Overview

You will certainly be using a wide variety of media and also biochemical tests to isolate and identify your unknown organisms. Your text has actually a great section top top enrichment, selective, and differential media. Please review it before getting started.

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You are just working through a small subset of the accessible media and also tests. The is enough yet to give you a an excellent idea the the trouble solving that goes on behind the scene to support patient care.

The information provided on this pages was derived from the DIFCO hand-operated of media, i beg your pardon is also obtainable in the lab.

Read the directions carefully.

You should pay close fist to the problems of the tests and media to stop erroneous results.

Things come watch for include:

type of media you pull her sample native (enrichment or selective).mixed or pure cultures for inoculation.type the inoculation - one or an ext colonies, needle prick from facility or totality colony, light or hefty innoculation.application - execute you stab, streak or smear the bowl or tube.length, temperature, and atmospheric problems for incubation; some tests operation for much longer times if you obtain a an unfavorable result in 24 hours, others have to run for 4-5 days, others should be check out at 24 hours because the colors adjust with storage.

Bacitracin/SXT Sensitivity

Intended Use

Bacitracin differential disks are used to presumptively identify group A, beta-hemolytic streptococci from various other beta-hemolytic streptococci. The mix of SXT sensitivity increases the accuracy that the results.

Principle

Bacitracin is an antibiotic isolated from Bacillus subtilis. It inhibits cell wall surface synthesis mainly through inhibiting the biosynthesis the peptidoglycan. SXT inhibits folate metabolism which interferes through bacterial DNA synthesis. Group A, beta-hemolytic streptococci are more sensitive to bacitracin than other beta-hemolytic streptococci.

Test Procedure

The typical protocol has been modified for our lab.

Using a loop, pick 3-4 fine isolated colonies, ideally from an 18-24 hour culture. Transfer to a tiny amount that sterile water.Adjust the turbidity come 0.5 McFarland standard.Use the procedure outlined in antimicrobial susceptibility trial and error to swab the entire plate to acquire confluent growth.Visually division the key in thirds, ar a bacitracin and SXT in their section of the plate. Making use of sterile forceps or a swab, lightly yet firmly press the disks come the agar surface ar to adhere them.Save the other section for the optochin disk.Invert the plates and incubate them for 18-24 hrs at 35°C in 5-10% CO2.Incubate one more 24 hours if the results are negative.ResultsAny zone that inhibition around the decaying is taken into consideration sensitive (S).No zone the inhibition with development up come the decaying is taken into consideration resistance (R).

This table is native MacFaddin, Biochemical Tests because that Identification of medical Bacteria.


BacitracinSXTPresumptive ID
SRGroup A b-streptococci
RRGroup B b-streptococci
RSNot team A or B b-streptococci
SSRule out team A or B v serologic tests

LimitationsOnly beta-hemolytic streptococci have to be tested.While this test is exact it is not very specific. Other biochemical or serological test are forced for specific identification.The development should it is in confluent. Too light the a expansion could cause some non-group A streptococci to show up susceptible come bacitracin.

Catalase

Intended UseDifferentiates Streptococcus (-) native Micrococcus (+)Differentiates Staphylococcus (V+) and also Bacillus (+) indigenous Clostridium (-)Principle

Hydrogen peroxide (H2O2) is the finish product of aerobic breakdown of sugars. Due to the fact that it is toxicity to bacter cells, most aerobic bacteria create catalase or peroxidase to safeguard themselves. Streptococcus, Enterococcus, and Lactobacillis are exceptions. Because they execute not use the cytochrome c pathway, they do not develop H2O2 and lack catalase.

Test ProcedureTransfer a well isolated nest to a clean glass slide and include 1 fall of 3% H2O2. Do not reverse the order and also do not mix.Observe for immediate bubble formation.Use 15% H2O2 for the detection of catalase in anaerobes.ResultsThe development of bubbles is taken into consideration a optimistic result.LimitationsDo not take your nest from a blood agar plate. The catalase current in the erythrocytes will provide a false positive result.H2O2 is unstable. You can do a quality manage test that the H2O2 reagent by place a autumn on a blood agar plate. Vigorous bubbling must result.

Coagulase Test

Intended Use

Differentiates Staphylococcus aureus from various other Staphylococcus species.

Principle

The coagulase test detects the visibility of complimentary and tied staphylcoagulase. This enzyme is excreted extracellularly by human being strains of Staph. Aureus. The mechanism of action is unknown.

Test ProcedureThaw a tube of 0.5 mL rabbit plasma.Inoculate a loop-full that organism right into the tube. Determined a well isolated colony.Ideally you must incubate the tube at 35°C for 4 hrs checking every 30 minute for gerean formation. Us incubate lock overnight and also put castle in the refrigerator until the following lab duration with equivalent results.Check for gerean formation.Dispose that the pipe in the biohazard container.Results

The formation of a clot in the bottom that the tube is considered a hopeful result. The clot will not relocate as you tilt the tube. Unclotted plasma will circulation in the tube.

LimitationsMethicillin resistant Staph. Aureus have lessened clumping factor.Do not shake or agitate the tube together this could break up the clot.Some staphylococci strains develop fibrolysin after an extensive incubation at 35°C that deserve to break increase the clot causing false negative. Incubate the pipe overnight at room temperature if you carry out not gain a clot in 4 hours.Some various other rarely encountered staph types are likewise coagulase positive by the tube method.
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Eosin Methylene Blue (EMB) Agar

Principle

A differential plating tool for the detection & isolation of the gram-negative enteric bacteria.

PurposeTo assist in the differentiation of lactose fermenting bacteria native non-lactose fermenting bacteria.To assist in the differentiation the Enterobacter aerogenes and Escherichia coli.Test ProcedureInoculate the organism straight onto the surface of one EMB agar plate and streak because that isolation.Incubate inoculated key aerobically at 37°C.Examine for expansion after 18-24 hours of incubation.InterpretationsColiforms that use the lactose and/or sucrose are blue/black through a greenish metallic sheen. Indicative of Escherichia coli.Coliforms such together Klebsiella pneumoniae have mucoid swarms that might be purple and/or exhibit a environment-friendly metallic sheen.Good come excellent, colorless swarms indicative that Proteus vulgaris, Salmonella choleraesuiss, and Shigella spp.

Fluid Thioglycolate tool (FTM)

Tests the oxygen demands of various microorganisms.

Intended Use

Characterizes microbes according to their oxygen requirements

Obligate aerobesFacultative anaerobesObligate anaerobesPrinciple

Various types of bacteria require miscellaneous oxygen (or oxygen-free) settings to thrive in.

Test ProcedureDilute her organism in a tube of sterile water to acquire a turbidity indistinguishable to the 0.5 McFarland check standard. Organize your diluted tube and also the 0.5 McFarland check standard against the black-lined McFarland referral card to accurately rate the turbidity.Using a sterile 1mL pipette, ar 1 mL the organism right into the middle of the tube.Cap tightly; perform not jostle.Incubate for 24 hrs at 37°C.ResultsStrict (obligate) aerobes grow at the surface of the medium where over there is a high concentration that oxygen.Obligate anaerobes thrive near the bottom of the broth tube whereby there is no oxygen.Facultative anaerobes grow finest where more oxygen is present, however growth will take place throughout the broth tube.
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Hemolysis - Blood Agar

Intended Use

Blood agar is supplied to support the growth of fastidious organisms and to determine the kind of hemolysis (destruction the red blood cell walls) an organism produces.

Principle

Blood agar is a rich medium that has been supplemented v fresh 5-10% blood. The hemolytic solution can be dependent upon the form of blood. Lamb blood is frequently used, yet some organisms require rabbit or bovine blood.

Test ProcedureStreak a plate of blood agar because that isolation.Optional: perform your critical streak through a needle and also poke into the agar. This usually offers clear, reputable zones that beta hemolysis and is especially important to see the effects of streptolysin O i beg your pardon is oxygen labile. See page 84 of the Difco/BBL Manual.Incubate the plates in ~ 37°C because that 24-48 hours. Strep organisms have to be incubated in the CO2 incubator.The plate will certainly be a brownish red shade after 48hours.Results

You can distinguish four types of hemolysis through the figure of the agar.

Beta hemolysis is suggested by a clean colorless zone bordering the colonies. There has been full lysis of the red blood cells.Alpha hemolysis is suggested by a small zone that greenish come brownish discoloration of the media. This is caused by the reduction of hemoglobin come methemoglobin and its succeeding diffusion right into the neighboring medium.Alpha prime hemolysis is shown by a ar of finish hemolysis, surrounding by a zone of partial hemolysis, a pink halo. This pattern have the right to be simpler to see if girlfriend scrape off the colony.Gamma hemolysis is suggested by no readjust in the media.LimitationsThe patterns of hemolysis have the right to vary with the incubation atmosphere and also the kind of blood in the media.Some Staph organisms will certainly only show hemolysis ~ they have been refrigerated complying with incubation.
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MacConkey (Mac) Agar

PrincipleA differential plating medium recommended for use in the isolation and also differentiation the lactose-fermenting organisms from lactose non-fermenting gram negative enteric bacteria.Selectivity of the tool is because of the presence of crystal violet and bile salts which markedly to completely inhibit the expansion of gram optimistic organisms.PurposeOrganisms capable of fermenting lactose develop a localized pH autumn which, followed by the absorption of neutral red, imparts a red/pink/purple color to the colony. A region of precipitated bile may likewise be present due to this localized drop in pH.Organisms that carry out not ferment lactose continue to be colorless and translucent. They are quickly detected bytransfer light and appear together colorless colonies against a red background.Test ProcedureInoculate the organism straight onto the surface ar of a MacConkey agar plate and streak because that isolation.Incubate inoculated plate aerobically in ~ 35-37°C.Examine for growth after 18-24 hours of incubation.InterpretationGood to terrific growth, red/pink/purple swarms with bile precipitate indicative of Escherichia coli.Good to great growth, red/pink/purple nests without bile precipitate indicative of Enterobacter aerogenes.Good come excellent, colorless colonies without bile precipitate indicative the Proteus vulgaris, Salmonella typhimurium, and Shigella spp.

Motility check Medium

Intended UseUsed for detecting motility of microorganisms.Principle

Motility is evident by the existence of diffuse development away indigenous the line of inoculation.

Test ProcedureInoculate with expansion from an 18-24 hour culture by stab inoculation with a needle.Incubate in ~ a temperature and also duration suitable for the organism gift tested.Examine tubes because that growth and signs that motility.ResultsMotility is obvious by the existence of diffuse development away from the heat of inoculation.Non-motile organisms only grow along the heat of inoculation.LimitationsThe motility that Proteus spp. Is temperature dependent.Due to the temperature suspended of motility in part organisms, a an adverse tube have to be incubated secondary 5 days at a lower temperature that 22-25°C.

Mannitol Salt Agar (MSA)

Principle

Selective and also differential media supplied for the isolation of Staphylococcus aureus.

Intended Use

Differentiates Staphylococcus aureus (+) from various other Staphylococcus species.

PrincipleContains 7.5 % NaCl (salt) which inhibits the expansion of many microorganisms, other than Staphylococcus species.Contains phenol red indicator i beg your pardon is red/pink under neutral/basic conditions, however turns yellow under acidic conditions.When mannitol is fermented there is a decrease in pH, turning the red/pink media yellow.Test ProcedureInoculate the organism directly onto the surface ar of one MSA plate and also streak for isolation.Incubate inoculated key aerobically in ~ 37°C.Examine for expansion after 18-24 hrs of incubation.Results

The yellowing of the red/pink media indicates a optimistic result.

Novobiocin

Intended Use

Novobiocin Differentiation Disks are useful for presumptively differentiating Staphylococcus saprophyticus from various other coagulase-negative staphylococci (CoNS) in clinical specimens.

Principle

Novobiocin Differentiation Disks are ready by impregnating 5µg of novobiocin ~ above high top quality 6mm diameter filter paper disks. Novobiocin Differentiation Disks are helpful in presumptively differentiating S. Saprophyticus from other CoNS. Novobiocin inhibits the synthesis of DNA and also RNA. A zone that inhibition is developed by contact with the novobiocin.

ProcedureAllow disks to equilibrate to room temperature.Using a pure 18-24 hour culture, prepare a suspension, identical to a McFarland 0.5 opacity standard, in Tryptic soybean beans Broth, Sterile Water, or mind Heart Infusion (BHI) Broth.Inoculate Mueller Hinton Agar, Blood Agar, or Tryptic soy Agar (TSA) plate through a sterile swab to obtain confluent growth.Aseptically use one novobiocin disk top top the inoculated agar surface and lightly push down to ensure full contact with the medium.Incubate aerobically for 18-24 hours at 35-37 degrees C.Measure (in millimeters) the diameter that the zone of inhibition roughly the novobiocin disk, and also record as vulnerable or resistant.Interpretation of ResultsSensitive - A zone of inhibition higher than 16mmResistant - A zone the inhibition much less than or equal to 16mmLimitationsIt is recommended that biochemical and/or serological tests be performed on colonies from pure society for complete identification.The novobiocin disc is no helpful and also can give misleading results if it is perform on isolates various other that those native urinary specimens.Occasional person isolates that space not S. Saprophyticus, S. Cohnii subsp., or S. Xylosis may additionally be resistant to novobiocin.

Oxidase Biochemical Assay

Tests because that the presence of the enzyme indophenol oxidase.

Intended Use

The oxidase check is based upon the production of an enzyme called indophenols oxidase. This enzyme oxidizes a redox dye (present in the reagent) which outcomes in a color adjust of yellow to dark purple.

Principle

Indophenol oxidase, in the existence of atmospheric oxygen, oxidizes the phenylenediamine oxidase reagent to kind a dark violet compound, indophenol.

Test ProcedureHave her instructor or IA crush the ampule inside the dropper.Tap bottom on tabletop a few times. Climate invert for practically drop-by-drop dispensing the reagentPreparation for testing:Colonies to be tested should be secluded from various other coloniesThe usage of new isolates (18-24 hr cultures) is encourage for routine testing.If refrigerated, cultures must be allowed to reach room temperature prior to testingPerforming the test – Filter document MethodAdd a couple of drops of oxidase check reagent to a piece of filter paper (Whatman No. 1 or equivalent).Streak a loopful that bacteria ~ above the reagent-saturated document with a platinum loop or wooden applicator stick. Usage of stole of nichrome loops may reason false-positive reactionsResults

Positive reactions revolve the bacteria violet to violet immediately, or as much as 30 seconds. An unfavorable reactions remain colorless or rotate light pink/light purple after 30 seconds. Delay reactions need to be ignored.

LimitationsAllow as much as 30 secs for a confident reaction.Any delayed reactions should be taken into consideration negative.Do not add excess reagent, at it may cause the reaction to fade on oxides-positive organisms.Steel loop, nichrome loop, and also wire loop containing stole may offer a false-positive reaction. A platinum loop or wood applicator rod is recommended.

Salt yongin Broth

Intended Use

Salt tolerance broth is to plan to differentiate non-beta-hemolytic strains the streptococci.

Principle that Use

Brain love Infusion (BHI) broth is supplemented v 6.5% sodium chloride and bromcresol purple as a pH indicator. The indicator is had to make analysis the test outcomes easier. The broth additionally includes dextrose. The fermentation that dextrose (glucose) results in the manufacturing of acid. This changes the pH of the media resulting in the media to rotate from violet to yellow.

Test ProcedureSelect no an ext than 2-3 colonies (preferably from an overnight culture) come inoculate a pipe of salt tolerance broth.It is vital to lightly inoculate the tube otherwise you may obtain a false positive.Loosen the cap and incubate aerobically because that 24 hours at 37°C.Continue incubation approximately 72 hours if you acquire a negative result at 24 hours.Results

A positive reaction is indicated by obvious turbidity in the media with or there is no a shade change. A an adverse result is suggested by no expansion after 72 hours. Enterococcus spp. Typically changes the media shade within 24 hours.

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LimitationsMany staphylococci can flourish in media include 10% salt. Mannitol salt agar has 7.5% salt.Salt yongin media was intended to differentiate catalase an adverse gram-positive cocci. Be certain to do a catalase test prior to you continue with the salt yongin broth test.Other species of catalase an unfavorable gram-positive organisms can prosper in this media.