysis lavender color, and it turns Tributyrene agar plate. 2. Test culture Procedure o Z 3. 1. Inoculate the test culture on the plate as spot or line, and incubate at 37 °C for 24-72 hour: 2. Observe for the clear zone of calcium carbonate solubillization and the change of color surrounding growth of organisms. Interpretation : : Formation of clear zone due to solubilization of calcium carbonate is a positive test indicat- ing the production of the enzyme lipase. CATALASE TEST Oxygen is both beneficial as well as toxic to living organisms, It is beneficial because it acts as terminal electron acceptor during aerobic respiration. However, oxygen is also a toxic substance, Toxicity of oxygen is basically due to formation of toxic derivatives of oxygen. Certain oxi- dative enzyme system interact with molecular oxygen to produce free superoxide radical (O,) 0, +e idan ony Oy seenteinettnncctecns (I) Superoxide radicals can inactivate vital cell components, Many organisms are protected from the toxic action of superoxide by their ability to produce the enzyme superoxide dismutase (SOD) whichy catalyzes the following reaction O;~ + O;~ % 2H> —suneroxide dismutase > ER BOs ace ay Hydrogen peroxide may further interact with superoxide to form Hydroxyl free radical O;- + H,0, ——> 0, + OH + OH wii.) Hydroxyl radicals ee highly reactive free radicals and can damage almost every kind of molecules found -in living cells. Hydrogen peroxide, on the other hand is a very powerful oxidizing agent highly toxic to cells, Aerobic and facultative organisms have developed protective mechanisms against the toxic forms of oxygen. One is the enzyme SOD which removes superoxide radicals by increasing the rate of reaction (2) leading to formation of hydrogen peroxide. H,0, so produced can be moved by catalase and peroxidase enzymes as follows: 2H,0, —— 2.0 + 0, .. @) H,0, + red. substrate Peroxidase > 24,0 + ox. substrate .......(5) Since superoxide and hydrogen peroxide are eliminated according to the reactions (2), @, (); the formation of hydroxy radical is inhibited, because both reactants are required for the reaction. Most anaerobes and microaerophiles do not have such protective mechanisms, so if they are exposed to oxygen, their growth is inhibited, hence they are found in oxygen free environ. ment only. Principle Catalase is an enzyme that splits up hydrogen peroxide into oxygen and water, Chemically catalase is a hemoprotein, similar in structure to hemoglobin, except that the four iron atoms 122in the molecule are in oxidized (Fe) rather than the reduced (Fe) state, Catalase is present, often in high concentrations in the majority of aerobic organisms, but is absent from most obligate anaerobes. Thus when H,0, is added externally in a medium, catalase activity results in the production of molecular gaseous oxygen. Catalase activity can be tested cither by slide test or tube test. Slide Test Requirements 1. Microscopic glass slide. 2. 3% Hydrogen peroxide solution. 3. Test culture. Procedure 1. Place one or two drops of hydrogen peroxide solution on a glass microscopic slide. 2. With a nicrome wire loop pick up cells from the center of a well isolated colony of the test culture, and transfer them into the drop of hydrogen peroxide. 3. Observe for the production of the gas bubbles or effervescence. Tube Test Requirements 1. Nutrient agar slant or nutrient broth. 2. 3% Hydrogen peroxide solution. 3. Test culture, Procedure \. Streak a loopful of the test culture on the nutrient agar slant or inoculate into the broth tube. 2. Incubate the medium at 37 °C for 24 hours. 3. After incubation, add 1 ml of hydrogen peroxide over the growth on agar slant or in broth. 4, Observe for the effervescence of oxygen. Interpretation Rapid appearance and sustained production of gas bubbles or effervescence constitute a positive test. Since some bacteria may possess enzymes other than catalase that decompose H,02, few tiny bubbles after 20-30 seconds is not considered as a positive test. * OXIDASE TEST When carbohydrates are oxidized via a respiratory mechanism under conditions in which oxygen is the final electron or hydrogen acceptor, energy is generated by passage of electrons through fa series of electron donors and acceptors. The path through which these electrons flow is called clectron transport chain (ETC). The components of ETC include flavoproteins, ubiquinone (coenzyme Q), and cytochromes, Electron transfer takes place in the following order: Substrate —H* + e- > NAD / NADP ——2=—> FP ——> CoQ.Cyt b —> Cyt ¢, —> Cyt ¢ —> Cyt(a + a;) ——> 0, cytochrome oxidase Cytochromes are of three types namely; cytochrome a, cytochrome b, and cytochrome c. Cytochromes act sequentially to transport electrons from ubiquinone to oxygen, thus forming last link in the respiratory chain. Cytochrome a and a, together are called cytochrome oxidase. This process of ATP generation during electron transport is known as oxidative phosphorylation. Principle ‘As a last link in the respiratory chain of oxidase positive bacteria, cytochrome takes up electrons and passes them to molecular oxygen which being terminal hydrogen acceptor, is 123ized r q me c to the oxi educed to hydrogen peroxide. The subsequent regeneration of cytochr’ form is catalyzed by the enzyme cytochrome oxidase. In oxidase test, p-phenylenediamine derivatives used as reagents are Onicha compounds by oxidized Cytochrome ¢ which in turn changes to reduced Cytol test proceeds only in air, since oxygen is necessary for production oxidized ¢y ae The oxidase test is useful procedure in the clinical Lee oe Oe: eee aad jc species of bacteria (such as Neisseria gonorrhoeae, Pseudo i ens ie ee pave, in contrast t0 species in the family Enterobacteriaceae, which are oxidase negative Requirements 1. Nutrient agar plate, 2. Filter paper, platinum wire loop. ; 3, Test culture, 4. 19% tetramethyl-p-phenylenediamine dihydrochloride solution. Procedure (Kovac’s method) * 1. Grow the test organism freely under aerobic conditions on nutrient agar medium for 18-24h 2. Take a filter paper strip and moisten it with 3-4 drops of tetramethyl-p-phenylenediamine dihydrochloride solution, \ 3. With the help of platinum wire pick up a colony and make a compact smear on moistened filter_paper. 4. Wait for 10-15 seconds and observe for formation of violet color. Interpretation |. Appearance of violet color on moist filter paper is an indication that the organism possess cytochrome oxidase. 2. The use of platinum wire loop for the test is important because the loop made from other materials lead to false positive reactions. 3. Organisms with less cytochrome oxidase ac DEHYDROGENASE TEST The biological oxidation of organic metabolites is the removal of electrons, In most cases it involves the removal of two electrons and thus simultaneous loss of two protons. This is equivalent to the cemoval of two hydrogen atoms and is called dehydrogenation. When a pair of electrons or hydrogen atoms from an oxidizable substrate is coupled with the reduction of an ultimate electron acceptor, such as oxygen, there is a generation of energy. Certain enzymes which removes electrons and hydrogen ions from reduced substrates are referred to as dehydrogenases. These enzymes have NAD* or NADP* as their coenzymes. The vitamin niacin (nicotinic acid) forms the part of the structure of NAD and NADP. Another class of dehydrogenases are known as flavoproteins and they contain either FAD or FMN as prosthetic group. One of the basic part of their coenzyme structure is the vitamin riboflavin. These enzymes are present in organisms which carry out aerobic respiration, Principle The test involves the detection of the dehydrogenase enzyme by using methylene blue as the compound which accepts the hydrogen released in the respiratory eleciron transport chain, and gets reduced (colorless form). Organic sub.ieq + Methylene blucjex -Setvdtogenase > Orpanic sub.) + Methylene bluciee blue coloriess dized to colored ome c. The ity can produce color change after long time. Requirements Nutrient broth. 2. Test culture. 3. Methylene blue solution (1% solution)Procedure 4 or al 1. Prepare a dense culture (~10° organistns/ml) of the test organism, overnight incubated culture. ternatively use the ove culfure in a sterile mutrient broth and mix it well 2. Inoculate 0..5-1.0 ml of the Saat 3 Add 1 ml of sterile methylene blue solution (1%) in nutrient Dro! 4, Incubate the nutrient broth at 37 °C for 24 hours. $ After incubation observe for the disappearance of blue color and record the results. Interpretation iepraarei of the blue color indicates the presence of enzyme dehydrogenas® and ue ie ie considered positive, The methylene blue remains blue in color at the top surface o' Berne to the oxidation of the methylene blue by air present at the surface. COAGULASE TEST Coagulase is an enzyme which adds to the invasiveness (the capacity 10 invade and maltiply e Trciayitismoas) of the pathogenic bacteria, It i @ thrombin like enzyme which 2289"! the formation of fibrin around the organisms and thus preventing phagocytosis. ee is produced by certain strains spp. of Staphylococcus, Yersenia and others. Organism, fim lee feo types of coagulase: free coagulase (soluble coagulase) is released in to the medium an a bound coagulase which is found on the external surface of the wall. Presence of coagulase can be detected by the following tests. “ eer Tw Test Principle 2 D Clotting requires interaction of free coagulase with coagulase reacting factors (CRF) in plasma, which is probably a derivative of prothrombin; a coagulase-CRF complex converts fibrinogen to fibrin, Although some fibrinopeptides are released as with thrombin, the pro- cess differs from normal clotting in that the multiple accessory factors including Ca™, are not required, and the clot is more friable and docs not retract. Citrate, oxalates and EDTA are usually added to act as anticoagulants and prevent false positive results. Procedure 1. Nutrient broth 2, Test culture. 3. Citrated or oxalated human or rabbit plasma (about 2 ml). Procedure fs |. Inoculate a loopful of test culture into the nutrient broth, Incubate at 37 °C for 18-24 hours. 2. Add about 0.5 ml of 18-24 hours broth culture to 2 ml of plasma in a small test tube. 3. Incubate the tube at 37 °C. 4. Examine for the clot formation at an hourly intervals up to 4 hours. Interpretation Clot or gel formation in a tube can be checked by tilting the tubes slightly to its sides. Clot formation is due to the production of free coagulase and it is considered as positive test. Slide Test Principle Bound coagulase act directly on fibrinogen ie, no auxiliary plasma factors appears to be necessary for coagulation. Fibrinogen is directly converted to fibrin in present \ce coagulase and plasma is coagulated, y ee Requirements 1, Test culture on the plate. 2. Distilled water. 3. Citrated or oxalated human or rabbit plasma (0.5 ml), # 49KProcedure i Pick up a well isolated colony from the plate and prepare @ 4 Place @ drop of thick bacterial suspension on a slide a 3. A loopful of citrated/oxalated plasma is added to a thick bacterial susp 4. Observe for clumping lense suspension. sion and stir it well of cells within 5-10 seconds Interpretation eee : by icrome: Coagulation is observed by formation of fibrin, which can be Biel oe Ee eeeueater appears as a fine, delicate strand. The test is considered positive indic bound coagulase HEMOLYSIN PRODUCTION TEST - Certain microorganisms produce biochemicals known as toxins, sear tions of the human cells and tissues. Hemolysins are such types ‘ oe certain pathogens which contribute to their virulence by causing the ly: eytes and releasing hemoglobin Etcineeatch Hemolysins are of various types and are protenic in nature, Bacteria See naa 6 hemolysin when cultivated on blood agar plate a clear zone of hemo! i pies cated RBCs) is observed. When such a zone is clear, colorless or pale yellow in color: © tt Cl f-hemolysis. Similarly when such zone has a partial clarity and is eee ie pel ans or ferred to as a-hemolysis. Alpha hemolysis is due to conversion of hemoglobin tf Wie! tet globin. The term y-hemolysis is sometimes used to describe the absence of e nonhemolytic colonies on blood agar. a Hemolytic activity is observed in several species of Streptococci, Staphylococci & Clostridium. Principle % Hemolysins produced by microorganisms diffuse in the blood agar and lyse RBCs, releasing hemoglobin from the cells, This results into the formation of clear zone surrounding the colony producing hemoglobin. Requirements 1, Blood agar plate. 2. Test culture, Procedure 1. Streak the test culture on blood agar plate and incubate the plate at 37 °C for 24-48 hours. 2. Observe the plate for the zone of hemolysis surrounding the colony. Interpretation The formation of clear colorless zone indicates the production of B-hemolysins while a zone f greenish discoloration is the indication of a-hemolysins. . © normal func at disrupt the norma! a ahs produced by f human erythro- TRIPLE SUGAR IRON (TSI) AGAR TEST All the biochemical tests described so far depend upon and demonstrate a particular meta- bolic reaction of the microorganism, However certain media may be deliberately formulated to demonstrate multiple reactions, Such media are of great help because they are economical; they also save time because more than one test can be performed in a single medium, Such media are of great importance in identification of an unknown organism. Principle ‘The principle and mechanism of reaction of TSI agar slant is des i i: eee ee 2% scribed in Appendix-l, along Requirements 1. TSI agar slant. 2. Test culture 126en 1 ‘ocedure Streak a loopful of test culture on slant; and stab the same culture into butt of the slant 2. Incubate the TSI slant at 37 °C for 24 hours. 3. After incubation observe the medium for presence of acid/gas/H,S in butt as well in the slant. Interpretation Interpret the results as described in Appendix-ll. LITMUS MILK TEST Pr Milk is a highly nutritious food for the growth of microorganisms. Milk contains carbohy- drates, proteins, fats and other growth factors required by microorganism. Among various different sugars and proteins, lactose, and casein are chief constituents of milk. Both, lactose and casein are readily attacked by microorganisms. Hence, microorganisms bring about sev- eral changes in milk. inciple Microorganisms present in milk can bring about one or more of the following changes in the litmus medium: = Certain bacteria containing the enzyme B-galactosidase, are capable of fermenting lactose to = In addition to being a pH indicator, litmus can also serve as an oxid: various types of organic acids (lactate, acetate, formate). These acids lower milk pH to ~ 4. At this pH the normal purple litmus turns pink and indicates acid production n-reduction indi- cator by acting as an electron acceptor. While in oxidized state, litmus is purple. If bacteria remove O; from the medium, the litmus is reduced by accepting electrons from the medium and turns white. This reduction is indicated by a white zone at the bottom of the tube and proceeds upward. The surface rim usually remains purple due to oxidation of litmus by atmospheric O; © Many bacteria possess an enzyme rennin, that causes casein to coagulate and form a rennet curd (clot) along with acid formation. = The end products of lactose fermentation may include gas formation (CO; and / or H,). The presence of these gases may be seen as separation of the curd, the presence of bubbles in the curd, or the development of tracts or fissures in the curd. Some bacteria, such as Clostridia, produce so much gas that the curd is torn to shreds. This is known as stormy fermentation. = Often bacteria that form a rennet curd are also proteolytic and possess enzyme that catalyze the digestion (proteolysis) of curd . When this occurs, the rennet curd is digested to soluble products (amino acids) that appear as a brownish, straw-colored fluid = An alkaline reaction is indicated by cither no change in the purple color of the litmus milk or by a change in the color from purple to deep blue. An alkaline reaction occurs from the decarboxylation or deamination of the casein amino acids with simultaneous release of alkaline end products that are responsible for the color change. Requirements . Procedure 1. Litmus milk medium, 2. Test culture. ( Wee ae 1, Inoculate litmus milk medium with the test culture. 2. Incubate the medium at 37 °C For 24-72 hours. 3. Observe for various different changes appearing in the medium. Interpretation Microorganisms may bring about one or more different types of changes in the milk as described in the principle; and interpret the results on the basis of the changes observed. 127