biochemical tests

Purpose: Purple Broth and Phenol Red Broth are used to detect bacterial ability to ferment carbohydrates. fermentation characteristics are useful in differentiating Gram-negative bacilli, especially members of Enterobacteriaceae.

• Principle:the principle behind purple broth and phenol red broth is the same. each medium consists of a basal recipe to which a single fermentable carbohydrate is added. both media include peptone, a pH indicator, and a specific carbohydrate. purple broth includes bromcresol purple as the pH indicator, which is yellow below pH 6.8 and purple above; pr broth includes phenol red, which is yellow below pH 6.8, pink above pH 7.4 and red in between.
• Acid production from fermentation of the carbohydrate lowers the pH below the neutral range of the indicator and turns the medium yellow. deamination of the peptone amino acids produces ammonia which raises the pH and turns PR broth pink or, in the case of Purple broth, produces no color change. during preparation of the media an inverted durham tube is placed inside the broth to trap gas that may be produced during fermentation. a positive indication of gas production is a bubble or pocket in the durham tube where the broth has been displaced.
• The ability of these media to detect acid production is largely dependent upon incubation time. if the medium is examined within 18 hours of inoculation, pink color(pr broth) or purple color(purple broth) indicate that the organism has not fermented the substrate and has likely deaminated the peptone amino acids. readings after 18 hours, however are unreliable because of the possibility of a reversion. a reversion is what happens in the medium when an organism switches metabolic modes from fermentation to deamination after the exhaustion of carbohydrate.
• testing for: ability to ferment sugar:by looking for acid production (A) &/or gas production (G).
• If pH is high, there was deamination, or ammonia production. This means the test is negative for fermentation, and the broth will be pink or have no color change
• if pH is low, there was acid production from fermentation of the carbohydrate and this will turn the medium yellow.

• Purpose: test most commonly used for presumptive identification of enterococci and members of the Streptococcus bovis group, all of which are positive.
• Ability to hydrolize esculin in the presence of bile
• Principle:Bile Esculin Azide Agar is both a selective and differential medium. it contains esculin, peptone, bile, sodium aside, and ferric citrate. bile inhibits growth of Gram-pos organisms (except Group D streptococci and enterococci), and sodium azide inhibits growth of Gram-neg organisms.
• esculin can be easily hydrolyzed under acidic conditions, but few bacteria outside of group D strep and enterococci are able to do so in the presence of bile.
• group D strep and enterococci grow well on the medium and usually darken it in 18-24 hours. therefore an organism that has not darkened more than half the medium after 48 hours is considered negative. a slant that is more than half darkened at any time within 48 hours can be recorded as a completed positive.

purpose: litmus milk is used primarily to differentiate members within the genus clostridium. it differentiates enterobacteriaceae from other Gram-neg bacilli based on enterics' ability to reduce litmus. Litmus milk is also used to cultivate and maintain cultures of lactic acid bacteria.

• principle: litmus milk is undefined medium consisting of skim milk and the pH indicator azolitmin. Skim milk provides nutrients for growth, lactose for fermentation, and protein in the form of casein. azolitmin is pink at pH 4.5 and blue at pH 8.3. between these extremes it is purple.
• there are four basic reactions that occur in litmus milk---- lactose fermentation, reduction of litmus, casein coagulation, and casein hydrolysis

• result*/* interpretation*/* symbol
• pink color*/*acid reaction*/*A
• pink and solid(white in the lower portion if the litmus is reduced)clot not movable; */* acid clot*/* AC
• fissures in clot*/* gas*/* G
• Clot broken apart*/* stormy fermentation*/* S
• White color (lower portion of medium)*/* Reduction of litmus*/* R
• semisolid and not pink; clear to gray fluid at top*/* curd*/* C
• classification of medium; loss of body digestion of peptone*/*; peptonization*/*P
• blue medium or blue band @ top*/* alkaline reaction */* K
• no change*/* none of the above reactions*/* NC

• purpose: used to detect the ability of an organism to reduce nitrate to nitrite, nitrogen gas, or other nitrogenous compound.
• principle:nitrate broth is an undefined medium of beef extract, peptone, and potassium nitrate. an inverted durham tube is placed in each broth to trap a portion of any gas produced. no color indicators are added. as the name implies, nitrate reduction is one form of anaerobic respiration. most nitrate-reducing bacteria contain the enzyme nitrate reductase and perform a single step reduction of nitrate converting it to nitrite. before a broth can be tested for nitrate reductase activity, it must be examined for evidence of denitrification, bacteria that contain other enzymes capable of reducin nitrate to molecular nitrogen other than nitrate reductase. to do this, it's simply a matter of examining for evidence of the presence of gas in the durham tube. if there is gas in the tube, and the organism is known not to be a fermenter, the test is complete and denitrification has taken place.
• if there is no gas in the tube then add sulfanilic acid and α-naphthylamine are added to the medium to test for nitrate reduction to nitrite. nitrite, if present, will produce a red, water-soluble compound. this will indicate that the organism reduced nitrate to nitrite. if no color change takes place, the nitrate either was not reduced or was reduced to one of the other nitrogenous compounds. so another test must be performed.
• a small amt of powdered zinc is added to the broth to catalyze the reduction of any nitrate present to nitrite. if nitrate is present, the medium will turn red which will indicate that nitrate was NOT reduced by the organism. no color change after zinc, indicates that the organism reduced nitrate.

• purpose: used to detect bacterial motility.
• principle: motility test medium is semisolid medium designed to detect bacterial motility. its agar concentration is reduced from typical 1.5% to 0.4%-just enough to maintain its form while allowing movement of motile bacteria. it is inoculated by stabbing with a straight transfer needle. motility is detectable as diffuse growth radiating from the central stab line.

• purpose: to differentiate members of Enterobacteriaceae, especially from sulfur-reducers such as salmonella, francissella, and proteus from non sulfur reducers such as morganella morganii and providencia rettgeri
• principle: performed using SIM medium that is formulated with casein and animal tissue as source of amino acids, an iron-containing compound, and sulfur in the form of sodium thiosulfate.
• sulfur reduction to H₂S is an anaerobic activity and can be accomplished by bacteria in two different ways, depending on the enzymes present. Cysteine desulfurase catalyzed the putrefaction of the amino acid cysteine to pyruvate. Thiosulfate reductase catalyzes the reduction of sulfur at the end of the anaerobic respiratory ETC. both systems produce hydrogen sulfide gas. when either reaction occurs in SIM media, the gas produced combines with iron to form ferric sulfide, a black precip. any blackening of the medium is an indication of sulfur reduction and a positive test. no blackening of the medium indicates no sulfur reductions and a negative reaction.

• purpose: identifies bacteria capale of producin indole using the enzyme tryptophanase, thus differentiating the Enterobacteriaceae.
• principle: is performed using SIM medium. hyrolysis of tryptophon in SIM medium can be detected by the addition of KOVACS' reagent. when a few drops of Kovacs' are added to the tube, quinoidal compound that turns the reagent layer a cherry red color is produced. the formation of red color in the reagent indicates a positive reaction and the presence of tryptophanase. no red color is indole-negative.

• purpose: used to differentiate the enterobacteriaceae by identifying it's ability to produce stable acid end products by means of a mixed-acid fermentation of glucose.
• principle: MR-VP broth is a simple solution containing only peptone, glucose, and a phosphate buffer. the peptone and glucose provide protein and a fermentable carbohydrate while the potassium phosphate is included to resist pH changes in the medium.
• designed to detect organisms capable of overcoming the buffer and lowering the pH. a few enterics can do this by mixed acid fermentation. acids produced by these organisms tend to be stable whereas acids produced by other organisms tend to be quickly converted to more neutral products.
• after incubation, some broth is removed and Methyl Red is added. Meth red is red at pH 4.4 and yellow at pH 6.2. between those two values it is various shades of orange. red is the only true indication of positive result; orange is neg or inconclusive; yellow is negative.

• purpose: identifies organisms able to produce acetoin from the degradation of glucose during a 2,3-butanediol fermentation.
• principle: designed for organism that are able to ferment glucose but quickly convert their acid products to acetoin and 2,3-butanediol. addition of VP reagents to the medium oxidized the acetoin to diacetyl, which in turn reacts with guanidine nuclei from peptone to produce a red color. a positive VP result is red. no color change is negative. copper color is result of interactions between the reagents and should not be confused with the true red color of a positive result.

• purpose: used to determine the ability of an organism to use citrate as its sole carbon source.
• principle: in many bacteria, citrate is produced as acetyl coenzyme A reacts with oxaloacetate at the entry to the Krebs cycle. citrate is then converted through a complex series of reactions back to oxaloacetate, which begins the cycle anew.
• in a medium where citrate is the only available carbon source, bacteria that possess citrate-permease can transport the molecules into the cell and produce pyruvate by a reversal of the above-described reaction. pyruvate can then be fermented to a veariety of products depending on the pH of the environment.
• simmon's citrate agar is a defined medium that contains sodium citrate as the sole carbon source and ammonium phosphate as the sole nitrogen source. bromthymol blue dye, which is green at pH 6.9 and blue at pH 7.6, is added as an indicator. bacteria that survive in the medium and utilize the citrate also convert the ammonium phosphate to ammonia and ammonium hydroxide both of which tend to alkalinize the agar. as the pH goes up, the medium changes from green to blue. thus, the conversion to blue is a positive citrate test result.

• purpose: used to determine the ability of a microbe to produce gelatinases.
• gelatinase-positive negative
• staphylococcus aureus staphylococcus epidermidis
• serratia most others of enterobacteriaceae
• proteus
• bacillus anthracis, cereus and several other members

• principle: gelatin is a protein derived from collagen. gelatinases comprise a family of extracellular enzymes produced and secredted by some microorganisms to hydrolyze gelatin. the individual amino acids can be taken up the the cell and used for metabolic purposes.
• nutrient gelatin is a simple test medium composed of gelatin, peptone and beef extract. nutrient gelatin differs from most other solid media in that the solidifying agent is also the substrate for enzymatic activity. when a tube of nutrient gelatin is stab inoculated with a gelatinase-positive organism, secreted gelatinase will liquify the gelatin. gelatinase-negative organisms do not secrete the enzyme and do not liquify the medium.
• a small disadvantage of nutrient gelatin is that it melts at 28^oC
• slostridium tetani
• c.perfringens

• purpose: used to diff organisms based on their ability to hydrolyze urea with the enzyme urease.
• many enteric bacteria and a few others possess the ability to metabolize urea, but only members of Proteus, Morganella, and providencia are considered rapid urease-positive organisms.
• principle: ureas is a product of decarboxylation of certain amino acids. it can be hydrolyzed to ammonia and carbon dioxide by bacteria contining the enzyme urease.
• urea agar was formulated to differentiate rapid urease-positive organisms from slower urease-positive and urease-negative bacteria. it contains urea, peptone, potassium phosphate, glucose, and phenol red. peptone and glucose proved nutrients for a broad range of bacteria. potassium phosphate is a mile buffer used to resist alkalinization of the medium from peptone metabolism. phinol red, whei is yellow or orange below pH 8.4 and red or pink above, is included as an indicator.
• urea hydrolysis to ammonia by urease-positive organisms will overcome the buffer in the medium and change it from orange to pink. rapid urease-positive organisms will turn the entire slant pink within 24 hours. weak positives may take several days. urease negative organisms either produce no color change in the medium or turn it yellow from acid products.
• urea broth differs from urea agar in two important ways. first its only nutrient source is a trace of yeast extract. second, it contains buffers strong enough to inhibit alkalinization of the medium by all but the rapid urease-positive organisms.
• pink color in the medium in less than 24 hours indicates a rapid rease-positive organism. yellow or orange are negative.

• purpose: used to differentiate bacteria based on their ability to hydrolyze starch with the enzyme α-amylase or oligo-1,6-lucosidase.
• principle:starch is too large to pass through the bacterial cell membrane. therefore, to be of metabolic value to the bacteria, it must first be split into smaller fragments or individual glucose molecules. organisms that produce and secrete the extracellular enzymes α-amylase and oligo-1,6-glucosidase are able to hydrolyze starch by breakding the glycosidic linkages between the sugar subunits.
• starch agar is a simple plated medium of beef extract, soluble starch and agar. when organisms that produce α-amylase and oligo-1,6-glucosidase are grown on starch agar they hydrolyze the starch in the medium surrounding the bacterial growth.
• the reagent iodine is used to detect the presence or absence of starch in the vicinity around the bacterial growth. iodine reacts with starch and produces a blue or dark brown dolor; therefore, any microbial starch hydrolysis will be revealed as a clear zone surrounding the growth

• purpose: detect cell morphology, size and arrangement
• principle: crystal violet, safranan, meth blue-all cells will the color of the primary stain used. no diff will be shown for g+ or g- cells. basic stains are applied to bacterial smears that have been heat fixed. heat fixing kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible.

• purpose: gram stain used to distinguish between g+ and g- cells and the the most important and widely used differential stain. also allows determination of cell morphology, size and arrangement.
• principle:primary stain - crystal violet(1 minute)
• mordant - iodine increases binding of primary stain(1 minute)
• decolorizing agent - acetone/alcohol - most critical step - g- cells decolorized by the solution whereas g+ cells are not. (30 seconds)
• counter stain/secondary stain - safranin - stains g- cells (1 minute)
• g+ cells stain purple while g- cells appear reddish-pink
• dead cells are pink while endospores are hollow
• g- walls have higher lipid content(due to outer membrane) and a thinner peptidoglycan layer than g+ walls. the alcohol/acetone in the decolorizer extracts the lipid, making the g- wall more porous and incapable of retaining the crystal violet-iodine complex.

differential stain to detect presence and location of spores in bacterial cells.

• schaeffer-fulton method
• primary stain - malachite green, cells and spores are green. heat or steam is used to force the stain into present spores. (steam 10-15 minutes)
• decolorizing agent - water will remove stain from cells, but not spores (about 30 seconds)
• counter stain - safranin is used to counterstain the cells red (1-2 minutes)

• differential stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol.
• the presence of mycolic acids in the cell walls of acif fast organisms is the cytological basis for this stain. mycolic acid is waxy substance that gives acif fast cells a higher affinity for the primary stand and resistance to decolorization.
• variety of acid fast staining procedures are employed, two of which are ziehl-neelsen and kinyoun method. they differ primarily in that the zn method uses heat as part of the staining process, whereas the k method is a "cold" stain.
• zn method
• primary stain - phenol carbolfuchsin
• steam heat to drive stain into the cell
• decolorizing agent - acid alcohol
• counterstain - meth blue
• acid fast cells are reddish-purple; nonacid-fast cells are blue

• kinyoun method
• primary stain - concentrated barbolfuchsin
• no heat
• decolorization - acid alcohol
• counter stain - meth blue or brilliant green

• used to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat fixing. also, in situations where accurate size determination is crucial, a negative stain can be used since it produces minimal cell shrinkage
• nigrosin or congo red are primary stains
• no mordant, decolorizing agents, or counterstains. this is a simple stain

• differential stain used to detect cells capable of producing an extracellular capsule.
• primary stain - acidic stain such as congo red or nigrosin or crystal violet to color the background, copper stain is decolorizing agent that decolorizes the capsules but not the cell "background"
• cell and background stain purple; capsule is clear