CARA PEMBUATAN CARA PEMBUATAN MEDIA PADA MEDIA PADA

MICROBIOLOGY MICROBIOLOGY

Selvianti P DjarudjuSelvianti P Djarudju

isolation

inoculation

incubation

Specimen collection

inspection

identification

5 “I” s

MediaMedia A microbiologist can A microbiologist can fine tunefine tune a media for a media for

almost any purpose.almost any purpose. General purpose mediaGeneral purpose media are designed to are designed to

grow a board spectrum of grow a board spectrum of microorganismsmicroorganisms e.g. nutrient agar/broth, e.g. nutrient agar/broth, brain and heart infusion, trypticase soy agar brain and heart infusion, trypticase soy agar (TSA)-casein, soy digest and NaCl.(TSA)-casein, soy digest and NaCl.

Enriched mediaEnriched media has has complex organic complex organic substratessubstrates such as blood serum and special such as blood serum and special growth factors (vitamins, amino acids).growth factors (vitamins, amino acids).

A bacteria that requires requires complex A bacteria that requires requires complex growth factors is termed growth factors is termed fastidiousfastidious..

Enrichment mediaEnrichment media facilitates the facilitates the extensive extensive growth of particular organismgrowth of particular organism..

Base Media Base Media

Breeding base medium simple Breeding base medium simple breeding that contains general breeding that contains general substances that need by large part substances that need by large part microorganism and put also as microorganism and put also as fundamental component to make fundamental component to make medium breeding other. medium breeding other.

Enrichment MediaEnrichment Media The addition of The addition of blood, serum extract or blood, serum extract or

tryptic soy agartryptic soy agar will support the growth of will support the growth of many fastidious microorganisms.many fastidious microorganisms.

Blood agarBlood agar has the addition of citrated blood has the addition of citrated blood to tryptic soy agar to make possible variable to tryptic soy agar to make possible variable hemolysis which permit the differentiation of hemolysis which permit the differentiation of some bacteria.some bacteria.

α α hemolysis: greenish brown halo around the colony hemolysis: greenish brown halo around the colony ( e.g. ( e.g. Streptococcus gordonii Streptococcus gordonii or or S. pneumoniaeS. pneumoniae))

ββ hemolysis: complete lysis of blood cells resulting in hemolysis: complete lysis of blood cells resulting in a clearing effect around the colony (e.g. a clearing effect around the colony (e.g. Staphylococcus aureus, Streptococcus pyogenes)Staphylococcus aureus, Streptococcus pyogenes)

Non hemolytic: no change in media (Non hemolytic: no change in media (Staphylococcus Staphylococcus epidermidis, Staphylococcusepidermidis, Staphylococcus saprophyticussaprophyticus

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Enriched MediaEnriched Media

Blood agar plate with bacteria from human throat. This media differentiates among different colonies by appearance

Chocolate agar, a medium that gets brown from heated blood. Used for isolation of N. gonorrhoeae.

Selective/Differential MediaSelective/Differential Media A selective mediaA selective media contains agents that contains agents that

inhibit inhibit the growth of certain the growth of certain microorganisms and microorganisms and selectselect for the growth of for the growth of others.others.

A selective media is important as a A selective media is important as a primary primary isolationisolation of specific organisms. of specific organisms.

E.g E.g mannitol salt agarmannitol salt agar (MSA) has a high (MSA) has a high NaCl concentration (7.5%) which will inhibit NaCl concentration (7.5%) which will inhibit the growth of most microbes but will select the growth of most microbes but will select for the growth of for the growth of StaphylococcusStaphylococcus..

MacConkey agarMacConkey agar which contains which contains bile saltsbile salts as a selective agent.as a selective agent.

Selective/Differential MediaSelective/Differential Media Bile saltsBile salts is a component of feces and is a component of feces and

inhibit the growth of inhibit the growth of Gram positive Gram positive bacteriabacteria and encourage the growth of and encourage the growth of Gram negative rodsGram negative rods..

Other selective agents:Other selective agents: Methylene blueMethylene blue and and crystal violetcrystal violet inhibit inhibit

Gram positivesGram positives Selenite and brilliant greenSelenite and brilliant green are used in are used in

media to isolate media to isolate Salmonella Salmonella from feces.from feces. Sodium azideSodium azide is used to isolate enterococci is used to isolate enterococci

from water and food.from water and food.

The Conditions at Microbial Can The Conditions at Microbial Can Grow Optimum In A Medium Grow Optimum In A Medium

That is:That is: Medium must contain all easy Medium must contain all easy

nutrients be used by microbial. nutrients be used by microbial. Medium must has pressure osmosis, Medium must has pressure osmosis,

surface and pH appropriate surface and pH appropriate Medium doesn't contain hindrance Medium doesn't contain hindrance

substance substance Medium must sterileMedium must sterile

General vs Selective MediaGeneral vs Selective Media

Differential MediaDifferential Media

Differential mediaDifferential media grow several types grow several types of organisms and of organisms and display visible display visible differencesdifferences among among organisms.organisms.

Differences may Differences may show up as colony show up as colony size, media colour, size, media colour, gas bubble formation gas bubble formation and precipitate and precipitate formation.formation.

Selective/Differential MediaSelective/Differential Media

Mannitol salt agar is used to isolate members of the genus Staphylococcus

MacConkey agar differentiates between lactose-fermenting bacteria and (pink-red centre) and lactose-negative bacteria ( no pink colouration).

Differential Differential MediaMedia

♦ Triple sugar iron agar Triple sugar iron agar (TSIA). (TSIA).

♦ This media contain This media contain fermentable carbohydratesfermentable carbohydrates

♦ Red phenol to indicate pH Red phenol to indicate pH change change

♦ Iron that indicate HIron that indicate H22S gas S gas production. production.

♦ Rxns (left to right) are: Rxns (left to right) are:

♦ No growthNo growth

♦ Growth with no acidGrowth with no acid

♦ Acid production in the Acid production in the bottom onlybottom only

♦ Acid production in the Acid production in the bottom and Hbottom and H22S gas S gas formation (blackformation (black))

DifferentiDifferential Mediaal Media

Chromagar orientation uses colour-formation to distinguish at least 7 common urinary pathogens.

This allow for rapid identification and treatment.

In this example, the bacteria were streaked as to spell their names.

Characteristic MediaCharacteristic Media

Characteristic mediaCharacteristic media are used to are used to test bacteria for particular activity, test bacteria for particular activity, product or requirement.product or requirement.

E.g. urea broth used to detect for E.g. urea broth used to detect for urease. urease.

Kinds of medium is Medium of Kinds of medium is Medium of Potato Dextrosa Agar (PDA), PDB, Potato Dextrosa Agar (PDA), PDB, NA, NB, LB, LA, TEA, AND TEB.NA, NB, LB, LA, TEA, AND TEB.

..

NA (Nutrien Agar) Untuk Bakteri

Peptone………….................................................. 5,00 gBeef Extract ..........................................................3,00Bacteriological Agar........................................... 15,00Final pH 6,8 ± 0,2 at 25ºC

PDA (Potato Dextrose Agar) untuk Jamur

Potato(solids) ...................................................... 4,00 g Dextrose............................................................. 20,00Bacteriological Agar ...........................................15,00Final pH 5,6 ± 0,2 at 25ºC

Pembuatan Media

Direct Microscopic Direct Microscopic ExaminationExamination

Direct microscopic examinationDirect microscopic examination of a of a stained specimen is often the stained specimen is often the most rapid most rapid methodmethod for the identification of for the identification of characteristics.characteristics.

Stains include Stains include Gram, acid fast, direct Gram, acid fast, direct fluorescent antibody testfluorescent antibody test (DFA). (DFA).

DFA can be used to highlight the presences DFA can be used to highlight the presences of microorganisms in a specimen. of microorganisms in a specimen.

DFA test are available for DFA test are available for Staphylococcus Staphylococcus aureus, Streptococcus pyogenes, Neisseria aureus, Streptococcus pyogenes, Neisseria gonorhoeae gonorhoeae and and Haemophilus influenza.Haemophilus influenza.

Direct Direct ExaminationExamination

E. coli (white), Micrococcus luteus (yellow), Serratia marcescens (red)

Micrococcus luteus

Serratia marcescens

Direct Microscopic Direct Microscopic ExaminationExamination

Direct Direct Fluorescent Fluorescent

Antibody Antibody TestTest

Biochemical TestsBiochemical Tests The microbe is cultured in a media with a The microbe is cultured in a media with a

special substrate and tested for an end special substrate and tested for an end productproduct..

Prominent biochemical tests include Prominent biochemical tests include carbohydrate fermentation, acid or gas carbohydrate fermentation, acid or gas production and the hydrolysis of gelatin production and the hydrolysis of gelatin or starch.or starch.

Many of these test in rapid miniaturized system Many of these test in rapid miniaturized system that can detect for 23 characteristics in small that can detect for 23 characteristics in small cups called cups called Rapid testRapid test..

The info from the rapid test are input into a The info from the rapid test are input into a computer to help in identification of the computer to help in identification of the organisms. organisms.

Carbohydrate Carbohydrate FermentationFermentation

. This medium show . This medium show ffermentationermentation ( (acid acid productionproduction) and ) and gas gas formationformation..

The small The small Durham Durham tubetube for collecting gas for collecting gas bubbles.bubbles.

Left- right:Left- right:

Uninoculated Uninoculated negative controlnegative control

Centre, positive for Centre, positive for acid (yellow) and acid (yellow) and gas (open space).gas (open space).

Growth but no gas Growth but no gas or acid.or acid.

Methyl Red TestMethyl Red Test This is a qualitative This is a qualitative

test for test for acid acid productionproduction..

The bacteria is grown The bacteria is grown in MR-VP broth.in MR-VP broth.

After addition of After addition of several drops of several drops of methyl red solutionmethyl red solution a bright red colour is a bright red colour is positive and yellow-positive and yellow-orange negative.orange negative.

Nitrate Nitrate ReductionReduction

♫ After 24-48 hrs of After 24-48 hrs of incubation, incubation, nitrate reagentsnitrate reagents are added.are added.

♫ Left to rightLeft to right::♫Gas formation Gas formation

(positive for (positive for nitrate reduction).nitrate reduction).

♫positive for nitrate positive for nitrate reduction to nitrite reduction to nitrite ( red colour).( red colour).

♫Negative controlNegative control

Starch Starch HydrolysisHydrolysis

After incubation on After incubation on starch agarstarch agar, plates , plates are flooded with are flooded with iodine iodine solution.solution.

Positive test Positive test indicated by indicated by colourless areacolourless area around growth.around growth.

Negative test Negative test indicated below.indicated below.

Catalase Catalase TestTest

Place a drop ofPlace a drop of HH22OO22 on the on the culture.culture.

A positive reaction A positive reaction show gas bubbles.show gas bubbles.

Often used to Often used to differentiate differentiate StreptococcusStreptococcus from from Staphylococcus.Staphylococcus.

Biochemical TestsBiochemical Tests Other biochemical tests of interest Other biochemical tests of interest

include:include:HH22S productionS productionIndole testIndole testOxidase testOxidase testOxidation fermentationOxidation fermentationPhenylalanine deaminase testPhenylalanine deaminase testAntibiotic susceptibility testsAntibiotic susceptibility tests

Principle, procedure, most common use.Principle, procedure, most common use.

Rapid TestsRapid Tests

Rapid testRapid test: a biochemical system for the : a biochemical system for the identification of identification of EnterobacteriacaeEnterobacteriacae and and other Gram –ve bacteria.other Gram –ve bacteria.

It consist of It consist of plastic strips with 20 plastic strips with 20 μl of μl of dehydrated biochemical substratesdehydrated biochemical substrates used to detect biochemical characteristics.used to detect biochemical characteristics.

The biochemical The biochemical substrates are substrates are inoculatedinoculated with pure cultures and with pure cultures and suspended in physiological saline.suspended in physiological saline.

After 5 hrs-overnight the 20 tests are After 5 hrs-overnight the 20 tests are converted to converted to 7-9 digital profile. 7-9 digital profile.

Rapid Rapid Test Test

ResultsResultsOXI--0-ARA-2 2+AMY-0-

MEL--4+SAC-2 7+RHA-1+

SOR--4+INO-0 5-MAN-1+

GLU--4+GEL-0 4-VP--0

IND--4+TDA-0 4-URE-0-

H2S--0

-CIT-0 1-ODC-1+

LDC--4+ADH-0 5-ONPG-1+

normal 7 digit code5 144 572 = E. coli

Bacteriophage TypingBacteriophage Typing

Bacteriophage typing is based on the Bacteriophage typing is based on the specificity of phage surface receptorspecificity of phage surface receptor for the cell surface receptor.for the cell surface receptor.

Only those phages that can Only those phages that can attach to the attach to the surface receptorssurface receptors can cause lysis. can cause lysis.

The procedure involves:The procedure involves: A plate is A plate is heavily inoculatedheavily inoculated so that so that

there is no uninoculated areas.there is no uninoculated areas. The plate is The plate is marked offmarked off in squares (15-20 in squares (15-20

mm) and each square mm) and each square inoculatedinoculated with a with a drop of suspension for drop of suspension for different phagesdifferent phages..

Heavily Inoculated PlateHeavily Inoculated Plate

Bacteriophage TypingBacteriophage Typing

The plate is incubated for 24 hrs then The plate is incubated for 24 hrs then observed for plaques.observed for plaques.

The phage type is reported as a specific The phage type is reported as a specific genus and speciesgenus and species followed by followed by the the typestypes that can infect the bacterium. that can infect the bacterium.

E.g. 10/16/24 means that the bacteria is E.g. 10/16/24 means that the bacteria is sensitive to phages 10, 16 and 24.sensitive to phages 10, 16 and 24.

Phage tying remain a Phage tying remain a tool for researchtool for research and and reference labsreference labs..

Unculturable OrganismsUnculturable Organisms

Environmental researchers estimate Environmental researchers estimate that that < 1%< 1% of microorganisms of microorganisms are are culturableculturable and therefore it is not and therefore it is not possible to use phenotypic possible to use phenotypic methods of identification.methods of identification.

These microorganisms are called These microorganisms are called viable nonculturable (VNC).viable nonculturable (VNC).

Immunological MethodsImmunological Methods

The study of antibody (Ab)- antigen (Ag) rxns The study of antibody (Ab)- antigen (Ag) rxns in vitroin vitro is called is called serologyserology..

Serological rxns are the basic of Serological rxns are the basic of immunological identificationimmunological identification and diagnostic and diagnostic methods.methods.

The usefulness of serological test is dependent The usefulness of serological test is dependent on its on its sensitivity and specificitysensitivity and specificity..

Sensitivity is the ability to Sensitivity is the ability to detect minute detect minute amountsamounts of Ab or Ag. of Ab or Ag.

Specificity is the ability to Specificity is the ability to detect a single Ag detect a single Ag or Abor Ab..

False Negatives/PositivesFalse Negatives/Positives

High sensitivity prevents false High sensitivity prevents false negatives.negatives.

False negativesFalse negatives occurs when there occurs when there is not reaction when the Ag or Ab is is not reaction when the Ag or Ab is present.present.

High specificity prevents false High specificity prevents false positives.positives.

False positivesFalse positives occurs when there is occurs when there is cross reaction with another molecule.cross reaction with another molecule.

Precipitation ReactionsPrecipitation Reactions

Precipitation (ppt) is the interaction of a soluble Ag Precipitation (ppt) is the interaction of a soluble Ag with an soluble Ab to for an with an soluble Ab to for an insoluble complexinsoluble complex..

The complex formed is an aggregate of Ag and Ab.The complex formed is an aggregate of Ag and Ab. Ppt rxns occurs maximally only when the Ppt rxns occurs maximally only when the optimal optimal

proportionsproportions of Ag and Ag are present. of Ag and Ag are present. Ppt can also be done in agar referred to as Ppt can also be done in agar referred to as

immunodiffusionimmunodiffusion.. Ppt test uses antibodies to detect for Ppt test uses antibodies to detect for

streptococcal group antigens.streptococcal group antigens.

Precipitation ReactionsPrecipitation Reactions

Agglutination ReactionsAgglutination Reactions Agglutination Agglutination (Aggn) is the (Aggn) is the visible visible

clumpingclumping of an Ag when mixed with a of an Ag when mixed with a specific Ab.specific Ab.

Aggn tests are widely used because Aggn tests are widely used because they are simple to perform, highly they are simple to perform, highly specific, inexpensive and rapid.specific, inexpensive and rapid.

Standardized tests are available for Standardized tests are available for the determination of the determination of blood groupsblood groups and and identification of pathogens identification of pathogens and their productsand their products..

Agglutination ReactionsAgglutination Reactions

Genotypic methodsGenotypic methods

Genotypic methods of microbe Genotypic methods of microbe identification include the use of :identification include the use of :Nucleic acid probesNucleic acid probesPCR (RT-PCR, RAPD-PCR)PCR (RT-PCR, RAPD-PCR)Nucleic acid sequence analysisNucleic acid sequence analysisrRNA analysisrRNA analysisRFLPRFLPPlasmid fingerprinting.Plasmid fingerprinting.

Nucleic Acid ProbesNucleic Acid Probes

A single stranded probe is added and if there A single stranded probe is added and if there is is sequence complementalitysequence complementality between the between the target and the probe a positive hybridization target and the probe a positive hybridization signal will be detected.signal will be detected.

Hybridization is detected by a Hybridization is detected by a reporter reporter moleculemolecule (radioactive, fluorescent, (radioactive, fluorescent, chemiluminescent) which is attached to the chemiluminescent) which is attached to the probe.probe.

Nucleic acid probes have been marketed for Nucleic acid probes have been marketed for the identification of many pathogens such as the identification of many pathogens such as N. gonorrhoeaeN. gonorrhoeae..

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

PCR is widely used for the identification of PCR is widely used for the identification of microorganisms.microorganisms.

Sequence Sequence specific primersspecific primers are used with are used with PCR in the amplification of DNA or RNA of PCR in the amplification of DNA or RNA of specific pathogens.specific pathogens.

PCR allows for the detection even if PCR allows for the detection even if only a only a few cells are presentfew cells are present and can also be used and can also be used on on viable nonculturablesviable nonculturables (see sensitivity table)(see sensitivity table)..

The presence of the The presence of the appropriate amplified appropriate amplified PCR productPCR product confirms the presence of the confirms the presence of the organisms.organisms.

PrimersPrimers are available for the identification of are available for the identification of Niesseria gonorrhoeae, Niesseria gonorrhoeae, and to monitor food for the and to monitor food for the presence ofpresence of Salmonella Salmonella andand Staphylococcus. Staphylococcus.

Sensitivity of Microbe Detection Sensitivity of Microbe Detection TestsTests

MethodsMethods Toxin or organismToxin or organism SensitivitySensitivity

Flow CytometryFlow Cytometry S. Typhimurium S. Typhimurium in in milkmilk

101033/ml in 40 /ml in 40 minmin

Fluorescent Fluorescent antibodyantibody

SalmonellaSalmonella 101066/ml/ml

Latex Latex agglutinationagglutination

E. coli E. coli enterotoxinenterotoxin 32 ng/ml32 ng/ml

ELISAELISA C. perfringensC. perfringens type A type A toxintoxin

1 ng/ml1 ng/ml

PCRPCR E. coliE. coli 1-5 cells/100 1-5 cells/100 ml of Hml of H22OO

Real Time PCR and RT-PCRReal Time PCR and RT-PCR Currently many PCR tests employ Currently many PCR tests employ real time PCRreal time PCR.. This involves the use of This involves the use of fluorescent primersfluorescent primers.. The PCR machine The PCR machine monitors the incorporation of monitors the incorporation of

the primersthe primers and display an and display an amplification plotamplification plot which which can be viewed continuously thru the PCR cycle.can be viewed continuously thru the PCR cycle.

Real time PCR Real time PCR yields immediate resultsyields immediate results.. Another application of PCR is Another application of PCR is RT-PCRRT-PCR (reverse (reverse

trancriptase PCR).trancriptase PCR). During RT-PCR an RNA template is used to During RT-PCR an RNA template is used to generate generate

cDNA and from this dsDNAcDNA and from this dsDNA is generated. is generated. The enzyme used is The enzyme used is reverse transciptasereverse transciptase.. RT-PCR is used to detect for HIV and to monitor the RT-PCR is used to detect for HIV and to monitor the

progress of the disease.progress of the disease.

RT-PCRRT-PCR

RAPD-PCRRAPD-PCR Random amplified polymorphic DNA PCR Random amplified polymorphic DNA PCR

uses uses a random primer a random primer (10-mer) to (10-mer) to generate a DNA profile.generate a DNA profile.

What are random primers?What are random primers?

The primerThe primer anneals to several places on the anneals to several places on the DNA template and generate a DNA profile DNA template and generate a DNA profile which is used for microbe identification.which is used for microbe identification.

RAPD has many advantages: RAPD has many advantages: Pure DNA is not neededPure DNA is not neededLess labour intensive than RFLP.Less labour intensive than RFLP.There is not need for prior DNA sequence data.There is not need for prior DNA sequence data.

RAPD has been used to fingerprint the RAPD has been used to fingerprint the outbreak of outbreak of Listeria monocytogenesListeria monocytogenes from from milk.milk.

PCR vs PCR vs RAPD-PCRRAPD-PCR

DNA sequencingDNA sequencing The determination of a small amount of DNA The determination of a small amount of DNA

sequence can be used for microbial sequence can be used for microbial identification.identification.

The most common sequence used for The most common sequence used for microbe identification is DNA sequence of microbe identification is DNA sequence of the the 16S rRNA gene16S rRNA gene..

PCR is used to amplify the 16S rRNA gene PCR is used to amplify the 16S rRNA gene and the sequence determined.and the sequence determined.

rRNA is a major component for ribosome and rRNA is a major component for ribosome and ribosome have the same functionribosome have the same function in in protein synthesis in all cells.protein synthesis in all cells.

DNA SequencingDNA Sequencing

Computer analysis of 16S rRNA sequence has Computer analysis of 16S rRNA sequence has revealed the presence of revealed the presence of signature signature sequencessequences, short oligonucleotides , short oligonucleotides unique unique to certain groups of organismsto certain groups of organisms and useful and useful in their identification.in their identification.

rRNA sequence can be used to fine tune rRNA sequence can be used to fine tune identity at the species level e.g differentiating identity at the species level e.g differentiating between between Mycobacterium Mycobacterium and and LegionellaLegionella..

16s rRNA sequence can also be used 16s rRNA sequence can also be used to to identify microorganisms from a identify microorganisms from a microbial communitymicrobial community..

Restriction Fragment Length Restriction Fragment Length PolymorphismPolymorphism

RFLP involves RFLP involves digestiondigestion of the genomic of the genomic DNA of the organism DNA of the organism with restriction with restriction enzymes.enzymes.

The restricted fragments are The restricted fragments are separatedseparated by agarose gel by agarose gel electrophoresis.electrophoresis.

The DNA fragments The DNA fragments are transferred to are transferred to a membranea membrane and and probed probed with probes with probes specific for the desired organisms.specific for the desired organisms.

A DNA profile emerges which can be A DNA profile emerges which can be used for microbe identification. used for microbe identification.

RFLP of RFLP of M. M. tuberculosis tuberculosis

from 17 from 17 patientspatients

Plasmid fingerprintingPlasmid fingerprinting What is a plasmid?What is a plasmid?

Plasmid fingerprinting identifies Plasmid fingerprinting identifies microbial microbial species or similar strainsspecies or similar strains as related as related strains often contain the same number of strains often contain the same number of plasmids with the same molecular weight.plasmids with the same molecular weight.

Plasmid of many strains and species of Plasmid of many strains and species of E. E. coli, Salmonella, Camylobacter coli, Salmonella, Camylobacter and and PsseudomonasPsseudomonas has demonstrated that this has demonstrated that this methods is methods is more accuratemore accurate than phenotypic than phenotypic methods such as biotyping, antibiotic methods such as biotyping, antibiotic resistance patterns , phage typing and resistance patterns , phage typing and serotyping.serotyping.

Plasmid fingerprintingPlasmid fingerprinting

The procedure involves:The procedure involves: The bacterial strains The bacterial strains

are are growngrown, the cells , the cells lysed and harvested.lysed and harvested.

The plasmids are The plasmids are separatedseparated by agarose by agarose gel electrophoresisgel electrophoresis

The gels are The gels are stained stained with EtBrwith EtBr and the and the plasmids located and plasmids located and compared.compared.